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Blackeye cowpea mosaic virus

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Title:
Blackeye cowpea mosaic virus purification, partial characterization, serology, and immunochemical and cytological techniques for detection of virus-infected legume seeds
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Lima, J. Albersio A., 1940-
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xiii, 154 leaves : ill. (some col.) ; 28 cm.

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Subjects / Keywords:
Antiserum ( jstor )
Cytoplasmic inclusions ( jstor )
Hypocotyls ( jstor )
Immunodiffusion ( jstor )
Phytopathology ( jstor )
Purification ( jstor )
Seedlings ( jstor )
Seeds ( jstor )
Soybeans ( jstor )
Viruses ( jstor )
Cowpea -- Diseases and pests ( fast )
Dissertations, Academic -- Plant Pathology -- UF
Mosaic diseases ( fast )
Plant Pathology thesis Ph. D
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis--University of Florida.
Bibliography:
Includes bibliographical references (leaves 138-153).
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Also available online.
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Typescript.
General Note:
Vita.
Statement of Responsibility:
by J. Albersio A. Lima.

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BLACKEYE COWPEA MOSAIC VIRUS: PURIFICATION, PARTIAL CHARACTERIZATION,
SEROLOGY, AND IMMUNOCHEMICAL AND CYTOLOGICAL TECHNIQUES
FOR DETECTION OF VIRUS-INFECTED LEGUME SEEDS














By

J. ALBERSIO A. LIMA











A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY











UNIVERSITY OF FLORIDA 1978








































To my wife, Diana, and my son, Roberto, who with understanding, friendship, and love helped to transform a goal into a reality.
















ACKNOWLEDGEMENTS


I wish to express my sincere gratitude and appreciation to Dr. Dan E. Purcifull, chairman of my supervisory committee, for his invaluable counsels, friendship, advice, and constant guidance during the course of this investigation.

Appreciation is extended to other members of my s upervisory committee, Drs. Ernest Hiebert, John R. Edwardson, Raghavan Charudattan, Francis W. Zettler, and Daniel A. Roberts for their helpful suggestions during the research and their efforts in criticizing the manuscript. I also wish to extend my appreciation to Mr. Richard G. Christie for his valuable help with the light microscope and for his constant enthusiasm for teaching useful cytological techniques for diagnosing plant virus diseases. The understanding and cooperation of Mr. S. Christie, Mr. W. Crawford, Mrs. J. Hill, and Mrs. D. Miller during the laboratory experiments are also greatly appreciated.

I further wish to extend my gratitude to Mrs. Maria I. Cruz for

her understanding and cooperation as the Secretary of the International Programs of the University of Florida, Gainesville, and for her time spent in typing this dissertation.

I was supported by funds from the United States Agency for International Development (USAID), Universidade Federal do Cearg, and Ford Foundation, to whom I wish to express my sincere thanks.

Special recognition is expressed to my wife, Diana, whose patience friendship, and love made this work possible.



i i i











TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS. ... .........................................iii

LIST OF TABLES ................................. ........... vi

L IST OF FIGURES....... ................ .. ..................... v ii

VIRUS ABBREVIATIONS.......................................... x

ABSTRACT ........................................... .... xi

CHAPTER I PURIFICATION, PARTIAL CHARACTERIZATION, AND
SEROLOGY OF BLACKEYE COWPEA MOSAIC VIRUS...........

Introduction.................................. I
Literature Review ............................. 2
MatE rials and Methods .......................... 10
Sources of Virus Isolate ................... 10
Virus and Inclusion Purification........... I1
Virus Particle Size Determination.......... 14 Stability of Virus in Sap.................. 15
Polyacrylamide Gel Electrophoresis of Viral and Inclusion Proteins..................... 16
Sedimentation Coefficient Determination.... 17 Serology... .. ................ ................. 18
Antiserum production for virus and cytoplasmic inclusions ................. 18
Serological tests ....................... 19
Serological relationships between B1CMV and other potyviruses............ ..... 21
Light and Electron Microscopy of Virus Induced Pinwheel Inclusions ............... 22
Host Range and Screening Cowpea Varieties for Resistance ............................ 23

Resu lts .......... ............................. 24
Purification and Properties of Blackeye Cowpea Mosaic Virus ........................ 24
Purified Inclusion Preparations............. 42
Virus Particle Size and Stability in Sap... 47 Serology .................................... 52
Light and Electron Microscopy ............. 64
Host Range and Resistant Cowpea Varieties.. 65 Discussion .............. ..................... 71





iv











Page

CHAPTER II IMMUNOCHEMICAL AND CYTOLOGICAL TECHNIQUES FOR
DETECTION OF LEGUME VIRUSES IN INFECTED SEEDS.... 84
l troduction ................ ................. 84
Literature Review............... .............. 86
Seed-Borne Viruses in Vigna spp ............ 87
Seed-Borne Viruses in Glycine max.......... 90
Seed-Borne Viruses in Phaseolus vulgaris... 92 Materials and Methods .....................9.. 94
Source of Seed and Seed Germination........ 94 Preparation of Antigens for Serology....... 95 Double Immunodiffusion Tests ............... 95
Single Radial Immunodiffusion Tests........ 96 Serologically Specific Electron Microscopy. 97 Double Immunodiffusion Tests and SSEM for Detection of Other Viruses in Germinated Legume Seeds........ ..................... 98
Serology and Microscopy of Cytoplasmic Inclusions Induced by BICMV and SoyMV in Hypocotyls of Germinated Seeds.......... 98 ResuIts........................................... 99
Preparation of Antigens for Serological Tests ...................................... 99
Double Inmunodiffusion Tests............... 102
Single Radial Immunodiffusion Tests ........ 107 Serologically Specific Electron Microscopy.................................... ill
Double Immunodiffusion Tests and SSEM for Detection of Other Viruses in Germinated Legume Seeds .... ........ ....... 116
Serology and Mycroscopy of Cytoplasmic Inclusions Induced by BICMV and SoyMV in Hypocotyls of Germinated Seeds............. 121
Discussion........................................ 132

LITERATURE CITED... .................... ,.,.............1.... ]13

BIOGRAPHICAL SKETCH................ ................. .. ......... 154












V











LIST OF TABLES


Table Page

I Symptoms and results of serological assays on
varieties of cowpea, Vignaunguiculata mechanically
inoculated with BICMV, BCMV-S, CAMV, and CPMV....... 70

II Comparison of immunodiffusion tests with hypocotyl
discs and growing-on tests for detection of virusinfected needs...................................... 108




















11 1 ,..~~.... ~












LIST OF FIGURES

Figure Page

I Systemic and localized symptoms induced by blackeye
cowpea mosaic virus (BICMV) in cowpea, V. unguiculata
'Knuckle Purple Hull' and C. amaranticolor............ 26

2 Flow diagram outlining the procedure of purification
of BICMV using n-butanol as clarifying agent......... 28

3 Flow diagram outlining the procedure for purification
of BICMV and its cytoplasmic inclusions, using chloroform and carbon tetrachloride as clarifying
agents .............. ..................... 30

4 Flow diagram outlining the steps carried out during
the purification of BICMV and its cytoplasmic inclusions by a combination of the first and second methods
for purification of virus and inclusions.............. 32

5 Absorption spectra of purified preparations of BICMV
in 0.02 M Tris buffer, pH 8.2, and BICMV cytoplasmic
inclusions in the same buffer........................ 35

6 Electron microscopy of BICMV in a purified preparation
and in cowpea leaf extracts .........................

7 Histograms of lengths of BICMV particles from purified
preparation negatively stained with phosphotungstate, and cowpea leaf extract using the serologically specific electron microscopy and uranyl acetate as a 39
positive stain.....................................

8 Histograms of BICMV particle lengths from two different
electron microscopic preparations to show particle
length distribution from 600 to 900 nm............... 41

9 Schlieren patterns from sedimentation velocity experiment with stored and fresh purified preparations of
BICMV.................. ............................... 44

10 Electrophoretic analyses of BICMV induced cytoplasmic
inclusions and BICMV capsid protein in 6% polyacrylam ide ge l...... ....................................... 46

11 Electron micrographs of purified preparations of
BICMV cytoplasmic inclusions stained with molybdate.. 49




vii











Figure Page

12 Double inmmunodiffusion tests in agar medium containing
0.8 Noble aqcar, I.0% NaN3, and 0.5% SDS............. 51

13 Single radial diffusion tests in agar media containing
different concentrations of SDS and antisera for BICMV
and cowpea mosaic virus (CPMV) ...................... 54

14 Single radial diffusion tests with SDS and pyrrolidine
degraded capsid protein of BICMV and CPMV............ 56

15 Reciprocal double immunodiffusion tests with BICMV
and other potyviruses in medium containing 0.8% Noble agar, 1.04 NaN, and 0.5% SDS prepared in 0.05 M TrisHCI buffr pH 7.2................................... 61

16 Immunodiffusion tests with BICMV, Moroccan isolate of
cowpea aphid-borne mosaic virus (CAMV), and siratro strain of bean common mosaic virus (BCMV-S) in agar
medium containing 0.8% Noble agar, 1.0% NaN and
0.5% SDS prepared in 0.05 M Tris-HCI buffer, pH 7.2. 63

17 Photomicrographs of cytoplasmic inclusions in
epidermal strips of cowpea leaves systemically infected with BICMV, stained with a combination of
calcomine orange and luxol brilliant green........... 67

18 Electron micrographs of ultrathin sections of cowpea
leaf cells infected with BICMV showing cross-sections
and longitudinal sections of pinwheel inclusions..... 69

19 Double irimunodiffusion tests with extracts from different portions of BICMV-infected and healthy
4-5-day-old cowpea seedlings......................... 101

20 Diagram showing methods for assaying legume seeds by single and double radial immunodiffusion.......... 104

21 Double immunodiffusion tests with hypocotyls from healthy and BlCMV-infected, 4-5-day-old cowpea seedlings in medium containing 0.8% Noble agar,
1.0% NaN3, and 0.5t SDS, prepared in water........... 106

22 Single radial immunodiffusion tests with hypocotyl extracts from healthy and BICMV-infected, 4-5-dayold cowpea seedlings................................. 110

23 Electron micrographs of BICMV in hypocotyl extracts from the same cowpea seedling using different
preparations................................ ......... 113


viii










Figure Page

24 Electron micrographs of serologically specific electron microscopy with BICMV, BCMV-S, and CPMV..... 115

25 Double immunodiffusion tests with hypocotyls of 4-5day-old Lean and soybean seedlings using antiserum
for BCMV-S and for SoyMV ............................. 118

26 Electron micrographs of serologically specific electron microscopy with extracts from BCMV- and
SoyMV-intected hypocotyls............................ 120

27 Double inmmnunodiffusion tests with hypocotyl extracts from 4-5-day-old seedlings of cowpea and soybean using antisera for BICMV, SoyMV, and their cytoplasm ic inclusions................................... 123

28 Photomicrographs showing different views of cytoplasmic inclusions induced by BICMV in epidermal strips
of cowped hypocotyl tissue stained with a combination
of calcomine orange and luxol brilliant green ........ 125

29 Photomiciographs showing different views of epidermal cells of hypocotyls from 4-5-day-old soybean seedlings
containing cytoplasmic inclusions induced by SoyMV... 127

30 Electron micrographs of ultrathin sections of cells from hypocotyls of 4-5-day-old seedlings infected
with BICMV .......................................... 129

31 Electron micrographs of ultrathin sections of hypocotyl cells of 4-5-day-old soybean seedlings
grown from SoyMV-infected seeds....................... 131





















ix











VIRUS ABBREVIATIONS

Virus Names Abbreviation

Bean common mosaic virus............................. BCMV
Bean common mosaic virus-siratro isolate............... BCMV-S
Bean pod mottle virus .................. ................ BPMV
Bean yellow mosaic virus......... .. ............... ..... BYMV
Bidens mottle virus............................................... BiMV
Blackeye cowpea mosaic virus ........................... BICMV
Clover yellow vein virus.............................. CYVV
Commelina mosaic virus.................................. CoMV
Cowpea aphid-borne mosaic virus................................. ..CAMV
Cowpea chlorotic mottle virus........................ CCMV
Cowpea mild mottle virus................................ CMMV
Cowpea mosaic virus..................................... CPMV
Cowpea ringspot virus ....... ......................... CpRV
Cowpea yellow mosaic virus ............................. CYMV
Cucumber mosaic virus ................................. CMV
Dasheen mosaic virus ................................... DMV
Iris mosaic virus ...................................... IMV
Lettuce mosaic virus.. ................................ LMV
Pea seed-borne mosaic virus............................ PSMV
Pepper mottle virus ........................................ PeMV
Pepper vein mottle virus............................... PVMV
Pokeweed mosaic virus .................................. PWMV
Potato virus X .......................... ............... PVX
Potato virus Y......................................... PVY
Southern bean mosaic virus ............................ SBMV
Soybean mosaic virus .................................... SoyMV
Sugarcane mosaic virus. ................................ SMV
Tobacco etch virus...................................... TEV
Tobacco mosaic virus................................... TMV
Tobacco ringspot virus ................................ TRSV
Turnip yellow mosaic virus............................ TuMV
Watermelon mosaic virus-I...... .. .. .... .. ......... WMV-1
Watermelon mosaic virus-2........ ..................... WMV-2


x











Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements foi the Degree of Doctor of Philosophy BLACKEYE COWPEA MOSAIC VIRUS: PURIFICATION, PARTIAL CHARACTERIZATION,
SEROLOGY, AND IMMUNOCHEMICAL AND CYTOLOGICAL TECHNIQUES
FOR DETECTION OF VIRUS-INFECTED LEGUME SEEDS By

J. ALBERSIO A. LIMA

March, 1978

Chairman; Dan E. Purcifull
Major Department: Plant Pathology

Blickeye cowpea mosaic virus (BICMV) was increased in cowpea, Vigna unguiculata (L.) Walp., 'Knuckle Purple Hull', and infected leaves were used for virus and cytoplasmic inclusion purification. Either n-butanol or a combination of
consisted of a main protein component with a MW of 34,000 daltons and two smaller proteins with MWs of 29,000 and 27,000 daltons. Purified BlCMV had a 260/280 nm absorption ratio of 1.2 and a modal length of 753 nm. Freshly purified BICMV preparations showed a single sedimenting peak with s20=157-159 S. The purified BICMV cytoplasmic inclusions had absorption spectra characteristic for proteins. Electron microscopy of purified inclusions revealed the presence of tubes showing striations with periodicities of approximately 5 nm.




Xi










Antisera reactive in SDS-immunodiffusion were obtained against

untreated virions, pyrrolidine degraded coat protein,and untreated BICMV cytoplasmic inclusions. Reciprocal double immunodiffusion tests with SDS-treated antigens showed that BICMV is serologically unrelated to seven potyviruses and serologically related to, but distinct from: bean common mosaic virus (BCMV), bean yellow mosaic virus (BYMV), cowpea aphid-borne mosaic virus (CAMV), dasheen mosaic virus (DMV), lettuce mosaic virus (LMV), potato virus Y (PVY), soybean mosaic virus (SoyMV), tobacco etch virus (iEV), and watermelon mosaic virus-2 (WMV-2). The intragel cross-absorption technique was also used to demonstrate distinction between closely related potyviruses. Agar medium impregnated with a mixture of antiseroi was used for serodiagnosis of BICMV and cowpea mosaic virus in cowpa.

Light and electron microscopy of cytoplasmic inclusions induced by BICMV, siratro (Macroptilium atropurpureum (D.C.) Urb.) strain of BCMV (BCMV-S) and CAMV revealed that they are similar to those induced by the potyviruses from Edwardson's subdivision-I. The different reactions induced by BICMV, BCMV-S, and CAMV in some cowpea varieties indicated that they can also be used as differential hosts for these three potyviruses. Sources of resistance for BICMV were found among the cowpea varieties tested. Based on its physical, biological, cytological, and immunochemical properties, BICMV can be differentiated from any other virus that infects cowpea.

Cytoplasmic inclusions induced by BICMV in cowpea and by SoyMV in soybean were detected by serology, light microscopy, and electron microscopy in hypocoiyls of 4-5-day-old seedlings grown from virusinfected seeds.

xii










Inmunodiffusion tests and serologically specific electron microscopy were used to d tect BICMV in hypocotyls of 4-5-day-old cowpea seedlings grown from BICMV-infected seeds. Discs of individual hypocotyls were embedded into the agar medium 4-5 mm away from the antiserum wells. Virus-specific precipilin lines formed between virusinfected hypocotyl discs and antiserum wells, whereas no reactions were observed with healthy hypocotyls. Precipitin lines were also observed with extracts of mixtures from infected (1 g) and healthy (up to 29 g) tissues These immunochemical techniques were also used for detecting BCMV ii hypocotyls of infected 4-5-day-old Phaseolus vulgaris L. seedlinys and for detecting SoyMV in infected Glycine max

(L.) Merr. seedlings. Single radial immunodiffusion tests with extracts or discs of cowpea hypocotyls were also useful for detecting BICMV in germinated ,eeds. The reliability and simplicity of the immunodiffusion tests make them suitable for use in routine seed health testing program in any laboratory.























xiii
















CHAPTER I

PURIFICATION, PARTIAL CHARACTERIZATION, AND
SEROLOGY OF BLACKEYE COWPEA MOSAIC VIRUS


Introduction

Cowpea, Vigna unguiculata (L.) Walp. (=Vigna sinensis (L.) Endl.), is grown as a crop in high-temperature areas of tropical and subtropical countries. Cowpea seeds constitute a source of good quality protein and dried seeds are an important part of the diet of many people in the tropical and subtropical world, particularly in Africa and the rural zone of northeastern Brazil. The fresh seeds and immature pods are also eaten and they can be frozen or canned as is sometimes done in the United States. Cowpeas are also grown as fodder plants for hay, silage or pasture and used as a green manure and cover crop. When grown under optimum conditions, cowpea can produce seed yields as high as 2,600 Kg/ha. However, several factors limit cowpea yields in most fields. Virus diseases are considered as a major limiting factor to the production of cowpeas in several countries (Dale, 1949; Wells and Deba, 1961; Toler et al., 1963; Brantley et al., 1965; Kuhn et al., 1966; Harrison and Gudauskas, 1968a; Harrison and Gudauskas, 1968b; Gay and Winstead, 1970; Zettler and Evans, 1972; Bock, 1973; Phatak, 1974; Haque and Persad, 1975; Kaiser and Mossahebi, 1975; and Lima and Nelson, 1977). Several viruses infect cowpea, and many of them can be transmitted through seeds from infected cowpea plants. The most important cowpea seed-borne virus in the southeastern United States is


1






2



an aphid-transmitted, filamentous virus approximately 750 nm long (Harrison and Gudauskas, 1968a; 1968b; Gay and Winstead, 1970; Zettler and Evans, 1912; and Uyemoto et al., 1973). This virus was first isolated in Florida by Anderson (1955a), who designated it "blackeye cowpea mosaic virus" (BICMV) (Anderson, 1955b), a name that has been retained by Zettler and Evans (1972) and Edwardson et al. (1972).

Because no antiserum specific for BICMV was available, and because only sparse information about the virus properties could be found in the literature, the first part of this research was undertaken to purify and characterize BICMV in vitro and in vivo. Antisera prepared for BICMV and its cytoplasmic inclusions were used for serological characterization of the virus. Some methods for virus and inclusion purification, as well as certain physical, biological, immunochemical and cytological properties of BICMV were described in the present investigation. An abstract of part of this research was already published (Lima et al., 1976).


Literature Review


Several viruses infect cowpea, V. unguiculata, causing different

types of mosaic. The first report about mosaic of cowpea was published in 1921 by Elliot, who reported a high incidence of cowpea virus disease in Arkansas (Elliot, 1921). Smith (1924) demonstrated experimentally that the cowpea virus was transmitted either by rubbing the leaves of diseased and healthy plants together or by the bean leaf beetle, Ceratoma trifurcata Forst. Subsequently, Gardner (1927) working with a cowpea virus, observed that it was transmitted through seeds of certain cowpea varieties.











A widespread mosaic disease was reported on different cowpea varieties in Trinidad (Dale, 1949). Dale (1949) observed that the virus responsible for the disease was not transmitted by Aphis medicaginis Koch, but that the leaf beetle, Ceratoma ruficornis (Oliv.) was a good vector and was probably responsible for transmitting the virus in the field. On the basis of his studies, he concluded that the virus was unrelated to those described by McLean (1941), Snyder (1942), and Yu (1946), but was more likely the virus studied by Smith (1924). Dale (1953) subsequently confirmed that the cowpea mosaic virus isolated from Trinidad was efficiently transmitted by C. ruficornis, but not by aphids.

Lister and Thresh (1955) isolated a virus from cowpea and identified it as a strain of tobacco mosaic virus (TMV). They observed that a purified preparation of the virus contained rod-shaped particles of varying lengths, indistinguishable from the particles of TMV, and was precipitated specifically with antiserum prepared against TMV. A cowpea strain of TMV was also isolated from a range of leguminous hosts at Ibadan, Nigeria (Chant, 1959). Chant (1959) also found another virus infecting cowpea in Nigeria and as its physical properties differed from other cowpea viruses, he proposed the name cowpea yellow mosaic virus (CYMV). The virus was purified and an antiserum prepared against it. Both TMV and CYMV were transmitted by the beetle Ootheca mutabilis Sahlb. In subsequent work, Chant (1960) studied the influence of TMV and CYMV on growth rate and yield of cowpea, and found that infection of cowpea with the cowpea strain of TMV did not affect yield as much as infection with CYMV. Wells and Deba (1961) tested 116 cowpea varieties and 342 indigenous pure lines against CYMV and observed










that 6 varieties and 16 pure lines were resistant. Robertson (1965) screened 79 cowpea varieties for resistance to CYMV in a screened greenhouse. Those varieties that showed no local or systemic reactions when inoculated with the virus were classified as immune; those that developed necrotic lesions but did not become systemically infected were classified as resistant; and those that showed systemic infection were classified as susceptible. Chant (1962) found that the cowpea virus from Trinidad caused local lesions on Chenopodium amaranticolor Coste and Reyn., Mucuna atterrina Holland, Petunia hybrida Vilm., and P. vulgaris, and that the virus was polyhedral with a mean diameter of approximately 25 nim.

Double-immunodiffusion tests showed that a cowpea virus from Arkansas and the Trinidad cowpea mosaic virus were closely related, but not identical serologically and that both were antigenically related to bean pod mottle virus (BPMV) (Shepherd, 1963). Studying other properties of the virus, Shepherd (1964) confirmed a close similarity of the Arkansas virus with the cowpea mosaic virus from Trinidad (Dale, 1949). Walters and Barnett (1964), working with a cowpea mosaic virus serologically identical to the Arkansas isolate, demonstrated also that it was efficiently transmitted by the bean leaf beetle, C. trifurcata. A detailed study of three cowpea mosaic virus isolates from Surinam (South America), along with the previously reported cowpea viruses from Trinidad (Dale, 1949) and Nigeria (Chant, 1959, 1960, 1962) revealed that they are strains of cowpea mosaic virus (Agrawal, 1964). Detailed descriptions of host range, biophysical, biochemical, and immunochemical properties of cowpea mosaic virus were reported, and the abbreviation CPMV was proposed to eliminate any possible confusion






5



with CMV (cucumber mosaic virus). Cowpea mosaic virus (CPMV) has been extensively studied in different laboratories and was fully described by van Kammen (1971, 1972). It was selected as the type member of the comovirus group (Fenner, 1976) and reported from several other parts of the world, including Brazil (Carner et al., 1969; and Lima and Nelson, 1977), Nigeria (Williams, 1975), Venezuela (Debrot and Rojas, 1967), and Puerto Rico (Perez and Cortes-Monllor, 1970; and Alconero and Santiago, 1973).

Kuhn (1964b) purified and characterized a new virus isolated from cowpea in Georgia and named it cowpea chlorotic mottle virus (CCMV), which was subsequently described by Bancroft (1971). This virus belongs to the bromovirus group (Fenner, 1976) and is physically similar to brome mosaic virus (Bancroft, 1970) and broad bean mottle virus (Gibbs, 1972), neither of which produces symptoms in cowpea (Bancroft, 1971).

Strains of cucumber mosaic virus (CMV) are also known to infect

cowpea. Cucumber mosaic virus strains have been isolated from naturally infected cowpeas showing mosaic symptoms in southeastern United States (Anderson, 1955a; Kuhn, 1964a; and Harrison and Gudauskas, 1968a), Italy (Vovlas and Avgelis, 1972), Morocco (Fischer and Lockhart, 1976b), and South Africa (Klesser, 1960). An aphid-transmitted, spherical virus, approximately 25 nm in diameter, was also reported from India by Chenulu et al. (1968). According to their descriptions, the virus closely resembles a strain of CMV.

Shepherd and Fulton (1962) identified a seed-borne virus of cowpea as a strain of southern bean mosaic virus (SBMV) (Shepherd, 1971). Although a virus isolated from naturally infected cowpea in Arkansas






6




had properties somewhat similar to the cowpea strain of SBMV, the two viruses were not serologically related (Shepherd, 1963).

A carlavirus isolated from cowpea in Ghana was described and designated as cowpec mild mottle virus (CMMV) by Brunt and Kenten (1973) and Brunt (1974). Cowpea mild mottle virus is seed-borne in cowpeas, is 650 nm in length and is apparently not transmitted by aphids.

A virus with small isometric particles, isolated from Iranian cowpea seeds was considered as new and named cowpea ringspot virus (CpRV) on the basis of symptomatology and particle morphology, which were similar to uther ringspot viruses (Phatak, 1974;and Phatak et al., 1976). According to Phatak (1974), the virus was not transmitted by aphids, induced intracellular inclusions in cowpea, had a wide experimental host range and was serologically unrelated to 40 other isometric viruses most of which commonly infect various legumes. Cowpea ringspot virus was also transmitted in 15-20% of the seeds of three cowpea cultivars (Phatak et al., 1976).

McLean (1941) studied some physical and biological properties of a cowpea virus and found that it was transmitted by the following species of aphids: Macrosiphum solani Ashm., Acynthosiphon pisum (Harris), Aphis gossypii Glover, Myzus persicae (Sulz.), but not by the bean leaf hopper (Empoasca fabae Le. B.), the tarnished plant bug (Lygus pratensis L.), the Mexican bean beetle (Epilachra corrupta Mls.), and the striped cucumber beetle (Diabrotica vittata Faba). Snyder (1942) described a mosaic disease of asparagus bean, Vigna sesquipedalis Wight, and also studied some biological and physical properties of the causal agent. His positive results obtained with aphid transmission








indicated that these viruses were not identical to the one described by Smith (1924). A cowpea virus similar to those described by McLean (1941) and Snyder (1942) was reported from China by Yu (1946). The virus which was transmitted by aphids was also seed-borne in cowpea. In addition to cowpea, the virus also infected lima bean and adzuki bean, Phaseolus angularis (Willd.) Wight (=Vigna angularis (Willd.) Ohwi. and Ohshi) (Yu, 1946), Cowpea viruses apparently similar to those were also reported from Ceylon (Abrygunawardena and Perera, 1964), Germany (Brandes, 1964), India (Nariani and Kandaswany, 1961), and New Guinea (van Velsen, 1962).

An aphid-borne virus isolated from cowpea in northern Italy was studied by Lovisolo and Conti (1966), and designated as cowpea aphidborne mosaic virus (CAMV). The virus was a rod, approximately 750 nm long, and was seed-Lorne in cowpea, but appeared to be clearly different from BICMV isolated in Florida (Anderson, 1955b), As reported by Lovisolo and Conti (1966), the virus was first recorded and described in Italy by Vidano (1959) and Rui (1960). The virus was transmitted in a non-persistent manner by M. Persicae, Aphis fabae Scop., A. medicaginis Koch, A. gossypii, and Macrosiphum euphorbiae (Thomas) (Vidano and Conti, 1965). A similar virus was later isolated in East Africa and three strains of this virus were differentiated by host range and serology (Bock, 1973). It was also observed that CAMV is distantly serologically related to bean common mosaic virus (BCMV) (Lovisolo and Conti, 1966; and Bock, 1973), but no direct serological relationship was detected with the African type strain of CAMV and potato virus Y (PVY), bean yellow mosaic virus (BYMV), pea seed-borne mosaic virus (PSMV), clover yellow vein virus (CYVV), soybean mosaic virus (SoyMV), sugarcane mosaic virus (SMV),










tobacco severe etch virus (TEV), and iris mosaic virus (IMV) (Bock, 1973; and Bock and Conti, 1974). A seed-transmitted virus tentatively identified as CAMV was considered to be responsible for the most important and widespread disease of cowpeas in Iran (Kaiser et al., 1968). Additional studies bout various properties of the Iranian isolate of CAMV indicated its similarity to the Italian and African isolates (Kaiser and Mossahebi, 1975). A CAMV isolate was also reported from Japan infecting adzuki bean, P. angularis, under natural conditions (Tsuchizaki et al., 1970). Fisher and Lockhart (1976a) isolated a rod-shaped virus from severely infected cowpeas in Morocco and identified it as a strain of CAMV on the basi:; of its particle length, aphid-transmission, host range, serology, and physical properties. The Moroccan isolate differed from those CAMV isolates previously described (Lovisolo and Conti, 1966; Bock, 1973; and Bock and Conti, 1974) by failing to infect Ocimum basilicum L., a diatlnostic species for CAMV (Bock and Conti, 1974), and other plants reported to be systemic hosts for CAMV. Padma and Summawar (1973) indicated the value of Chenopodium murale L. as a good indicator host for differentiation, screening and isolation of a rodshaped cowpea virus and the icosahedral CPMV. Cytoplasmic inclusions were observed in plant cells infected with CAMV (Inouye, 1973; and Nicolaeseu et al., 1976).

A virus isolated from cowpea in India (Khatri and Singh, 1974) was reported to be a strain of CPMV. However, the authors reported aphid transmission of this virus, so its identification as a strain of CPMV is questionable. A filamentous virus approximately 750 nm in length isolated froiii cowpeas in Ghana did not react with antisera specific for CAMV, peanut mottle virus, BCMV, and BYMV (Brunt, 1974).











An aphid-transmitted virus was responsible for complete loss of cowpea in irrigated areas of northern Nigeria (Raheja and Leleji, 1974), Based on the fact that the virus was neither mechanically transmitted nor seed-borne in cowpe,,, Raheja and Leleji (1974) concluded that it was either an atypical strain of CAMV or a new virus not previously described.

A virus isolated from Crotalaria spectabilis Roth in a field at Gainesville, Floridi, was studied by Anderson (1955b) and designated blackeye cowpea mosaic virus (BICMV). Anderson (1955b, 1955c) reported that BICMV infected plants of cowpea, Crotalaria and Desmodium in the field, but considered Crotalaria and Desmodium as secondary hosts for the virus. In a subsequent study, Anderson (1959) observed that BICMV was transmitted by M. persicae but not by the bean leaf beetle, C. trifurcata. Cobett (1956) found that BICMV was serologically related to BYMV and identified it as a strain of BYMV. Based on Corbett's conclusion, several subsequent reports have referred to BICMV as a cowpea strain of BYMV (Brierly and Smith, 1962; Kuhn, 1964a; Kuhn et al., 1965; and Harrison and Gudauskas, 1968a).

Light and electron microscopic studies of BICMV and BYMV showed marked cytological differences between these two flexuous rod-shaped viruses (Edwardson et al., 1972). According to Edwardson et al. (1972), the cytoplasmic inclusions induced by BICMV were consistently different from those induced by BYMV. In the light microscope, groups of plates were observed in cells of BYMV-infected tissues, whereas groups of tubes were seen in cells of epidermal strips obtained from BlCMV-infected tissue. Electron microscopy of ultrathin sections indicated that BICMV-induced cytoplasmic inclusions consisted of pinwheels with scrolls, whereas BYMV-induced cytoplasmic inclusions were made of pinwheels and






10



laminated aggregates. Light and electron microscopic investigations revealed that BICMV induces nuclear inclusions in C. spectabilis (Zettler et al., 1967; Edwardson et al., 1972; and Christie and Edwardson, 1977), while no such inclusions were observed in cells of C. spectabilis infected with BYMV. Based on those cytological distinctions, Edwardson et al. (1972) concluded that BICMV and BYMV are distinct members of the potyvirus group Subsequently, Zettler and Evans (1972) demonstrated that BICMV and BYMV had dissimilar host ranges, providing additional evidence that they are distinct viruses.

In host range Itudies, BICMV was shown to be very similar to BCMV, but different from watermelon mosaic virus-2 (WMV-2), (Uyemoto et al., 1973). Leaf-dip preparations of BICMV-infected tissue revealed the presence of flexuou rods, 750 nm long, and double immunodiffusion tests with BCMV and WMV-2 antisera indicated that BICMV was serologically identical to BCMV and related to, but distinct from WMV-2 (Uyemoto et al., 19/3).


Materials and Methods


Source of Virus isolate

The blackeye cowpea mosaic virus used in this study was isolated from infected seeds of cowpea V. unguiculata 'Knuckle Purple Hull' harvested from a field in Gainesville, Florida. The virus was transmitted by aphids from infected cowpea plants grown from infected seeds to non-infected 'Knuckle Purple Hull' plants. Two aphids (M. persicae) were used per test plant and each aphid was allowed to have an acquisition period of 30 to 60 sec. A single test plant showing typical mosaic was assayed by leaf-dip electron microscopy for the presence of






11



rod-shaped virus particles and used as the initial source of inoculum for virus propagation. The virus was mechanically transmitted from the selected infected plant to healthy 'Knuckle Purple Hull' seedlings, where it was increased for virus and inclusion purification, and other studies.


Virus and Inclusion Purification

Blackeye cowpea mosaic virus was propagated in either V. unguiculata or Nicotiana benthamiana Domin, and systemically infected leaves were used for virus and inclusion purification. Either n-butanol or a combination of chloroform and carbon tetrachloride was used in the clarification process. The adaxial surface of the primary leaves of 5 to 7-day old cowpea seedlings were inoculated with BICMV obtained by grinding infected lef tissue in 0.05 M potassium phosphate (KPO4) buffer, pH 7.5 (1/2, w/v). The first trifoliolate leaves showing typical mosaic were collected 15 to 18 days later and subjected to the following purification procedures based on previous works (Hiebert et al., 1971; Hiebert arid McDonald, 1973; and McDonald and Hiebert, 1975).

n Butanol clarification method. Two hundred to 400 g of leaf tissue were homogenized in a blender with two parts (w/v) of 0.5 M KPO4 buffer, pH 7.5, containing 0.5 to 1.0% sodium sulfite (Na2SO ). The resulting extract was filtered through a double layer of cheesecloth and enough n-butanol was added to make a final concentration of 8% (v/v). This mixture was stirred overnight at 4 C and the coagulated green debris obtained was removed by a low speed centrifugation at 11,700 9 in a Sorvall Centrifuge (Sorvall Superspeed RC2-B Automatic Refrigerated Centrifuge) for 10 min, Virions were precipitated from the supernantant by the addition of 6 8% (w/v) of polyethylene glycol






12



MW 6000 (PEG) followed by stirring for 60 min. The precipitated virions were collected by certrifugation at 13,200 g for 10 min. The resulting pellet was resuspended in 0.02 M KPO4, pH 8.2, containing 0.1% 2-mercapthoethanol (2-ME) (v/v) and the virus was separated from the host components by equilibrium density gradient centrifugation (120,000 g for 16 18 hr in a beckman SW 50.1 rotor) in 30% cesium chloride (CsCl) prepared in the same buffer. The virus zone, located at 12 to 15 mm from the bottom of ihe tube, was collected dropwise through a hole punched in the bottojii of the tube and diluted with 0.02 M KPO4, pH 8.2, containing 0.1% 2-ME. The virus preparation was clarified by centrifugation at 11,700 g for 10 min and reconcentrated by centrifugation at 85,000 y for 90 min. The final pellet was resuspended in 0.02 M Tris buffer, pH 8.2, and the virus concentration was determined spectrophotometrically using an ,xtinction coefficient of 2.4 mg/ml (Purcifull, 1966). The optical density (0.0.) readings for the virus at wavelengths of 260 and 280 nm were corrected for light scattering before estimating the 260/280 ratio and concentrations of virus in purified preparations. The correction for light scattering was done by plotting the log of the optical densities against the wavelengths of 320, 340, and 360 nm and extrapolating these values to 230 300 nm range of wavelength.

Chloroform-carbon tetrachloride clarification method. This clarification process was selected when it was desirable to purify both the virus and inclusions from the same batch of tissue. Systemically infected tissue (200 400 g) were homogenized in a solution containing 1.30 ml of 0.5 M KPO4 (pH 7.5), 0.35 ml of chloroform, 0.35 ml of carbon tetrachloride, and 5.0 mg of Na2SO3 per gram of tissue.






13


The homogenized mixture was centrifuged in a Sorvall Centrifuge at 5,000 rpm for 5 min and the pellet containing the organic solvents was discarded. The aqueous phase was centrifuged at 13,200 g for 15 min to precipitate the virus induced inclusions. The supernatant was treated as previously described for virus purification and the pellet containing the inclusions was resuspended in 0.05 M KPO4, pH 8.2, and

0.1% 2-ME. The inclusion suspension was homogenized in a Sorvall Omni-mixer homogenizer for 2 min and enough Triton X-100 was added to make a final concentration of 5% (v/v) After stirring for one hour at 4 C this mixture was subjected to a low speed centrifugation of 27,000 g for 15 min to precipitate the inclusions. The pellet was resuspended in 10 to 20 ml of 0.02 M KPO4, pH 8.2, containing 0.1% 2-ME, and homogenized for 30 sec. The inclusions were sedimented again by centrifugation of 27,000 g for 15 min. The pellet was homogenized for 30 sec and the homogenate was layered on a sucrose step gradient made up of 10 ml of 80%, 7 ml of 60%, and 7 ml of 50% (w/v) sucrose in 0.02 M KPO4, pH 8.2. The gradient was centrifuged for one hour at 27,000 rpm in a Beckman SW 25.1 rotor. The inclusions layered on top of the 80% sucrose zone and were collected by droplet from the bottom of the tube. To remove the sucrose, the inclusions were diluted in 0.02 M KPO4, pH 8 2, and precipitated by a centrifugation at 27,000 g for 15 min. The pellet was resuspended in 0,02 M Tris, pH 8,2, and inclusion yield was estimated spectrophotometrically after being disrupted in 2% sodium dodecyl sulfate (SDS). The inclusion preparations were either immediately used for immunization of rabbits or stored at

-20 C by either freezing directly or by freeze-drying.






14



Clarification with n-butanol and chloroform-carbon tetrachloride.

Because n-butanol resulted in virus preparations of higher purity, but chloroform-carbon tetrachloride was superior for preservation of inclusion proteins (Hit-bert, unpublished), these solvent systems were combined for purification of virus and inclusions from the same batch of tissue. Infected tissue was homogenized with two parts (w/v) of 0.5 M KPO4, pH 7.5, ontaininq 0 5 1.0% Na2SO3. The homogenate was filtered through cheesecloth and subjected to centrifugation at 11,700 g for 10 nin. The supernatant was used for virus purification as described previously using n-butanol for clarification. The pellet was resuspended in approximately 2 volumes of 0,5 M KPO4, pH 8.2, 0.5% Na2SO homogenized with one volume of chloroform-carbon tetrachloride (1:1, v/v) and centrifuged at 5,000 rpm for 5 min in a Sorvall Centrifuge. The aqueous phase was subjected to a centrifugation at 11,700 g for 15 min. The supernatant was collected for additional virus purification using PEG, equilibrium density-gradient centrifugation and differential centrifugation. The pellet was resuspended in 0.05 M KPO 4, pH 8.2, containing 0.1% 2-ME and treated with 5% Triton X-100. The inclusions were then purified by sucrose step gradient centrifugation as described above.


Virus Particle Size Determination

Crude leaf extracts from systemically infected cowpea plants and purified virus preparations were negatively stained in 2% potassium phosphotungstate (PTA), pH 6.5, containing 0.1% bovine serum albumin (BSA) prior to photography in an electron microscope. The procedure used was similar to those previously described (Edwardson et al., 1968 and Purcifull et al., 1970). Small pieces of B1CMV-infected leaf were






15

chopped with a razor blade in 2% PTA, pH 6.5, containing 0.1% BSA on a glass slide and a small quantity of the resulting cell extract was deposited on a carbon coated Formvar film supported by 75 x 300 mesh copper grids. Excess liquid was then removed by touching momentarily the edge of the grid with a filter paper and the specimen was allowed to air-dry. The purified virus was stained directly on the grid. A small drop of virus solution was deposited on the grid. After I 2 min, the virus solution was partially blotted with a piece of filter paper and a small drop of 2% PTA solution was added. The grid was blotted and allowed to air-dry. The grids were then examined in a Philips Model 200 electron microscope. The virus particles were observed, photographd and their sizes were estimated by comparing projected micrographs to micrographs of a diffraction grating (2160 lines/mm). Twenty-five virus particles from leaf extracts and 190 particles from a purified preparation were measured and classified according to their length at intervals of 50 and 20 nm.

Blackeye cowpea mosaic virus-grids prepared according to the

serologically specific electron microscopic technique (SSEM) developed by Derrick and Brlansky (1976) were also used for virus particle measurements. Parlodion film grids sensitized with BICMV-antiserum (BICMV-As) were treated with cowpea leaf extract containing BICMV and positively stained with 1% uranyl acetate in 50% ethanol. The SSEM technique will be described in more detail in Chapter II. Stability of Virus in Sap

Thermal inactivation point (TIP), longevity in vitro (LIV), and

dilution end point (DEP) were determined for BICMV using C. amaranticolor as an assay plant. The TIP was determined by heating crude sap of






16



BICMV-infected cowpea leaves to 45, 50, 55, 60, 65, 70, and 75 C for 10 min. All treated saps as well as unheated sap of BlCMV-infected tissue were rubbed on the test plants, which were maintained in greenhouse conditions for at least three weeks for observation of symptoms.

Crude sap of infected leaves obtained in deionized water was

placed in test tubes and assayed for infectivity after storage at room temperature for 0, 8, 16, 24, 48, and 72 hr. For the DEP determination, crude juice was extracted from BICMV-infected leaves, and the extract was diluted to 10 10-2 10 10 10, and 10-6 with deionized water prior to assay.


Polyacrylamide Gel [lectrophoresis of Viral and Inclusion Proteins

The polyacrylamide gel electrophoresis studies were performed according to the method of Weber and Osborn (1969) as modified by Hiebert and McDonald (1973). Running gels of approximately 75 mm in height were prepared with 6% acrylamide (7.5 ml sodium phosphate buffer, pH 7.2; 15.0 ml water; 0.15 ml 10% SDS; 6.0 ml of 30% acrylamide;

0.045 ml N, N, N', N'-tetramethylenediamine (TEMED) and 1.2 ml ammonium persulphate 15 mg/ml), and a well-forming gel of 8% acrylamide with onefifth the electrophoresis buffer concentration (1.2 ml buffer; 7.2 mil H20; 0.2 ml 10% SDS; 3.0 ml of 30% acrylamide; 0.04 ml TEMED, and 0.3 ml ammonium persulphate 15 mg/ml) was cast on top of them. Disassociated protein solutions, 20 50 P samples in approximately 20% sucrose and one-fifth the electrophoresis buffer concentration, were placed into the formed wells. The top of the samples were covered with a cap gel of composition similar to the well-forming gel. The electrophoresis was performed in a vertical slab electrophoresis apparatus, Ortec, Model 4010/4011, Ortec, Incorporated,Oak Ridge, Tenn.,






17



for 1.5 to 4.0 hr at 160 V with a pulsed constant power supply at 300 pulses per second and about 90 mA current,

Prior to being used for electrophoresis, the protein was disassociated by mixing 0.2 ml of protein solution with 0.1 nil of 10% SDS and 10 20 pl 2-ME and heating this mixture in boiling water for 1 to 2 min. Samples of 20 50 pl of disassociated proteins were added to

0.1 ml of one-fifth of the electrophoresis buffer concentration, containing 30% sucrose and 0.15% SDS.

Serum albumin (MW 67,000), glutamate dehydrogenase (MW 53,000), ovalbuniin (MW 43,000), carbonic anhydrase (MW 29,000), and TMV coat protein (MW 17,500) were used as protein markers to estimate the molecular weight values for inclusions and virus coat protein subunits.

After electrophoresis, the gel slabs were stained and fixed

overnight in a staining solution containing 50% methanol, 10% glacial acetic acid, and 0.1/ Coomassie brilliant blue R250. Before photography, the gels were destained by soaking them for 8 hr in a solution made up of 10% methanol and 7.5% acetic acid followed by several changes in the solution over a period of several days. The distances migrated by the protein subunits into the running gels were measured from the photographs of the stained gels. Sedimentation Coefficient Determination

The sedimentation rates of fresh and stored purified BICMV in either 0.02 M Tris buffer, pH 8.2 or 0.05 M borate buffer, pH 8.2 were measured with a Beckman Model E analytical ultracentrifuge according to the method of Markham (1960). After the rotor reached a speed of 27,690 rpm photographs were taken at 4 min intervals using






18




Schlieren optics. The data were corrected for standard water viscosity conditions at 20 C, but not for the effect of virus concentration. The virus concentration: used varied from 0.5 to 1.0 mg/ml.


Serology

Antiserum production for virus and cytoplasmic inclusions. Antisera were obtained by injecting a New Zealand white rabbit with untreated virions and a second rabbit with pyrrolidine-degraded virus protein. All rabbits selected for immunization were first bled to produce normal sera. The concentrations of untreated B1CMV in 0.02 M Tris buffer, pH 8.2, used in the immunization process varied from 1.0 to 2.0 mg of nucleoprotein per ml of purified solution. BICMV used for pyrrolidine degradation was suspended in 0.005 M borate buffer, pH 8.2. The virus protein was degraded according to the method used by Shepard (1972). A virus solution was mixed with an equal volume of 5% pyrrolidine in distilled water (v/v). The mixture was then immediately dialyzed against two liters of 0.05 M borate buffer, pH 8.2, containing 0.37% actual formaldehyde for approximately 48 hr at 4 C to remove the pyrrolidine and fix the protein subunits.

A series of 4 to 5 intramuscular injections was given to each

rabbit with an interval of 10 to 15 days between the injections. Each injection consisted of 1.0 to 2.0 ml preparations of virus or degraded viral protein vigorously emulsified with equal volume of Freund's complete or incomplete adjuvants (Difco). Booster injections were given at intervals of about 2 months.

The immunized animals were bled every week, starting 10 to 15

days after the last injection of the initial series of 4 5 injections.






19



The rabbits were fasted for 4 12 hr prior to each bleeding and 30 50 ml of blood were collected into glass tubes according to the procedure described by Purcifull and Batchelor (1977). Blood samples were allowed to clot for approximately 45 min at 37 C in a waterbath. The clotted blood was subjected to a centrifugation of 2,000 rpm in a Sorvall table centrifuge for 10 min. The antisera were transferred with a Pasteur pipette to conical-bottomed tubes and clarified by a second centrifugation at 5,000 rpm for 10 min. Antiserum specificity and titer were determined by Ouchterlony (1962) double-diffusion tests in SDS-agar plates. The antisera were stored at -20 C by either freezing directly or after freeze-drying.

The BICMV-indued cytoplasmic inclusions (BICMV-1) used for antiserum production were purified from N, benthamiana. Freshly purified cylindrical inclusions, which were unreactive with antiviral sera, were used for immunization and the foot pad route of immunization (Ziemiecki and Wood, 1975) was used. The rabbit received three injections into the foot pad, each ,ontaining 0.1 ml of purified inclusions (0.1 0.2

0.D. units/ml at 280 nm) in 0.02 M Tris, pH 8.2, emulsified with an equal volume of either Freund's complete or incomplete adjuvants.

Serological tests. Both double and single immunodiffusion tests

in agar gel were used in the present study. Most double immunodiffusion tests were performed in agar medium containing 0.8% Noble agar (Difco);

0.5% SDS (Sigma) and i.0% NaN3 (Sigma) in deionized water (Purcifull and Batchelor, 1977), or 0.05 M Tris-HCI buffer pH 7.2. Reactant wells were punched in the solidified agar medium with an adjustable gel cutting device made by Grafar Corp., Detroit, Mich. Routinely the wells (7 mm in diamn!ter) were punched in an hexagonal arrangement






20



consisting of a center well with six peripheral wells spaced 4 5 mm from the center well as measured from the edges of the wells. Different gel patterns were also used in certain tests. Antigens used as reactants were prepared either in deionized water or in 1.5% SDS solution, according to Purcifull and Batchelor (1977). In the first case, fresh tissue was ground with a mortar and pestle in deionized water (1/2, w/v) and expressed through cheesecloth. The second method which was more commonly used, consisted of grinding fresh tissue in 1.0 ml of water per gram of tissue and adding 1.0 ml of 3.0% SDS per gram of tissue prior to expressing the sap through cheesecloth. The antigens and undiluted antisera were pipetted directly into the appropriate wells, and the plates were incubated in a moist chamber at 24 C for 24 48 hr. The development of precipitation patterns was observed by looking at the plates, which were illuminated from the bottom with indirect lighting. The reactants were removed and 15% charcoal (Norit A) in water (w/v) was added into the wells before photographs were taken.

Single radial immunodiffusion tests were conducted in agar media containing 0.8% Noble agar, 1.0% NaN3, 0.3 or 0.5% SDS, and 10, 15, or 20% BICMV antiserum. Media were prepared either with antiserum obtained for untreated BICMV and antiserum for pyrrolidine degraded BICMVprotein. Each SDS concentration in the media was tested with antigens prepared in distilled water or in 1.5% SDS. During medium preparation, care was taken to avoid heating the antisera over 50 C and while exposed to SDS, the antisera were maintained at 50 C for less than 2 min.

Single radial diffusion plates were also prepared with a mixture of antisera to BICIV and CPMV. The CPMV-antiserum was prepared by






21




immunizing a rabbit with CPMV degraded by SDS according to a procedure described by Purcifull and Batchelor (1977). A lyophilized, purified preparation containing approximately 3 mg of CPMV was resuspended in I ml of 1.0% SDS solution containing 2.0% 2-ME, and boiled for approximately 5 min before emulsification with Freund's adjuvant and intramuscular injection into a rabbit. Three similar injections were given into the same rabbit with 7-day intervals between injections.

Serological relationship between BICMV and other potyviruses. Reciprocal double immunodiffusion tests with BICMV and the following potyviruses were conducted in SDS-containing media: bean yellow mosaic virus (BYMV), bean common mosaic virus (BCMV-BV-1), bean common mosaic virus-siratro isolate (BCMV-S), bidens mottle virus (BiMV), dasheen mosaic virus (DMV), lettuce mosaic virus (LMV), pepper mottle virus (PeMV), potato virus, Y (PVY), soybean mosaic virus (SoyMV), tobacco etch virus (TEV), turnip mosaic virus (TuMV), watermelon mosaic virus-i (WMV-1), and watermelon mosaic virus-2 (WMV-2). The source of each antiserum was as follows: BYMV (Jones and Diachun, 1977); BCMV-BV-1 (J. K. Uyemoto, New York State Agricultural Experiment Station); BCMV-S (Lima et al., 1977); DMV (Abo El-Nil et al., 1977); BiMV, LMV, PeMV, PVY, SoyMV, TEV, TuMV, WMV-1, and WMV-2 (D. E. Purcifull, University of Florida, Gainesville).

Using BICMV-As, the serological relationship of BICMV with

commelina mosaic virus (CoMV) (Morales and Zettler, 1977), a Moroccan isolate of CAMV (Fischer and Lockhart, 1976a), pepper veinal mottle virus (PVMV) and pokeweed mosaic virus (PWMV) were also studied in double diffusion tests with SDS-treated antigens. In all serological tests, the reactants were arranged so that BICMV was always placed in






22



a well adjacent to the other virus-well. Sap extracts from appropriate healthy host tissues were included as controls in all serological tests, and all antigens were also tested against normal serum.

The intragel cross-absorption technique described by van Regenmortel (1966) was also used to study the serological relationships of 81CMV with BCMV-S and CAMV. Purified preparations of heterologous antigens (BCMV-S or CAMV) were placed in the center well and allowed to diffuse for approximately 24 hr. The excess of the antigen preparations were then removed and the BICMV antiserum was added in the same well. At the same time, the homologous and the heterologous antigens were positioned in the outer wells.


Light and Electron Microscopy of Virus Induced Pinwheel Inclusions

Epidermal leaf strips obtained from systemically infected cowpea, V. unguiculata, were floated on a 5% solution of Triton X-100 for 5 to 10 min and subsequently stained with a combination of calcomine orange and "luxol" brilliant green as described by Christie (1967). The stained leaf strips were mounted in euparal on glass slides and examined with a light microscope for the presence of cytoplasmic inclusions. Similarly, strips from noninoculated V. unguiculata were also stained and examined in the light microscope as controls.

Cylindrical inclusions were examined in situ in ultrathin sections with an electron microscope. Small pieces were taken from symptomatic areas of systemically infected cowpea leaves and fixed for 2 to 3 hr at room temperature in Karnovsky's formaldehydeglutaraldehyde fixative prepared in 0.1 M cacodylate buffer, pH 7.2 (Karnovsky, 1965). After washing with 0.1 M cacodylate buffer, the small leaf pieces were postfixed for I to 2 hr at room temperature






23



in 2% osmium tetroxide and progressively dehydrated in an increasing ethanol solution series. The leaf pieces were maintained for 5 to 15 min in each ethanol solution at room temperature. The pieces were stained overnight at 4 C in a solution of 75% ethanol containing 2% uranyl acetate and subsequently dehydrated in a second series of ethanol solutions (75 100Z) followed by 100% acetone or propylene oxide. They were then embedded in plastic containing Epon 812, Araldite 502, and dodecenylsuccinic anhydride. Ultrathin sections were cut with a diamond knife in a Sorvall MT-2 ultramicrotome and mounted on copper grids with carbon-coated Formvar film. The specimens mounted on the grids were poststained with 9% potassium permanganate (2 min), 1% uranyl acetate (2 nmin), and lead citrate (2 min). These sections as well as those obtained from noninoculated cowpea plants were examined with a Philips Model 200 electron microscope.

Purified BICMV-1 preparations were mounted on carbon-coated Formvar film supported by copper grids and stained with either 1% ammonium molybdate or 2% uranyl acetate, before examination by electron microscopy.


Host Range and Screening Cowpea Varieties for Resistance

Test plants were inoculated with crude sap from 'Knuckle Purple Hull' systemically infected with BICMV. The inoculum was prepared by grinding leaf tissue in 0.05 M KP04, pH 7.5 (1/2, w/v). The inoculations were done by rubbing the inoculum on carborundum-dusted leaves of the test plants which were maintained in greenhouse conditions for at least one month for observation of symptoms. All inoculated plants, Including those that did not show any symptoms were checked serologically for the presence of BICMV.






24



The cowpea varieties were also inoculated with CPMV, CAMV, and BCMV-S. Crude sap from all inoculated cowpea plants were also tested in double immunodiffusion against antisera specific for CPMV, BICMV, and BCMV-S, respectively. Since CAMV was shown to be serologically related to BICMV, the serological tests to detect its presence in the inoculated plants were done with BICMV antiserum.


Results


Purification and Properties of Blackeye Cowpea Mosaic Virus

Purified preparations of BICMV were obtained from systemically infected leaves of either V. unquiculata 'Knuckle Purple Hull' (Fig. 1-A) or N. benthamiana using the purification procedures diagrammed in Figures 2, 3, and 4. The best yield with the highest degree of purity was obtained using the first method of virus purification (Fig. 2) and infected cowpea leaves (Fig. I-A) as a source of virus. The first trifoliolate cowpea leaves collected 15 to 18 days after inoculations gave the highest yield of virus (8 10 mg) per 100 g (fresh weight) of infected tissue and n-butanol proved to be the best clarifying agent for cowpea tissue. An opalescent,sharp virus-band was usually obtained after equilibrium density gradient centrifugation in 30% CsCI. The virus zone was located at 12 to 15 nm from the bottom of the tube while most of the green host components stayed at the top portion of the gradient. The clear pellet obtained after a high speed centrifugation of virus removed from CsCI gradients confirmed the absence of colored host components. The combination of chloroform and carbon tetrachloride,although necessary for inclusion purification, was an inferior method of clarification for obtaining virus from cowpea


































Figure 1 Systemic and localized symptoms induced by blackeye cowpea
mosaic virus (BICMV) in cowpea, V. unguiculata 'Knuckle
Purple Hull' and C. amaranticolor.

A) Typical mosaic on secondary trifoliolate leaf of cowpea plant inoculated with BICMV (1), and primary
trifoliate leaf showing vein clearing (2).

B) Local lesions on leaf of C. amaranticolor inoculated
with BICMV.





26




























0t




































Figure 2 Flow diagramn outlining the procedure of purification of
BICMV using n-butanol as clarifying agent, polyethylene
glycol (PEG) for virus concentration, CsCl gradient
centrifugation for separation of virus from host components, and differential centrifugation for further virus purification. For details, see description in
materials and methods section.





28











SYSTEMICALLY INFECTED TISSUE

0.51 KPO4 pH 7.5 + 0.5-1.0% Na2SO3

GRIND

FILTER 8% PUTANOL STIr: OVERNIGHT CENTRIFUGATION: 11700g 10min PELLET (Discard) SUP1RNATANT 8% fEG STIR : 60min CENfRIFUGATION: 11700 g lOmin SUPERNATANT (Discard)

PELLET O.O2M KPO4 pH 8.2 + 0.1% 2-ME CsC1 GRADIENT CENTRIFUGATION: d=1.28g/cc 120000g 18 hr COL ECT VIRUS ZONE CENTRIFUGATION: 11700g 10min PELLET (Discard) SUP RNATANT CENTRIFUGATION: 85000g 90min SUPERNATANT (Discard)
PEL ET



0.02M TRIS pH 8.2 V I R U S





































Figure 3 Flow diagram, outlining the procedure for purification
of BICMV and its cytoplasmic inclusions, using chloroform
and carbon tetrachloride as clarifying agents. The procedure is described in the text.





30















SYSTEMICALLY INFECTED TISSUE

0.5M KPO4 pH 7.5 + CHC13+ CC14+ 1 Na2SO3

CENTRIFUGATION: 4,000g 5min PELLET
(Discard)

SUPERlNATANT

CENTRIFUGATION: 11,700g 15min


SUPERNATANT I PELLET (Virus) (Inclu ions) 8' EG 0.0 M KPO4 pH 8.2 + 0.1' 2-ME

STIR : 60min HOMOGENIZATION

CEN RIFUGATION: 11,700g 10min 5% jRITON-X

SUPERNATANT CENTRIFUGATION: 27,000g 15min (Discard)
PELLET SUPERNATANT (Discard)
O.O.M KPO4 pH 8.2 + 0.1% 2-ME PELET PEL ET
CsC1 GRADIENT CENTRIFUGATION:
d=l 28g/cc-120000g 18hr SUCROSE STEP GRADIENT CENTRIFUGATION: I 45,00g 60min COLECT VIRUS ZONE I
COLLECT INCLUSION ZONE CEN RIFUGATION: 11,700g 10min
CENTRIFUGATION: 27,000g 15min PELLET
(Discard) SUPERNATANT
SUPERNATANT (Discard)

CEN RIFUGATION: 85,000g 90min PELLET

SUPERNATAN I
0.02M TRIS pH 8.2 PELLET I N C L U S I O N S

0.02M TRIS pH 8.2 V I R U S





































Figure 4 Flow diagrami outlining the steps carried out during the
purification of BICMV and its cytoplasmic inclusions by a combination of the first (Fig. 2) and second (Fig. 3)
methods for purification of virus and inclusions.





32











INFECTED TISSUE
0. M KPO4 pH 7.5 + 0.5-1.0% Na2SO3

GRIND
FI TER
VIRUS ONLY FI

CENTRIFUGATION: 11700g-10min

SUPERNATANT ELLET
(Virus) (Inclu ions + Some Virus)
8% UTANOL 0.5M KPO4 pH 8.2 + Na2SO3+ CHC3 + CC14
STI : OVERNIGHT I
CENTRIFUGATION: 4000g-5min CEN RIFUGATION: 11700g-1Omin
PELLE
PELLET (Discard) (Discard)
AQUEOUS PHASE
SUPgRNATANT I
RNATANT CENIRIFUGATION: 11700g-15min 8% EG o l SUPERNATN
STI : 60min (Virus)
CENTRIFUGATION: 11700g-1Omin PEL ET
SUPERNATANT HOM GENIZATION
(Discard)
5% RITON-X
CEN RIFUGATION: 27000g-15min
0.0 M KPO4 pH 8.2 + 0.1% 2-ME SUPERNATAN
CsC GRADIENT CENTRIFUGATION: (Discard) 1
d=l.28g/cc 120000g 18hr PELLET PELLET
COLLECT VIRUS ZONE
COLT VIRUS ZONE 0.0 M KPO4 pH 8.2 + O.It 2-ME CENTRIFUGATION: 11700g-1Omin SUC
SUC OSE STEP GRADIENT PELLET CENjRIFUGATION: 45000g-60min
COLLECT INCLUSION ZONE SUPERNATANT
I CENTRIFUGATION: 27000q-15min CENTRIFUGATION: 85000g-90min SUPERNATANTSUPERNATAN (Discard) (Discard) PELEPEL ET
0.02M TRIS pH 8.2 0.02M TRIS pH 8.2 I N C L U S I O N S
V I R U S






33


tissues. With this method, a clear sap was obtained after the first low speed centrifugation but the virus zone in the CsCl gradient was

not very well separated from the host components,

Plants of C. amaranticolor and V. unguiculata mechanically

inoculated with purified preparations of BICMV showed the first symptoms of local lesions and systemic mosaic (Fig. 1) 4 and 7 days after inoculation, respectively. The ultraviolet absorption curve (Fig. 5) obtained for the purified preparations of BICMV had a maximum between 260 and 262 nm, and a minimum at 244 to 245 nm. The ratio between the absorption at wavelengths of 260 and 280 nm was approximately 1,2 after correction for light scattering, as would be expected for a member of the PVY gr)up. This value is consistent and agrees with those of other long flexuous rod-shaped viruses (Shepherd and Purcifull, 1971; Tosic et al., 1974; and Barnett and Alper, 1977), The virus solutions showed strong stream birefringence and electron microscopic examinations indicated that 73% of the 190 virus particles examined were between 700 and 800 nm (Figs. 6, 7, 8). The rods observed in the purified preparations (Fig. 6) indicated a low percentage of virus fragmentation during the purification processes. As the result of end-to-end virus aggregation, a few particles with 1400 to 1500 nm were also observed, Purified virus preparations usually were relatively free of normal plant constituents when examined with the electron microscope and in the spectrophotometer.

Sedimentation coefficients determined for the virus at 20 C either in 0.02 M Tris buffer, pH 8.2, or in 0.05 M borate buffer, pH 8.2, indicated that BICMV sedimented as a single species with the s20 values of 157 159 S. On the other hand, the Schlieren pattern (Fig. 9)



































Figure 5 Absorption spectra of purified preparations of BICMV
in 0.02 M Tris buffer, pH 8.2, and BICMV cytoplasmic
inclusionis in the same buffer.





35













0.8 B 1 I0.40



BI CMV
0.7 0.35
.... B1CMV-I




0.6 I 0.30








4- 4
0.5 0.25




0.4 0.20
'4- '40.3 0.15o






0.1 0.150







0.1 0.
0.0




0 0 220 240 260 280 300 320 340 Wave Length (nm)





























Figure 6 Electron microscopy of BICMV in a purified preparation
and in cowpea leaf extracts.

A) Purified preparation of BICMV negatively stained
with 2% phosphotungstic acid, pH 6.5, containing
0.1% BSA;

B) Serologically specific electron microscopy (SSEM)
of leaf extract from cowpea plants systemically
infected with BICMV. Antiserum for BICMV diluted
1/1000 in 0.05 M Tris buffer, pH 7.2, was used
to sensitize the grid and the virus particles
were positively stained with 1% uranyl acetate.
Note the considerable increase in virus concentration compared with the normal leaf-dip preparation
(C);

C) Leaf-dip preparation of cowpea leaf tissue
systemically infected with BICMV, negatively
stained with 2% phosphotungstate.







37





















-u










; r, i a i~"- 6 i ~c~i r:










I'-":"' i ? tC"




1~
: I
31 i,
~ c. x i ..
'' 7: rtr J,~ i~
.r 't ri, Iv~~in t r
i* 4, "d i .* *. ~~ I i
A c.
3; i bf X ,1 1 C .. .. ~~
R
~
i: ~5L,
i-I "' ".~:
'~' -:V ~a
c- I* t~~ i- r r ::~: ~RI -r ~ ;,i
'i: +L
i; :e ~ rt:+ca: eZ~ r I,
:
~ 2"1."~: Rlr ,z;;i 5:~ ~
I~. '"

X
J '"
i' *i 14 ~t~I ''' 'V" -L; r i



i '
;f-Y~t_ ~ ~ r"
~ r
:
r ~i ~ ; LY
X ~ B I 6:
i r- :T ~T:
~~~l~i t *w~ x
.tii~-~s Bli;r
o""C i- ;I 6~"i

4: ;
i~l i






1; 5-' 8:
t I ~ ii PEI
ii ~~~ 2~_: : 1, 9;~ i E*
r- 11


"~~,

1(1 ; *
.a i, irl I;,
~dl
I~ ,
ali :~
i ..
I~ I X
11 ,






































Figure 7 Histograms of lengths of BICMV particles from purified
preparation negatively stained with phosphotungstate (A), and cowpea leaf extract using the serologically specific electron microscopy and uranyl acetate as a
positive stain (B). Class interval for both histograms
50 nm.





39










I

120



100




10O 60




40 20



100

8-j










60 40
0







80 60 40 20




200 400 600 800 1000 1200 1400 1600 PARTICLE LENGTH (nm)



































Figure 8 Histograms of BICMV particle lengths from two different
electron microscopic preparations to show particle length distribution from 600 to 900 nm. Class interval = 20 nm.

A) Particle length distribution of BICMV from purified
preparation negatively stained with phosphotungstate;

B) Particle length distribution of BICMV from cowpea
leaf extract prepared on grids sensitized with BICMV antiserum and positively stained with uranyl acetate.





41




















80 [E




60 40 L 20



80
U













40 20




600 700 800 900 PARTICLE LENGTH (nm)






42



revealed a difference in S values between BICMV in fresh preparations and BlCMV in purified preparations stored at 4 C for more than 30 days. Both virus preparations showed a single sedimenting peak, but the s20 values for BICMV in fresh preparations and at a concentration varying from 0.5 to 1.0 mg/ml ranged from 157 to 159 S while the s20 values for the virus in the stored preparations and at the same concentrations ranged from 140 to 142 S (Fig. 9). The lower sedimentation coefficients obtained for the stored virus suggested that a change in virus mass (MW) had occurred. Hiebert and McDonald (1976) observed some possible enzymatic degradation of capsid protein of purified turnip mosaic virus. Proteolytic degradation of capsid protein of stored purified preparations of BICMV was also observed by polyacrylamide gel electrophoresis (PAGE) studies. Polyacrylamide gel electrophoresis analysis of SDS-degraded virus of a freshly purified preparation of BICMV revealed a main protein component with an estimated molecular weight (MW) of 34,000 daltons and two smaller ones with MWs of 29,000 and 27,000 daltons (Fig. 10). These smaller components may have arisen by degradation of the slow moving component during storage (Hiebert and McDonald, 1976). Stored BICMV preparations contained only the faster moving protein components with MWs of 29,000 and 27,000 daltons (Fig. 10), presumably derived from 34,000 daltons component.


Purified Inclusion Preparations

Using either of the methods outlined in Figs. 3 and 4, purified cylindrical inclusions induced by BICMV were obtained from the same batches of systemically infected leaf tissue of V. unguiculata or



































Figure 9 Schlieren patterns from sedimentation velocity experiment
with stored (A), and fresh (B), purified preparations
of BICMV. Photograph was taken 8 minutes after the rotor
reached a speed of 27,690 rpm. Sedimentation is from
left to right.






44

































































4 ;Zr

Ttj~ V4x~'


NIN I Movo

















0
0* L

Do Q- -C (a
C a-- 0-c 1 W) O 0
i 1Q r -a)0 CO O n (A 0)( 0U 4 L 4.- C L SLLru -0(Uu 0n0 CL' L a C 0 a) L E E >- w c t) -- ) 0 c C oD ct C J() U n 0 U 4 > ) ( 0 c r O C I m 3 O4 (D ru r rr, 0 w C .-- 3 (Oa c -a 0 O >> LQ) L (U rO -C O m U

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46






































(,01 OX) 1H913M Vn3310I1









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47


N. benthamiana used for virus purification. Electron microscopy of purified BICMV-1 negatively stained with molybdate revealed the presence of tubular inclusions with only trace amounts of host components (Fig. 11). At high magnification, striations of protein subunits were observed on individual tubes (Fig. 11-D). These regularly spaced striations were estimated to have a periodicity of approximately

5 nm. Striations with similar periodicity have been observed in cytoplasmic inclusions induced by several other potyviruses (Edwardson et al., 1968; Hiebert and McDonald, 1973; and Morales and Zettler, 1977). Few virus particles were observed in the purified preparations of BICMV-1 which were not reactive to BICMV-As (Fig. 12). Purified preparations of BICMV-1 with the highest degree of purity were obtained from N. benthamiana, with yields of 5 to 20 A280 units were usually obtained from 100 g of fresh weight of N. benthamiana or V. unguiculata tissues. The ultraviolet absorption spectrum obtained for SDS disassociated BICMV-I was typical of proteins, with a maximum at 277 nm and a minimum at 246 248 nm (Fig. 5). Polyacrylamide gel electrophoresis of SDS-disrupted inclusion proteins revealed a single subunit component estimated to have a MW of 70,000 daltons (Fig. 10). Virus Particle Size and Stability in Sap

Electron microscopic examinations of purified preparations of BICMV negatively stained with PTA indicated that 73% of 190 virus particles measured were between 700 and 800 nm with a modal length of 753 nm. Particle measurements of several leaf-dip preparations negatively stained with PTA and of grids prepared for SSEM with infected cowpea leaf tissue gave modal lengths of 758 and 780 nm,


































Figure 11 Electron micrographs of purified preparations of BICMV
cytoplasmic inclusions stained with molybdate. All
purified preparations consisted of tubes, most of which were fragmented during the purification process. Note
striations (St) on high magnification (D).




49
















a I







10 1000lnm St















5200 nm


















C v -3
0 0) VC u- 3 1 Cuvi
L- m 0 00002 CL u ( .. a) 0 3 d Z r a) c E > 0 SL .- > 0 L- 0 >_ 0- 0 0) ) C
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C ) r s U) c c -- 0a
O-- O > 0 0 o -m- cu Eco0u

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.- m L m E E> 0 L o0O > o c z E c D 2 re-- ma 6 L LL 00


O a E 30rE tE n E 0E I o U) L 0LU L 0 L.) 00 L
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E*


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51



























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52



respectively, with 90% of the particle lengths ranging from 700 to 800 nm (Figs. 7 and 8). Some variation was observed with the particle size of purified virus stained with PTA and virus particles in leaf extracts prepared by SSEM and stained with uranyl acetate (Figs. 7 and 8). On the other hand, grids with less plant debris and higher concentrations of virus particles were obtained with SSEM than with the conventional leaf-dip preparation (Fig. 6). Using normal leaf-dip preparations at least four grids were prepared and 10 electron micrographs were taken to measure a maximum of 25 virus particles. On the other hand, 132 virus particles were measured by examining two micrographs obtained by SSEM.

In cowpea leaf extracts, BICMV had a TIP of 65 C, LIV of 48 hr, and DEP of 10 4. Blackeye cowpea mosaic virus was still infectious after 10 min at 60 C: but not at 65 C and lost its infectivity after 48 hr at room temperature, but not at 24 hr. Sap of cowpea leaves systemically infected with BICMV lost infectivity when diluted more than 103 with distilled water.


Serology

Antisera specific for BICMV were obtained against untreated

virions and pyrrolidine degraded viral protein. Both antisera reacted with SDS- or pyrrolidine-treated BICMV in purified preparations or in plant sap in double and single radial diffusion tests (Figs. 12, 13, 14). Most bleedings were specific for viral antigens; however, some bleedings also reacted with extracts from healthy plants, suggesting the presence of antibodies specific for normal plant components. To remove these antibodies the antiserum was absorbed with plant






















Figure 13 Single radial diffusion tests in agar media containing
different concentrations of SDS and antisera for blackeye cowpea nsaic virus (BICMV-As) and cowpea mosaic
virus (CPMV-As)

The media in (A, B, C) contain 0,8% Noble agar,
1.0% NaN3, 0.3% SDS, and 10% BICMV-As (A), 15% BICMV-As
(B), and 20% BICMV-As (C). The media in (D, E, F)
contain 0.81 Noble agar, 1.0% NaN3, 0.5% SDS, and 10% BICMV-As (0), 15% BICMV-As (E), and 20% BICMV-As (F).
The wells in (A, B, C, D, E, F) were charged with:
(1) extracts from BICMV-infected cowpea prepared in
1.5Z SOS 1/2 (w/v), (2) solution used in "I" diluted
1/2 with 1.54 SDS, (4) solution used in "l" diluted 1/4 with 1.55 SDS, (8) solution used in "1" diluted
1/8 with 1.5 SDS, and (H) extract from healthy cowpea
prepared in 1.5% SDS.

The media in (G, H) contain 0.8% Noble agar,
1.0% NaN 0.51 SDS, and 15% BICMV-As + 15% CPMV-As (G), and 10 BICMV-As + 10% CPMV-As (H). The media
in (I, J) contain 0.8% Noble aqar, 1.0% NaN3, 0.3%
SDS, and 10% BICMV-As + 10% CPMV-As (1), and 8%
BICMV-As + 8Y CPMV-As (J). The wells in (G, H, 1, J) were charged with SDS-treated extracts from: BICMVinfected cowpea (row no. i), CPMV-infected cowpea
(row no. 2), cowpea leaf tissue containing both
BICMV and CPMV (row no. 3), and healthy cowpea (row
no. 4).




54









OEO
o B0i i






0H 0j



2r























Figure 14 Single radial diffusion tests with SDS and pyrrolidine
degraded capsid protein of blackeye cowpea mosaic virus
(BICMV) and cowpea mosaic virus (CPMV).

The media in (A, B, C) contain 0.8% Noble agar,
1.0% NaN3, 0.5 SDS, and 15% BICMV-As (A), 15% CPMV-As
(B), and l0:< BICMV-As + 10% CPMV-As (C). The wells
were charged with SDS-treated extracts from: BICMVinfected coupea (row i), CPMV-infected cowpea (row 2),
BICMV and CPMV in cowpea (row 3), and healthy cowpea
(row 4).

The media in (D, E, F) contain 0.8% Noble agar,
0.20 NaN, 0.85% NaCl, and 15% BlCMV-As (D), 15%
CPMV-As (E), and 10% BICMV-As + 10% CPMV-As (F)
prepared in 0.05 M Tris-HCI buffer, pH 7.2. The
wells were charged with pyrrolidine-treated extract from: BICMV-infected cowpea (row 1), CPMV-infected
cowpea (row 2), BICMV and CPMV in cowpea (row 3),
and healthy cowpea leaves (row 4).




56































27*











components purified from V. unguiculata by high speed centrifugation according to the method used by Purcifull et al. (1973). The high specificity of most of the antisera obtained against purified BICMV preparations confirmed the efficiency of the methods used for its purification. The titers of antiserum varied depending on the bleeding date and on the rabbits, but 32 was the highest antiserum titer estimated by SDS-gel double immunodiffusion tests with a series of dilutions (1/2, i/4, 1/8, 1/16, and 1/32) of BlCMV-infected cowpea tissue prepared in 1.5% SDS.

Antiserum specific for cytoplasmic inclusions induced by BICMV was obtained from a rabbit injected with preparations of BICMV-I purified from infected tissue of N. benthamiana. The BICMV-I antiserum reacted specifically with purified preparations of BICMV-I and crude sap of BICMV-infected cowpea, but not with either purified preparations of BICMV or crude sp of noninoculated plants (Fig. 12-B). The positive reactions with BICMV-I were more evident after 48 hr of incubation. The results obtained with BICMV antiserum also indicated that BICMV was not serologically related to its cytoplasmic inclusions (Fig. 12-A). Attempts to obtain specific antiserum by injecting rabbits with BICMV-I purified from infected cowpea tissue were unsuccessful. All three rabbits injected with BICMV-I purified from infected cowpea developed high titers of antibodies specific for normal plant tissue antigens.

Single radial immunodiffusion studies in SDS-agar medium impregnated with the virus antiserum indicated that appropriate SDS and antiserum concentrations need to be previously selected for highest sensitivity and to avoid spurious reactions. The best results were






58



observed when the antigens were prepared in 1.5% SDS and the medium used had 0.3% SDS and 10% antiserum (Fig. 13-A) or 0.5% SDS and 15% antiserum (Fig. 13-E). The same results were consistently observed with different batches of plates with the same medium compositions. Similar results were also observed with CPMV using antiserum obtained for SDS-treated virus. On the other hand, different results were observed in SDS medium containing a mixture of BICMV and CPMV antisera. All media containing 0.3% SDS were cloudy with all combinations of BICMV and CPMV antisera used (Fig. 13-1, -J),indicating some type of interaction between SDS and antiserum proteins. However, even with the cloudy appearance, some virus-specific reactions were still observed (Fig. 13-1, -J). Clearer media were obtained with 0.5% SDS and 10 or 15% of each antiserum. The best reactions, however, were observed when both BICMV and CPMV antisera were used at concentrations of 10% in media containing 0.5% SDS (Fig. 13-H). Strong precipitin rings were observed around the wells containing BICMV or a combination of BlCMV and CPMV whereas weaker reactions were observed around the wells containing only CPMV (Figs. 13-H, 14-C). Unexpectedly, no reactions were observed around the wells containing only CPMV in a medium containing 15% of each antiserum and 0,5% of SDS (Fig. 13-G).

Virus-specific reactions using BICMV and CPMV antisera were also obtained with the single radial diffusion method described by Shepard (1972). Precipitin rings around the virus-wells were observed when the antigens were prepared in 1.5 or 2.5% pyrrolidine and the medium used contained 0.8Z Noble agar, 0.2% NaN3, and 10 to 15% virus antisera prepared in 0.05 M Tris-HCI buffer, pH 7.2, containing 0.85% NaCI. Sharp, white precipitin rings were formed close to edges of






59


the wells containing BICMV in agar medium prepared with BICMV antiserum, whereas whitish halos with greater diameters were formed around the wells containing CPMV in agar medium impregnated with CPMV antiserum (Fig. 14-D, -E, -F). The same distinction between these two types of precipitin rings was observed when both antisera were added into the same medium, so that two concentric rings were formed around the wells containing both viruses (Fig. 14-F). The inner ring was the result of B1CMV-antibody specific reactions and the larger halos resulted from CPMV-specific reactions. This difference in types of precipitin rings could be related to the concentration of the antigens placed in the wells and to the reciprocal of antibody concentration (Shepard, 1972). Stronger and more compact rings were observed with CPMV when the antigens were diluted or the antiserum concentration was increased.

Reciprocal double immunodiffusion tests with SDS-treated antigens showed that BICMV is serologically related to, but distinct from, the following potyviruses: BCMV-BV-1, BCMV-S, BYMV, DMV, LMV, PVY, SoyMV, TEV, and WMV-2 (Fig. 15). No reactions were detected, however, with certain potyviruses, including BiMV, PeMV, TuMV, WMV-1, CoMV, PVMV, and PWMV. Antiserum for BICMV also reacted specifically with CAMV forming a distinct spur which extended past the heterologous reaction (Fig. 16-A). In all positive serological relationships observed in the reciprocal serological tests, spurs were formed in both directions (Fig. 15).

The serological distinctions observed between BICMV and BCMV-S, and CAMV by spur foriiation were demonstrated by the intragel cross absorption technique (Fig. 16-B, -D). The heterologous antigens,






















Figure 15 Reciprocal double immunodiffusion tests with BICMV and
other potyviruses in medium containing 0.8% Noble agar
1.0% NaN3, and 0.5% SOS prepared in 0.05 M Tris-HCI
buffer, pH 7.2. All the antigenic solutions were prepared in 1.5Z SDS. The center wells were charged with:
(1) BICMV antiserum, (2) PVY antiserum, (3) TEV antiserum,
(4) WMV-2 antiserum, (5) DMV antiserum, (6) BCMV-BV-1 antiserum, (7) BCMV-S antiserum, (8) SoyMV antiserum, (9) BYMV antiserum, (10) BiMV antiserum, and (II) IMV
antiserum. The top rows of wells in all cases were charged with SDS-treated extracts from: (a) BICMVinfected cowpea, and (c) healthy cowpea. The bottom rows
of wells were charged with SOS-treated extracts from: A) PVY-infected tobacco (b), and healthy tobacco (d); B) TEV-infected tobacco (b), and healthy tobacco (d);
C) WMV-2-infected pumpkin (b), and healthy pumpkin (d);
D) DMV-infected dasheen (b), and healthy dasheen (d); E) BCMV-DV-1 infected bean (b),and healthy bean (d);
F) BCMV-S-infected bean (b), and healthy bean (d); G) SoyMV-intected N. benthamiana (b), and healthy N. benthamiana (d); HF BYMV-infected pea (b), and
healthy pea Td) ; I) BiMV-infected Nicotiana hybrid (b),
and healthy Nicotiana hybrid (d); and J) LMV-infected
pea (b), and healthy pea (d).




61



















*
CC ; JF


10 ........ I0 .
iii i 0 iiirH]

RI:











Figure 16 luMnunodiffusion tests with BICMV, Moroccan isolate of
CAMV and siratro strain of bean common mosaic virus (BCMV-S) in agar medium containing 0.8% Noble agar, 1.0% NaN, and 0.5% SDS prepared in 0.05 M Tris-HCI
buffer, H 7.2.

A) Serological tests with BICMV and CAMV. The center
wells were charged with: BICMV antiserum (Bls), and normal serum (Ns). The peripheral wells were filled
with SDS-treated extracts from: (81) BICMV-infected cowpea, (Ca) CAMV-infected cowpea, and (H) healthy
cowpea.

B) Intragel cross-absorption test with B1CMV and CAMV.
The center wells were charged with: (1) BICMV antiserum, (2) purified CAMV and 20 hr later BICMV antiserum. The peripheral wells were filled with SDStreated extracts from: (Bl) BICMV-infected cowpea, (Ca) CAMV-infected cowpea, and (H) healthy cowpea.

C) Serological tests with BICMV and BCMV-S. The center
wells wore charged with: (Bis) BICMV antiserum, (Ss)
BCMV-S antiserum. The peripheral wells were filled with SDS-treated extracts from: (81) BICMV-infected cowpea, (S) BCMV-S-infected bean, (Hb) healthy bean, and (Hc) healthy cowpea. The arrows point to spurs.

D) Intragel cross-absorption test with BICMV and BCMV-S.
The center wells were filled with: (1) BICMV antiserum, and (2) purified BCMV-S and 20 hr later
BlCMV antiserum. The peripheral wells were charged with SDS-treated extracts from: (Bl) BICMV-infected cowpea, (S) BCMV-S-infected bean, (Hb) healthy bean,
and (Hc) healthy cowpea.

E) Serological tests with BCMV-S and CAMV using two
different antisera for BCMV-S. The center wells were
charged with: (1) BCMV-S antiserum from a rabbit inoculated with freshly purified preparations of
BCMV-S, and (2) BCMV-S antiserum obtained from the
same rabbit after a booster injection with purified
BCMV-S stored at 4 C for 30 days. The peripheral
wells were charged with SDS-treated extracts from:
(S) BCMV-S-infected bean, (Ca) CAMV-infected cowpea,
(Hc) healthy cowpea, and (Hb) healthy bean.

F) Serological test with fresh and stored purified
preparations of BICMV. The center well was charged with BI(IMV antiserum, and the peripheral wells with SDS-treated new purified preparation of BICMV (Np),
old purified BICMV (Op), and healthy cowpea extracts
(H).




63














..,, e~i~ :, ii
~"" IB I









CBI s

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64



which were placed in the center well prior to the antiserum, crossreacted with and fully precipitated the cross-reacting antibodies at the region of optimal proportions close to the center well.

Serological distinction was also observed between a freshly purified preparation of BICMV and purified BICMV stored at 4 C for more than 30 days (Fig. 16-F). This suggested some enzymatic degradation of certain BICMV antigenic determinants during the storage period.

Serological relationship studies between CAMV and BCMV-S using BCMV-S antisera obtained by different bleedings of the same rabbit indicated that the antiserum specificity varied according to the immunization program and the conditions of the antigenic solution used. A highly specific arntiserum for BCMV-S was obtained from a rabbit injected with approximately 8 mg of freshly purified preparations of BCMV-S. Using this specific antiserum it was possible to show a complete serological distinction between BCMV-S and CAMV (Fig. 16-E). About three months after the initial immunization, a booster injection with a purified preparation of BCMV-S stored at 4 C for more than one month was given to the same rabbit. All antisera obtained 15 days or more after the booster injection reacted with CAMV, forming a spur between CAMV and BCMV-S when they were placed into adjacent antigen wells around the antiserum well (Fig. 16-E). Light and Electron Microscopy

Light microscopic observations of epidermal leaf strip preparations from plants systemically infected with BICMV revealed the presence of tubular cytoplasmic inclusions similar to those described by Edwardson et al. (1972) and Edwardson (1974) for BICMV. Side views of groups of tubular inclusions were easily observed in BICMV-infected





65



leaves (Fig.17), and at high magnification end views of them could be seen as small dots by changing the microscope focus. In ultrathin sections of BICMV-infected tissue, these inclusions consisted of tubes attached to a central core, forming pinwheels (Fig. 18), similar to those induced by the potyviruses from Edwardson's subdivision-I (Edwardson, 1974). As reported by Edwardson (1974), the pinwheels contained arms with pronounced curvatures and tight scroll-like tubular inclusions. Only tubes were observed in purified preparations of cytoplasmic inclusions induced by B1CMV (Fig. 11). Host Range and Resistant Cowpea Varieties

Blackeye cowpe mosaic virus was readily transmitted mechanically from cowpea 'Knuckle Purple Hull' to the following plants in which it was detected serologically and caused the following symptoms: Crotalaria spectabilis (mosaic); Glycine max (L.) Mer. (mild mottle and chlorotic spots); Macroptilium atropurpureum (DC.) Urb. (mosaic); Macroptilium bracteatum (L.) Urb. (mosaic); Nicotiana benthamiana (mottle); Ocimum basilicum (local lesions); Phaseolus vulgaris 'Black Turtle-2' (epinasty, necrosis, yellowing) and 'Bountiful' (chlorotic spots on inoculated leaves); Vigna unguiculata 'Black Local' (mosaic), 'Early Ramshorn' (mottle), 'Knuckle Purple Hull' (mosaic), and 12 Brazilian cowpea cultivars in which the reactions varied from symptomless to mosaic (Table I). Small chlorotic lesions were found on the leaves of Chenopodium amaranticolor inoculated with purified preparations of BICMV or cowpea sap containing BICMV (Fig. I-B).

Based on failure to induce symptoms and on negative serological

results, BICMV did not infect Arachis hypogaea L. 'Florunner', Capsicum
































Figure 17 Photomicrographs (A, B, C, D) of cytoplasmic inclusions
in epidermal strips of cowpea leaves systemically infected with BICMV, stained with a combination of calcomine orange
and luxol brilliant green. A) cells with masses of cytoplasmic inclusions, B) details of mass of inclusions seen
in "A", C) cieneral view of the inclusion distribution in epidermal cells, and D) phase contrast micrograph of the
area photographed in "A". (Ci) cytoplasmic inclusions, (CW) plant cell wall, (G) guard cells, and (Nu) nucleus.





67











~, d -~1 18
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1 t r; ri H R ::,~i re jiZ~ ~ t ~I

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r P
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Figure 18 Electron micrographs of ultrathin sections of cowpea leaf
cells infected with BICMV showing cross-sections (A, B, C) and longitudinal sections (D) of pinwheel inclusions.





69



























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70


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71



annuum L. 'Early Calwonder'; Cucumis sativus L.; Cucurbita pepo L. 'Small Sugarl; Lupius anus angustifolius L. 'Bitter Blue'; Lupinus luteus L. 'Sweet Yellow'; Phaseolus vulgaris 1Black Turtle-I', 'Green Northern 11401, 'Improved Tendergreen', 'Lake Shasta', 'Michelite 62', 'Pink Rosa', !Pink Viva', 'Puregold Wax', 'Red Mexican U-34', 'Red Mexican U-35', 'Top-cropl and 'VC 18221; Pisum sativum L. 'Alaska', 'Bonneville' and 'Ranger'; and Vicia faba L.

The reactions uf cowpea varieties to mechanical inoculations of

BICMV, BCMV-S, CAMV, and CPMV are indicated in Table I. All inoculated plants were assayed serologically for the presence of the viruses (Table I).


Discussion


Blackeye cowpei mosaic virus and its cytoplasmic inclusions were

successfully purified from systemically infected cowpea or N. benthamiana leaf tissues with the procedures outlined herein. The first method of virus purification (Fig. 2) gave good yields of highly purified BICMV, and the combination of n butanol and chloroform-carbon tetrachloride (Fig. 4) was the better procedure for purification of BICMV and its cytoplasmic inclusions from the same batch of tissue. The high degree of purity of the BICMV preparations indicated by spectrophotometry, analytical centrifugation and PAGE analyses, as well as serological studies and electron microscopic observations, confirmed the efficiency of the purification procedures.

Aggregation of virus particles and virus and host components

during purification appears to be a limiting factor for obtaining high






72



yields of viruses in the PVY group (Shepherd and Pound, 1960; van Oosten, 1972; Hiebert and McDonald, 1973; Uyeda et al., 1975; and Barnett and Alper, 1977). Hiebert and McDonald (1973) reported aggregation of virus particles after PEG precipitation. The losses of BICMV by low speed centrifugation due to aggregation of virus particles were reduced by maintaining the virus in KPO4, buffer, pH

8.2, after precipitation with PEG.

Another critical aspect on purification of potyviruses for obtaining maximum virus yield is the host used for virus increase. In order to obtain a good yield of BICMV from the 'Knuckle Purple Hull' variety of cowpea the virus was inoculated into the source plants at the age of 3 to 4 days after emergence and the systemically infected leaf tissues were harvested 15 to 18 days after inoculation. Attempts to purify the virus from plants inoculated later than that or from tissue harvested more than 30 days after inoculation resulted in very poor yields of virus and cytoplasmic inclusions.

Electron microscopic examinations of purified preparations of BICMV indicated a low percentage of virus fragmentation during the purification processes (Fig. 6-A). Particle measurements of purified BICMV and of BICMV particles on grids prepared for SSEM with infected cowpea leaf tissue gave two modal lengths (Figs. 7, 8) which differed by approximately 30 nm. Variations in lengths of virus particles have been extensively observed (Edwardson, 1974). As reviewed by Edwardson (1974), virus length variations may be attributed to several factors, including sample preparation, host influence, virus strain differences, and normal fluctuations in the electron microscope magnification. Increase of 50 to 100 nm in certain potyvirus particle lengths induced






73




by magnesium ions were reported by Govier and Woods (1971). They indicated that in the presence of Mg ions the particles were straight, contrasting with the flexuous particles observed in the absence of Mg ions. On antiserum-coated grids several antibodies combine with a single virus particle and, possibly, increase its length. Because of the specific antigen-antibody reaction the BICMV particles were so strongly attached to the surface of the antiserum-coated grids that they could not be removed by repeated washing. On the other hand, positive staining of BICMV particles with ethanolic uranyl acetate may have induced some changes in their lengths. Measurements of 25 BICMV particles on irids prepared by conventional leaf-dip preparation with PTA gave a modal length similar to that estimated for purified BICMV negatively stained with PTA. Milne and Luisoni (1977) emphasized that negative staining gives better preservation and better resolution of viral capsids than positive staining. Using SSEM with uranyl acetate as a negative stain, Milne and Luisoni (1977), observed no change in the normal lengths of TMV and a potexvirus. However, leafdip preparations often contain too few particles to photograph conveniently for virus particle measurements, whereas relatively large numbers of particles can be photographed by using the serologically specific electron microscopic technique. As indicated by Derrick and Brlansky (1976), the addition of sucrose in the extracting and washing buffers greatly reduced the amount of plant debris on the SSEM grids. High amounts of plant debris in electron microscopic preparations are frequent problems in establishing the dimensions of a virus.






74


Polyacrylamide gel electrophoresis of SDS dissociated cytoplasmic inclusions and viral coat proteins clearly indicated that tha viral coat protein subunit was smaller than the inclusion subunit (Fig. 10). The PAGE results revealed that the inclusions were made of a single kind of protein with an estimated molecular weight of 70,000 daltons. Polyacrylamide gel electrophoresis of cytoplasmic inclusion preparations conducted by Hiebert and McDonald (1973) showed one protein component with molecular weight of 67,000 daltons for PVY; 67,000 for PeMV; 69,300 for BiMV; 69,600 for TEV; and 70,300 for TuMV. The PAGE studies also indicated that freshly purified BICMV consisted of a main protein component with a molecular weight around 34,000 daltons. Two smaller protein components were also revealed by PAGE analysis of SDS denatured viral coat protein (Fig. 10). Since only traces of the faster moving proteins were observed with fresh purified BICMV, and greater amounts of these proteinaceous components were revealed by PAGE analyses of stored purified BICMV preparations (Fig. 10), it is assumed that the smaller components are due to the degradation of the slow moving protein during purification and storage. Hiebert and McDonald (1976) observed that some possible enzymatic degradation of TuMV capsid protein occurred during storage of purified virus preparations. The lower sedimentation coefficient estimated for stored purified BICMV preparations (Fig. 9) is further evidence of proteolytic degradation of viral coat protein during storage at 4 C. According to Hiebert and McDonald (1976), it is likely that "s20 values reported for potyviruses that are near 140 S represent virus with partially degraded capsid protein, whereas those near 160 S represent virus with intact capsid protein." This proteolytic degradation also changes






75



the antigenic properties of viral coat protein (Hiebert and McDonald, 1976; and Purcifull and Batchelor, 1977). Using antiserum obtained for freshly purified BICMV, serological distinction was observed between freshly purified preparations of BICMV and purified BICMV stored at 4 C for more than 30 days (Fig, 16-F). The serological distinctions between different antigen preparations of the same virus observed herein are of great significance for serological identification and characterization of potyviruses as pointed out by Hiebert and McDonald (1976) and Purcifull and Batchelor (1977). It is important to keep in mind that purification, storage of either purified virus preparations or crude sap containing virus, and mailing of virusinfected fresh plant tissues may all result in modifications in the antigenic properties of viral coat protein. To solve this problem, the preservation of plant virus antigens by lyophilization of crude extracts from infected plants (Purcifull et al., 1975) or purified virus preparations is recommended. Blackeye cowpea mosaic virus has been maintained in lyophilized condition either in crude sap or purified preparation over two years during the course of this study without any perceptive change in its antigenic properties.

Another factor that should be considered during serological

relationship studies between viruses in the PVY group is the specificity of antisera. Variations in the degree of cross reactivity exhibited by different antisera obtained against the same virus have been attributed to differences between individual animals (van Regenmortel and von Wechmar, 1970), route and number of injections used in the immunization program (Hollings and Stone, 1965) and time of bleeding






76



(Tremaine and Wright, 1967; and Koenig and Givord, 1974). The results of the present study indicated that the immunization program and the conditions of the arntigenic solution used for rabbit immunization may also affect the antiserum specificity. A highly specific antiserum for BCMV-S was obtained from a rabbit immunized with freshly purified BCMV-S, whereas antiserum with a broader cross-reactive spectrum was obtained from the same rabbit after a booster injection with a purified preparation of BCMV-S stored at 4 C for more than one month. The use of such antisera would make it difficult to distinguish between certain plant viruses in SDS immunodiffusion tests. The serological distinction between BICMV and BCMV-S was impossible to detect in SDS doubleimmunodiffusion when the BCMV-S antiserum with a wider cross-reactivity was used. On the other hand, an antiserum with a wide spectrum of activity should be useful for identification of virus-infected tissue used for plant propagation and possibly for identification of virus at the group level. As any virus-infected plant organ is undesirable for plant propagation the specific virus identification may not be necessary in such cases. For example, the BCMV-S antiserum was successfully used to identify cowpea seeds infected with BICMV.

Unilateral serological relationships observed between BlCMV and SoyMV (Fig. 15-G) and with BlCMV and BYMV (Fig. 15-H) showed the nenessity of reciprocal tests for demonstrating the absence of serological relationship between two viruses. According to Matthews (1970) "to demonstrate that two viruses are serologically unrelated, reactive antisera must be prepared against each of the viruses under test." Reciprocal tests are also important to show distinction between two closely related viruses. It was more difficult to observe a spur





77



between BICMV and WMV-2 when both viruses were tested against antiserum to WMV-2 than when they were tested against BICMV antiserum (Fig. 15-C). Similar results were observed with BCMV isolates and BICMV (Fig. 15-E,

-F) which may explain the identical reaction reported by Lyemoto et al. (1973).

It is noteworthy that BICMV and BYMV are serologically distinct,

though related. This supports the contention of Edwardson et al. (1972) and Zettler and Evans (1972) that BICMV and BYMV should be considered distinct viruses.

Serological differences between closely related viruses are better detected with antisera of fairly low titer (Matthews, 1970). On the other hand, he also stated that a high titer antiserum is preferable for demonstrating distant serological relationship. This can be illustrated by the serological tests carried out with BICMV and CAMV isolates using a BICMV antiserum with a titer of 32. By diluting the antiserum to 1/4, no reaction was observed with the heterologous virus (CAMV) whereas a fairly good reaction was still detected with the homologous antigen. The absence of reaction between BICMV and LMV-antiserum (Fig. 15-J) may be a result of the low titer of the antiserum.

The intragel cross-absorption test was effective for demonstrating distinctions between two closely related viruses (Fig. 16-B, -D). This is additional evidence that serological distinctions that are undetectable in conventional double-immunodiffusion tests may be clearly revealed by intragel cross-absorption. Using this test, Matthews (1970) revealed a serological difference between type TMV and a nitrous acid induced mutant which showed a reaction of identity when tested against






78



unabsorbed TMV antiserum. For a full precipitation of the crossreacting antibodies close to the center well, a fairly high concentration of the heterologous antigen should be used to fill the antiserum well. This is illustrated by the intragel cross-absorption tests with BICMV antiserum shown in Figure 16. A precipitin ring was formed very close to the center well when a highly concentrated purified preparation of BCMV-S (0.5 1.0 mg/ml) was used to absorb BICMV antiserum (Fig. 16-0) whereas the ring formed approximately 2 mm away from edge of the well when BICMV antiserum was absorbed with a less concentrated preparation of CAMV (0.01 0.05 mg/mi) (Fig. 16-B), In both cases, though, the intragel cross-absorption test showed serological distinction between the viruses. The intensity of the reaction between the homologous antigen and the cross absorbed antiserum may give some information about the degree of relationship between the viruses. Weaker homologous reaction indicates closer serological relationship. Based on this, the results of the present study clearly indicate that BICMV is more closely related serologically to BCMV-S than to CAMV (Fig. 16-B, -D). The different degrees of serological relationships are also indicated by the intensity of the precipitin lines spurring over the heterologous virus reactions in straight diffusion tests (Fig. 16-A, -C). Serological relationship between different potyviruses has been commonly observed (Bercks, 1960; Purcifull and Shepherd, 1964; Purcifull and Gooding, 1970; Uyemoto et al., 1972; and Shepard et al., 1974), and the cross absorption of an antiserum with heterologous viruses has also been used to study serological relationship between plant viruses in tube precipitin tests (Wetter, 1967; and Alba and Oliveira, 1977), and in combination with gel diffusion tests (van Regenmortel, 1966; Wetter,






79



1967; Nelson and Knuhtsen, 1973; Shepard et al., 1974; and Jones and Diachun, 1977). The intragel cross-absorption technique was also observed to be useful in demonstrating cross-protection between serologically distinct strains of plant viruses (Lima and Nelson, 1975).

The fact that most of the antisera obtained against purified

BICMV preparations did not react with extracts of noninfected cowpea tissue can be added to confirm the efficiency of the virus purification procedures described herein, On the other hand, the high population of antibodies for normal plant antigens developed by the rabbits injected with BICMV-I purified from infected cowpea was an indication that virus-infected cowpea tissue may have a high concentration of host antigens, which were difficult to separate from the BICMV cytoplasmic inclusions. However, using N. benthamiana as a source plant for BICMV-1 purification, antiserum specific for BICMV-I was obtained. This is an additional indication of the useful application of N. benthamiana in plant virus research. Nicotiana benthamiana has been artificially infected with more than 50 plant viruses (Quacquarelli and Avgelis, 1975; and Christie and Crawford, in press), showing its great potential for cytological, serological, and physiological studies of different viruses in the same biological system.

The foot-pad route of rabbit immunization (Ziemiecki and Wood,

1975) used to obtain the antiserum specific for B1CMV-I was an efficient procedure. The high yield of antibodies obtained for BCMV-S using the same route of immunization (Lima et al., unpublished) is additional evidence that a high titer antiserum can be obtained at the expense of very little antigen.






80


Reciprocal immnunodiffusion tests with antisera specific for

BICMV and BICMV-1 (Fig. 12-A, -B) confirmed the findings of Hiebert et al. (1971), Purcifull et al. (1973), Batchelor (1974), and McDonald and Hiebert (1975) that the inclusion body proteins are immunochemically distinct from viral coat protein and host proteins.

The results of single radial immunodiffusion tests indicated that agar-media impregnated with mono-specific antiserum or with a mixture of antisera can be used for serodiagnosis of two morphologically distinct legume viruses. Single radial immunodiffusion tests were first used in plant virology by Shepard (1969) for serodiagnosis of potato virus X in potato tuber sprouts. Subsequently the same method was successfully used to identify plant tissue infected with carlaviruses (Shepard, 1970; and Shepard et al. 1971) potyviruses (Uyemoto et al., 1972; and Casper, 1974), a cucumovirus (Richter et al., 1975), a hordeivirus (Slack and Shepherd, 1975), and tobamovirus (Granett and Shalla, 1970; and Clifford, 1977). Radial-immunodiffusion plates containing a mixture of antisera to two or three filamentous viruses have been used for detection of potato viruses X, S, and M (Shepard, 1972). This, however, appears to be the first report of a multiple-antisera medium for detection of both an isometric and a rod-shaped plant viruses.

Shepard (1969) observed that single radial immunodiffusion was

more sensitive than double immunodiffusion for detection of PVX in infected plant tissue, but Richter et al. (1975) obtained better results with double diffusion tests than with single diffusion for serological detection of CMV in naturally infected herbaceous plants. No attempts to compare these two serological techniques were made in the present study. Some observations, however, indicated that single radial






81



immunodiffusion requires fairly large amounts of antiserum and that the proper antiserum concentration needs to be previously determined for highest sensitivity and to avoid spurious reactions in SDS-agar media. Better results in multi-antisera media were obtained when pyrrolidine was used as denaturant of virus coat protein.

The three serologically related but distinct legume viruses,

BICMV, BCMV-S, and CAMV can also be differentiated by some biological properties. The CAMV isolate was well adapted to cowpea, infecting and causing symptoms in all 20 inoculated cowpea varieties. On the other hand, five cowpea varieties showed immunity to BICMV, and only two were infected with BCMV-S,which caused very mild symptoms (Table I). The different symptomatological reactions induced by CAMV and BICMV in some of the varieties (Table I) clearly indicate that they can be used to distinguish these two potyviruses. It was observed, however, that some of the symptoms induced by the viruses varied with temperature, light conditions, and age at which the plants were inoculated, but no variation was observed with the immunity of any cowpea variety. The cowpea varieties that showed immunity to BICMV (Table I) should be included in a cowpea breeding program or in a control program for this virus in the southeastern United States. Cowpea lines with resistance to other viruses have been selected in different parts of the world (Williams, 1977a; and Beier et al., 1977). Virus-resistant lines with resistance to other plant pathogens have also been identified (Williams, 1977a and 1977b).

Attempts to compare BICMV with the East African type of CAMV were impossible because all samples of virus-infected leaf-tissue arrived in high degree of decomposition with the virus already inactivated.






82



Serological studies with such decomposed leaf tissue and BICMV

antiserum gave results similar to those obtained with the CAMV isolate (Fig. 16-A) obtained originally from Morocco. As the inactivation of the virus in the decomposed tissue may have destroyed some of its antigenic determinants, no conclusive results about its serological relationship with BICMV can be derived from these tests.

Light and electron microscopy of cowpea and other host cells infected with any one of these three legume potyviruses revealed that their cytoplasmic inclusions are morphologically similar. In ultrathin sections, their inclusions consisted of pinwheels similar to those induced by the potyviruses from Edwardson's subdivision-I (Edwardson, 1974). The cytoplasmic inclusions induced either by BCMV-S or CAMV, however, failed to react with antiserum for BICMV induced inclusions. The low titer of the inclusion antiserum, however, may be one of the reasons for the absence of reactions. Despite the great similarity in the ultrastructures of pinwheel inclusions induced by BICMV and CAMV, they showed some difference at the light microscope level. Whereas BICMV induced big masses of cytoplasmic inclusions in 'Knuckle Purple Hull' (Fig. 17), only scattered small bundles of inclusions were observed in the cells of this host infected with CAMV. This is also an indication of no direct correlation between the severity of the symptoms and abundance of cytoplasmic inclusions induced by these potyviruses, since 'Knuckle Purple Hull' is more susceptible to CAMV than to BICMV (Table I). A similar phenomenon was observed with these two viruses in C. spectabilis. In addition to this, the nuclear inclusions readily observed in cells of C. spectabilis systemically






83



infected with BICMV (Christie and Edwardson, 1977) were not seen in leaf tissue of this host infected with CAMV.

In summary, BICMV is a potyvirus that belongs to Edwardson's

subdivision--I (Edwardson, 1974) and has a modal length of approximately 750 nni. The BICMV particles have a single sedimenting peak with s20 157 159 S and have a main protein component with a MW of 34,000 daltons. Its cytoplasmic inclusions are made of tubes which show striations with periodicities of approximately 5 nm and consist of a single type of protein estimated to have a MW of 70,000 daltons. The virus also induces nuclear inclusions in certain hosts including C. spectabilis. Blackvye cowpea mosaic virus is serologically unrelated to seven potyviruses and serologically related to, but distinct from eight other potyviruses in SDS-immunodiffusion. The virus has a narrow host range outside Leguminosae, is seed-borne in at least two cowpea varieties and is transmitted by aphids in a nonpersistent manner. Based on its physicl, biological, cytological and immunochemical properties, BICMV can be differentiated from any other virus that infects cowpea. The antisera prepared for BICMV and its cytoplasmic inclusions were essential tools for the development of the serological techniques for detection of virus-infected seeds described in Chapter II.















CHAPTER II

IMMUNOCHEMICAL AND CYTOLOGICAL TECHNIQUES FOR DETECTION OF LEGUME VIRUSES IN INFECTED SEEDS


Introduction


The transmission of plant viruses through seed of infected host plants was first demonstrated by Reddick and Stewart (1919), who showed that bean common mosaic virus (BCMV) was transmitted by approximately 50% of seeds from infected Phaseolus vulgaris. Since then, the phenomenon of seed transmission of plant viruses has received considerable attention and an appreciable number of viruses have been demonstrated to be seed-borne to some extent (Fulton, 1964; Bennett, 1966; Shepherd, 1972; and Phatak, 1974). Virus can be introduced into a crop at an early stage of plant development through infected seeds. Thus, the production of virus-free seeds, or seed lots with very low virus content may provide a very effective control of seed-borne plant viruses. Seed certification programs have been developed to test seed lots for the presence of viruses and to select virus-free seeds. Barley stripe mosaic virus, which is responsible for a serious disease in Montana (Afanasiev, 1956), and lettuce mosaic virus(LMV), the causal agent of an important disease of lettuce (Grogan et al 1952), are good examples of virus diseases against which seed certification programs have been successful (Zink et al., 1956; Hamilton, 1965; Phatak, 1974; and Slack and Shepherd, 1975).


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Barley stripe mosaic virus has no known vector, such as insects or mites, but it has a high rate of seed transmission, which severely reduces crop production. On the other hand, LMV is transmitted by a low percentage of seeds, but is regularly spread further in the field by aphids, resulting in substantial losses (Zink et al., 1956).

Several methods (Phatak, 1974) have been developed to detect the presence of infected seeds to control plant diseases caused by seedborne viruses: Growing-on tests, Seeds are planted in greenhouses or under other inse.t-proof conditions and the first leaves of the seedlings are observed for the characteristic symptoms which vary according to the host-virus combination This method can fail under environmental conditions that adversely affect the symptom development and with latent strains of a virus that do not produce visible symptoms. Indicator-inaculation tests. Seeds are ground up with buffer solution and mechanically inoculated in the indicator hosts. Although this method has been used extensively for LMV (Phatak, 1974), it is very time consuming and requires considerable greenhouse space. Serological tests. Immunochemical tests have also been developed for detecting virus in extracts from single seeds (Scott, 1961, and Lister, 1977), and individual seed embryos (Hamilton, 1965). However, no successful results have been obtained in double immunodiffusion systems with long flexuous rod viruses such as those from the potyvirus group (Phatak, 1974). Electron microscopy. A serologically specific electron microscopic technique developed by Derrick and Brlansky (1976) has been successfully used to detect the presence of virus particles in extracts of groups of seeds (Brlansky and Derrick, 1976).





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The main purpose of the present investigation was to develop efficient and rapid serodiagnostic techniques to assay legume seed lots for the presence of virus-infected seeds. Seeds of cowpea, V. unguiculata 'Knuckle Purple Hull' infected with BICMV were used as a model hostvirus combination. Immunochemical techniques for detection of BICMV, BCMV, and SoyMV in hypocotyls of germinated virus-infected seeds of V. unguiculata, P. vulgaris, and G. Max, respectively, are described in this chapter. Abstracts of portions of this research have already been published (Lima and Purcifull, 1977a, 1977b).


Literature Review


The phenomenon of seed transmission of plant viruses was first demonstrated by Reddick and Stewart (1919), who presented strong evidence of seed transmission of BCMV in Phaseolus vulgaris. Since then, a large body of information has been accumulated about the transmission of numerous plant viruses through the seeds of infected host plants (Fulton, 1964; Bennett, 1966; Shepherd, 1972; Baker, 1972; and Phatak, 1974). Among the 183 plant viruses described in the Commonwealth Mycological Institute up to September, 1977, (Doi et al., 1977), 51 viruses have been experimentally demonstrated to be seedborne to some extent. Several plant viruses are known to be seed-borne in many leguminous host plants, but this review will cover only those viruses transmitted through seeds of Vigna spp., Glycine max, and Phaseolus vulgaris.






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Seed-Borne Viruses in Vigna spp.

Gardner (1927) apparently was the first to report the transmission of a cowpea virus through seeds of cowpea. Since then, many viruses which naturally infect cowpea have been demonstrated to be seed-borne in this host.

A virus isolated from cowpea in Trinidad was demonstrated to be

transmitted through 8% of seeds of asparagus-bean (Vigna sesquipedalis) obtained from virus-infected plants (Dale, 1949). The virus is believed to be a representative strain of cowpea mosaic virus (Agrawal, 1964; and van Kammen, 1971, 1972). It seems that the seed transmissibility of CPMV is erratic and depends on the type of virus isolate and the cowpea variety involved. A cowpea mosaic virus isolated from cowpea grown in Arkansas was seed-borne in this host (Shepherd, 1964). Approximately 620 'Blackeye' cowpea plants grown from seeds harvested from plants artificially inoculated with CPMV failed to develop mosaic symptoms (Perez and Cortes-Monllor, 1970). On the other hand, Haque and Persad (1975) observed that the rate of seed transmission of CPMV varied from zero to 5.8% depending on the cowpea varieties and selections.

Anderson (1957) reported the seed transmission of three cowpea viruses, including a strain of CMV, which was transmitted through

4 28% of cowpea seeds from artificially infected plants. A virus closely resembling a strain of CMV was seed-borne in cowpea with a transmission rate of 5 to 16% (Chenulu et al., 1968). On the basis of symptoms observed on cowpea plants grown from commercial seeds, Gay and Winstead (1970) reported seed transmission of CMV, a cowpea




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in fields of 'Knuckle Purple Hull* cowpeas in Gainesville, Florida,
or from Gilvan Pio Ribeiro, University of Georgia, Athens. As controls,
virus-free 'Knuckle Purple Hull' seeds produced in Texas were obtained
from a local commercial source.
Seeds were surface sterilized in 0.5% sodium hypochlorite for
10 min, rinsed thoroughly with sterile deionized water and placed in
moistened paper towels to germinate. The towels were rolled up, placed
upright in 30 ml beakers and the seeds were allowed to germinate for
3 to 5 days at 25 27 C in an incubator.
Preparation of Antigens for Serology
Hypocotyls from germinated seeds, singly or in groups of 5 or 10
were tested in double or single radial immunodiffusion tests. If in
formation about the percentage of infected seeds was wanted, I 2 mm
thick discs from individual hypocotyls were cut with a razor blade
which was rinsed with 95% ethanol and deionized water after cutting
each hypocotyl, These hypocotyl discs were tested individually in
double or single immunodiffusion tests by embedding them directly
into the agar. Groups of 5 or 10 hypocotyls were ground in water or
1.5% SOS (1/1 w/v) with a mortar and pestle as described previously
(Purcifull and Batchelor, 1977) and tested against B1CMV and B1CMV-I
antisera in double and single radial diffusion. Single or bulked
hypocotyls were checked by SSEM for the presence of B1CMV particles.
Double Immunodiffusion Tests
Individual hypocotyls or groups of hypocotyls from germinated
cowpea seeds were tested against B1CMV antiserum in double diffusion


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Dan E. Pure i fu 11 tha i rman
Professor of Plant Pathology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
?
Charudattan
Assistant Professor of Plant Pathology
I certify that I have read this study and that in my opinion*it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
J^hn R. Edwardson
Professor of Agronomy
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Ernest Hiebert
Associate Professor of Plant Pathology


Immunodiffusion tests and serologically specific electron micro
scopy were used to detect B1CMV in hypocotyls of 4-5-day-old cowpea
seedlings grown from B1CMV-infected seeds. Discs of individual hypo
cotyls were embedded into the agar medium 4-5 mm away from the anti
serum wells. Virus-specific precipitin lines formed between virus-
infected hypocotyl discs and antiserum wells, whereas no reactions
were observed with healthy hypocotyls. Precipitin lines were also
observed with extracts of mixtures from infected (] g) and healthy
(up to 29 g) tissues These immunochemical techniques were also used
for detecting BCMV in hypocotyls of infected 4-5-day-old Phaseolus
vulgaris L. seedlings and for detecting SoyMV in infected Glycine max
(L.) Herr, seedlings. Single radial immunodiffusion tests with ex
tracts or discs of cowpea hypocotyls were also useful for detecting
B1CMV in germinated seeds. The reliability and simplicity of the
immunodiffusion tests make them suitable for use in routine seed health
testing program in any laboratory.
x i i i


3
A widespread mosaic disease was reported on different cowpea
varieties in Trinidad (Dale, 1949). Dale (1949) observed that the
virus responsible for the disease was not transmitted by Aphis
medicaginis Koch, but that the leaf beetle, Ceratoma ruficornis (01iv.)
was a good vector and was probably responsible for transmitting the
virus in the field. On the basis of his studies, he concluded that
the virus was unrelated to those described by McLean (1941), Snyder
(1942) and Yu (1946), but was more likely the virus studied by Smith
(1924). Dale (1953) subsequently confirmed that the cowpea mosaic
virus isolated from Trinidad was efficiently transmitted by ruficornis,
but not by aphids.
Lister and Thresh (1955) isolated a virus from cowpea and identi
fied it as a strain of tobacco mosaic virus (TMV). They observed that
a purified preparation of the virus contained rod-shaped particles of
varying lengths, indistinguishable from the particles of TMV, and was
precipitated specifically with antiserum prepared against TMV. A cow
pea strain of TMV was also isolated from a range of leguminous hosts
at Ibadan, Nigeria (Chant, 1959). Chant (1959) also found another
virus infecting cowpea in Nigeria and as its physical properties
differed from other cowpea viruses, he proposed the name cowpea yellow
mosaic virus (CYMV). The virus was purified and an antiserum prepared
against it. Both TMV and CYMV were transmitted by the beetle Ootheca
mutabi1 is Sahib. In subsequent work, Chant (i960) studied the influence
of TMV and CYMV on growth rate and yield of cowpea, and found that
infection of cowpea with the cowpea strain of TMV did not affect yield
as much as infection with CYMV. Wells and Deba (1961) tested 116 cow
pea varieties and 342 indigenous pure lines against CYMV and observed


Figure I Systemic and localized symptoms induced by blackeye cowpea
mosaic virus (BICMV) in cowpea, ungu i cu lata 'Knuckle
Purple Hull' and C. ama rant¡color.
A) Typical mosaic on secondary trifol¡oate leaf of cow
pea plant inoculated with BICMV (l), and primary
trifoliate leaf showing vein clearing (2).
B) Local lesions on leaf of C. amarant¡color inoculated
with BICMV.


gure 10 Electrophoretic analyses of BlCMV induced cytoplasmic inclusions (BICMV-l) and B1CMV
capsid protein in a 6% polyacrylamide gel slab containing 0.1% sodium dodecyl sulfate
(SDS) and sodium phosphate buffer, dH 7.2.
A) Polyacrylamide gel electrophoresis of SDS dissociated proteins of BICMV-l
(a), viral coat protein of freshly purified preparation of BlCMV (b) viral coat
protein of purified BlCMV after 30 days of storage at A C, (c), and marker
proteins (d-h). The migrations of the marker proteins on the gel are repre
sented by the arrows: (d) bovine serum albumin, (e) glutamate dehydrogenase,
(f) ovalbumin, (g) carbonic anhydrase, and (h) TMV capsid protein.
B) Molecular weight determination of SDS dissociated proteins of BICMV-l and capsid
protein of fresh and old purified. BlCMV preparations in polyacrylamide gel electro
phoresis using marker proteins. The unnumbered circles along the standard line
represent the following marker proteins with their molecular weights in paren
theses from top to bottom: bovine serum albumin (67,000 daltons), glutamate
dehydrogenase (53,000 daltons), ovalbumin (A3,000 daltons), carbonic anhydrase
(29,000 daltons), and TMV capsid protein (17,500 daltons). The distances from
the top, correspond to the distances measured in photograph positioned as in (A).
The estimated molecular weights for the virus and its cytoplasmic inclusion
proteins are as follows: (1) 70,000 daltons (BICMV-l), (2) 3A,000 daltons
(undegraded BlCMV coat protein), (3) 29,000 daltons (degraded BlCMV coat protein),
and (A) 27,000 daltons (degraded virus protein).


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immunodiffusion requires fairly large amounts of antiserum and that
the proper antiserum concentration needs to be previously determined
for highest sensitivity and to avoid spurious reactions in SDS-agar
media. Better results in multi-ant¡sera media were obtained when
pyrrolidine was used as denaturant of virus coat protein.
The three serologically related but distinct legume viruses,
BlCMV, BCMV-S, and CAMV can also be differentiated by some biological
properties. The CAMV isolate was well adapted to cowpea, infecting
and causing symptoms in all 20 inoculated cowpea varieties. On the
other hand, five cowpea varieties showed immunity to BlCMV, and only
two were infected with BCMV-S, which caused very mild symptoms
(Table I). The different symptomato 1ogicaI reactions induced by CAMV
and BlCMV in some of the varieties (Table l) clearly indicate that
they can be used to distinguish these two potyviruses. It was ob
served, however, that some of the symptoms induced by the viruses
varied with temperature, light conditions, and age at which the plants
were inoculated, but no variation was observed with the immunity of
any cowpea variety. The cowpea varieties that showed immunity to
BlCMV (Table I) should be included in a cowpea breeding program or in
a control program for this virus in the southeastern United States.
Cowpea lines with resistance to other viruses have been selected in
different parts of the world (Williams, 1977a; and Beier et al.,
1977). Virus-resistant lines with resistance to other plant pathogens
have also been identified (Williams, 1977a and 1977b).
Attempts to compare BlCMV with the East African type of CAMV were
impossible because all samples of vi rus-infected leaf-tissue arrived
in high degree of decomposition with the virus already inactivated.



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BLACKEYE COWPEA MOSAIC VIRUS: PURIFICATION, PARTIAL CHARACTERIZATION, SEROLOGY, AND IMMUNOCHEMICAL AND CYTOLOGICAL TECHNIQUES FOR DETECTION OF V 1 BUSI NFECTED LEGUME SEEDS By J. ALBERSIO A. LIMA A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1978

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To my wife, Diana, and my son, Roberto, who with understanding, friendship, and love heiped to transform a goal into a reality.

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ACKNOWLEDGEMENTS I wish to express my sincere gratitude and appreciation to Dr. Dan E. Purcifull, chairman of my supervisory committee, for his invaluable counsels, friendship, advice, and constant guidance during the course of this investigation. Appreciation is extended to other members of my supervisory committee, Drs. Ernest Hiebert, John R. Edwardson, Raghavan Charudattan, Francis W. Zettler, 3nd Daniel A. Roberts for their helpful suggestions during the research and their efforts in criticizing the manuscript. I also wish to extend my appreciation to Mr. Richard G. Christie for his valuable help with the light microscope and for his constant enthusiasm for teaching useful cytological techniques for diagnosing plant virus diseases. The understanding and cooperation of Mr. S. Christie, Mr. W. Crawford, Mrs. J. Hill, and Mrs. D. Miller during the laboratory experiments are also greatly appreciated. I further wish to extend my gratitude to Mrs. Maria I. Cruz for her understanding and cooperation as the Secretary of the International Programs of the University of Florida, Gainesville, and for her time spent in typing this dissertation. I was supported by funds from the United States Agency for International Development (USAID), Universidade Federal do Ceara, and Ford Foundation, to whom I wish to express my sincere thanks. Special recognition is expressed to my wife, Diana, whose patience friendship, and love made this work possible. i i i

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TABLE OF CONTENTS Page ACKNOWLEDGEMENTS iii LIST OF TABLES vi LIST OF FIGURES vi i VIRUS ABBREVIATIONS x ABSTRACT xi CHAPTER I PURIFICATION, PARTIAL CHARACTERIZATION, AND SEROLOGY OF BLACKEYE COWPEA MOSAIC VIRUS I Introduction 1 Literature Review 2 Materials and Methods 10 Sources of Virus Isolate 10 Virus and Inclusion Purification II Virus Particle Size Determination \k Stability of Virus in Sap 15 Pol yacryl amide Gel Electrophoresis of Viral and Inclusion Proteins I6 Sedimentation Coefficient Determination.... 17 Serology |8 Antiserum production for virus and cytoplasmic inclusions |8 Serological tests 19 Serological relationships between BICMV and other potyviruses 21 Light and Electron Microscopy of Virus Induced Pinwheel Inclusions 22 Host Range and Screening Cowpea Varieties for Resistance 23 Results 2^* Purification and Properties of Blackeye Cowpea Mosaic Virus 2k Purified Inclusion Preparations k2 Virus Particle Size and Stability in Sap... k7 Serology 52 Light and Electron Microscopy 6^* Host Range and Resistant Cowpea Varieties.. 65 Discussion 7 1 iv

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Page CHAPTER II IMMUNOCHEMICAL AND CYTOLOGICAL TECHNIQUES FOR DETECTION OF LEGUME VIRUSES IN INFECTED SEEDS 84 liitroducr ion 8k Literature Review 86 Seed-Borne Viruses in Vigna spp 87 Seed-Borne Viruses in Glycine max 90 Seed-Borne Viruses in Phas eo 1 us vulgaris... 32 Materials and Methods 3k Source of Seed and Seed Germination 3k Preparation of Antigens for Serology 95 Double Immunodiffusion Tests 95 Single Radial Immunodiffusion Tests 96 Serologically Specific Electron Microscopy. 97 Double Immunodiffusion Tests and SSEM for Detection of Other Viruses in Germinated Legume Seeds 98 Serology and Microscopy of Cytoplasmic Inclusions Induced by BICMV and SoyMV in Hypocotyls of Germinated Seeds ,.. 98 Results 99 Preparation of Antigens for Serological Tests 99 Double Iniinunod i f f us ion Tests 102 Single Radial Immunodiffusion Tests 107 Serologically Specific Electron Microscopy Ill Double Immunodiffusion Tests and SSEM for Detection of Other Viruses in Germinated Legume Seeds 116 Serology and Mycroscopy of Cytoplasmic Inclusions Induced by BlCMV and SoyMV in Hypocotyls of Germinated Seeds 121 D iscuss ion 1 32 LITERATURE CITED , I38 BIOGRAPHICAL SKETCH 15/4 V

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LIST OF TABLES Table Page I Symptoms and results of serological assays on varieties of cowpea Vigna unguicu lata mechanically inoculated with BICMV, BCMV-S "CAMV and CPMV 70 li Comparison of immunodiffusion tests with hypocotyl discs and growing-on tests for detection of virusinfected beeds V i

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LIST OF FIGURES Figure Page 1 Systemic and localized symptoms induced by blackeye cowpea mosaic virus (BICMV) in cowpea V. unguiculata 'Knuckle Purple Hull" and C. amarant i color 26 2 Flow diaqram outlining the procedure of purification of BICMV using n^-butanol as clarifying agent 28 3 Flow diagram outlining the procedure for purification of BICMV and its cytoplasmic inclusions, using chloroform and carbon tetrachloride as clarifying agents 30 A Flow diagram outlining the steps carried out during the purification of BICMV and its cytoplasmic inclusions by a combination of the first and second methods for purification of virus and inclusions 32 5 Absorption spectra of purified preparations of BICMV in 0.02 M Tris buffer, pH 8.2, and BICMV cytoplasmic inclusions in the same buffer 35 6 Electron microscopy of BICMV in a purified preparation and in cowpea leaf extracts 37 7 Histograms of lengths of BICMV particles from purified preparation negatively stained with phosphotungstate and cowpea leaf extract using the serologically specific electron microscopy and uranyl acetate as a 39 pos i t i ve stain 8 Histogram^ of BICMV particle lengths from two different electron inicroscopic preparations to show particle length distribut ion f rom 600 To 900 nm Z^] 9 Schlieren patterns from sedimentation velocity experiment with stored and fresh purified preparations of BICMV 4/, 0 Electrophoret ic analyses of BICMV induced cytoplasmic inclusions and BICMV capsid protein in (>% polyacrylamide ge I i^^ 1 Electron micrographs of purified preparations of BICMV cytoplasmic inclusions stained with molybdate.. ^9

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Figure Page 12 Double immunodiffusion tests in agar medium containing 0.8^ Noble aqar, \ ,01 NaN^, and 0.5^ SDS 51 13 Single radial diffusion tests in agar media containing different concentrations of SDS and antisera for BICMV and cowpea mosaic virus (CPMV) 5^ ]k Single radial diffusion tests with SDS and pyrrolidine degraded capsid protein of BICMV and CPMV 56 15 Reciprocal double immunodiffusion tests with BICMV and other potyviruses in medium containing 0.8^ Noble agar, 1.0/c NaN^, and 0.5^?. SDS prepared in 0.05 M TrisHCI buffer, pH^7.2 61 16 Immunodiffusion tests with BICMV, Moroccan isolate of cowpea aphid-borne mosaic virus (CAMV) and siratro strain of bean common mosaic virus (BCMV-S) in agar medium containing 0.8;^ Noble agar, 1.0^ NaN., and 0.5^ SDS prepared in 0.05 M Tris-HCI buffer, pH 7.2. 63 17 Photomicrographs of cytoplasmic inclusions in epidermal strips of cowpea leaves systemically infected with BICMV, stained with a combination of calcomine orange and luxol brilliant green 67 18 Electron micrographs of ultrathin sections of cowpea leaf cells infected with BICMV showing cross-sections and longitudinal sections of pinwheel inclusions 69 19 Double immunodiffusion tests with extracts from different portions of Bl CMVinfected and healthy '4-5-day-old cowpea seedlings 101 20 Diagram showing methods for assaying legume seeds by single and double radial immunodiffusion 10^ 21 Double immunodiffusion tests with hypocotyls from healthy and B 1 CMVi nf ected ^-5-day-old cowpea seedlings in medium containing 0.8% Noble agar, 1.0^ NaN^, and 0.5% SDS, prepared in water 106 22 Single radial immunodiffusion tests with hypocotyl extracts from healthy and B 1 CMVi nf ected k-S-dayold cowpea seedlings I 10 23 Electron micrographs of BICMV in hypocotyl extracts from the same cowpea seedling using different preparations 113 v i i i

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Figure Page 2k Electron micrographs of serologically specific electron microscopy with BlCMV, BCMV-S, and CPMV 115 25 Double immunodiffusion tests with hypocotyls of k-Sday-old bean and soybean seedlings using antiserum for BCMV-S and for SoyMV II8 26 Electron micrographs of serologically specific electron microscopy with extracts from BCMVand SoyMVinfected hypocotyls 120 27 Double immunodiffusion tests with hypocotyl extracts from ^-5-day-old seedlings of cowpea and soybean using antisera for BlCMV, SoyMV, and their cytoplasmic inclusions 123 28 Photomicrographs showing different views of cytoplasmic inclusions induced by BlCMV in epidermal strips of cowpeu hypocotyl tissue stained with a combination of calcoiiiine orange and luxol brilliant green 125 29 Photomicrographs showing different views of epidermal cells of hypocotyls from 'i-S-day-old soybean seedlings containing cytoplasmic inclusions induced by SoyMV... 127 30 Electron micrographs of ultrathin sections of cells from hypocotyls of '4-5-day-old seedlings infected with BlCMV 129 31 Electron micrographs of ultrathin sections of hypocotyl cells of '^-S-day-old soybean seedlings grown from SoyMVinfected seeds 131 ix

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VIRUS ABBREVIATIONS Virus Names Abbreviat ion Bean common mosaic virus BCMV Bean common mosaic v i rus -s i ra c ro isolate BCMV-S Bean poiJ mottle virus BPMV Bean yellow mosaic virus BYMV Bidens mottle virus BiMV Blackeye cowpea mosaic virus BICMV Clover yellow vein virus CYVV Commel ina mosaic virus CoMV Cowpea aphid-borne mosaic virus CAMV Cowpea chlorotic mottle virus CCMV Cowpea mild mottle virus CMMV Cowpea mosaic virus CPMV Cowpea ringspot virus CpRV Cowpea yellow mosaic virus CYMV Cucumber mosaic virus CMV Dasheen mosaic virus DMV Iris mosaic virus IMV Lettuce mosaic virus LMV Pea seed-borne mosaic virus PSMV Pepper mottle virus PeMV Pepper vein mottle virus PVMV Pokeweed mosaic virus PWMV Potato virus X PVX Potato V i rus Y p\/Y Southern bean mosaic virus SBMV Soybean mosaic virus SoyMV Sugarcane mosaic virus SMV Tobacco etch virus j£\i Tobacco mosaic virus Tobacco ringspot virus TRSV Turn i p ye 1 low mosa ic virus TuMV Watermelon mosaic virus-l WMV-1 Watermelon mosaic viru5-2 WMV-2 X

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Abstract of Dissertation Presented to the Graduate Council of tht; University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy BLACKEYE COWPEA MOSAIC VIRUS: PURIFICATION, PARTIAL CHARACTERIZATION, SEROLOGY, AND IMMUNOCHEMICAL AND CYTOLOGICAL TECHNIQUES FOR DETECTION OF V I RUSI NFECTED LEGUME SEEDS By J. ALBERSIO A. LIMA March, 1978 Chairman: Dan E. Pure i full Major Department: Plant Pathology Bhickeye cowpea mosaic virus (BICMV) was increased in cowpea V i gna unguicu l ata (L.) Walp., 'Knuckle Purple Hull', and infected leaves were used for virus and cytoplasmic inclusion purification. Either n-butanol or a combination of chloroform and carbon tetrachloride was used in the clarification procesb. Pol yacry 1 am i de gel electrophoresis of sodium dodecyl sulfate (SDS) dissociated inclusions and virus revealed that the inclusions were made of a single protein estimated to have a molecular weiyht (mW) around 70,000 daltons whereas freshly purified BICMV consisted of a main protein component with a MW of 3^,000 daltons and two smaller proteins with MWs of 29,000 and 27,000 daltons. Purified BICMV had a 260/280 nm absorption ratio of 1.2 and a modal length of 753 nm. Freshly purified BICMV preparations showed a single sedimenting peak with S2q=157-159 S. The purified BICMV cytoplasmic inclusions had absorption spectra characteristic for proteins. Electron microscopy of purified inclusions revealed the presence of tubes showing striations with periodicities of approximately 5 nm. xi

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Antisera reactive in SDSimmunod i f f us i on were obtained against untreated virions, pyrrolidine degraded coat protein^ and untreated BICMV cytoplasmic inclusions. Reciprocal double immunodiffusion tests with SDS-treated antigens showed that BICMV is serologically unrelated to seven potyviruses and serologically related to, but distinct from: bean common mosaic virus (BCMV) bean yellow mosaic virus (BYMV) cowpea aphid-borne mosaic virus (CAMV) dasheen mosaic virus (DMV) lettuce mosaic virus (LMV) potato virus Y (PVY) soybean mosaic virus (SoyMV) tobacco etch virus (lEV)^ and watermelon mosaic virus-2 (WM\/-2). The intragel cross-absorption technique was also used to demonstrate distinction between closely related potyviruses. Agar medium impregnated with a mixture of antisera was used for serod iagnos i s of BICMV and cowpea mosaic virus in cowpea. Light and electron microscopy of cytoplasmic inclusions induced by BICMV, siratro (Mucropt i I i urn atropurpureum (D.C.) Urb.) strain of BCMV (BCMV-S) and CAMV revealed that they are similar to those induced by the potyviruses from Edwardson's subd i v i s ionI The different reactions induced by BICHV, BCMV-S, and CAMV in some cowpea varieties indicated that they can also be used as differential hosts for these three potyviruses. Sources of resistance for BICMV were found among the cowpea varieties tested. Based on its physical, biological, cytological, and immunochemical properties, BICMV can be differentiated from any other virus that infects cowpea. Cytoplasmic inclusions induced by BICMV in cowpea and by SoyMV in soybean were detected by serology, light microscopy, and electron microscopy in hypocoiyls of 'i-S-day-old seedlings grown from virusinfected seeds. X i i

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Immunodiffusion tests and serologically specific electron microscopy were used to detect BICMV in hypocotyls of 't-S-day-old cowpea seedlings grown from B 1 CMVinfected seeds. Discs of individual hypocotyls were embedded into the agar medium ^-5 mm away from the antiserum wells. Virus-specific precipitin lines formed between virusinfected hypocotyl discs and antiserum wells, whereas no reactions were observed with h.-althy hypocotyls. Precipitin lines were also observed with extracts of mixtures from infected (1 g) and healthy (up to 29 g) tissues These immunochemical techniques were also used for detecting BCMV in hypocotyls of infected ^-5-day-old Phaseolus vulgari s L. seedling^ and for detecting SoyMV in infected Glycine max (L.) Merr. seedlings. Single radial immunodiffusion tests with extracts or discs of ccjwpea hypocotyls were also useful for detecting BICMV in germinated seeds. The reliability and simplicity of the immunodiffusion test;, make them suitable for use in routine seed health testing program in any laboratory. X i i i

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CHAPTER I PURIFICATION, PARTIAL CHARACTERIZATION, AND SEROLOGY OF BLACKEYE COWPEA MOSAIC VIRUS I nt roduct ion Cowpea Vigna u nguiculat a (L.) Walp. (=V iqna sinensis (L.) Endl.), is grown as a crop in h i ghtempera tu re areas of tropical and subtropical countries. Cowfiea seeds constitute a source of good quality protein and dried seeds are an important part of the diet of many people in the tropical and subtropical world, particularly in Africa and the rural zone of northeastern Brazil. The fresh seeds and immature pods are also eaten and i hey can be frozen or canned as is sometimes done in the United States. Cowpeas are also grown as fodder plants for hay, silage or pasture and used as a green manure and cover crop. When grown under optimum conditions, cowpea can produce seed yields as high as 2,600 Kg/ha. Hov/ever, several factors limit cowpea yields in most fields. Virus diseases are considered as a major limiting factor to the production of cowpeas in several countries (Dale, 19^9; Wells and Deba, I96I; Toler et al., 1963; Brantley et al., I965; Kuhn et al., 1966; Harrison and Gudauskas, 1968a; Harrison and Gudauskas, 1968b; Gay and Winstead, 1970; Zettler and Evans, 1972; Bock, 1973; Phatak, 197^; Hague and Persad, 1975; Kaiser and Mossahebi, 1975; and Lima and Nelson, 1977). Several viruses infect cowpea, and many of them can be transmitted through seeds from infected cowpea plants. The most important cowpea seed-borne virus in the southeastern United States is 1

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2 an aphid-tr
PAGE 16

3 A widespread mosaic disease was reported on different cowpea varieties in Trinidad (Dale, \ShS) Dale {]3^3) observed that the virus responsible for the disease was not transmitted by Aphis med i cag in i s Koch, but that the leaf beetle, Ceratoma ruficorni s (Oliv.) was a good vector and was probably responsible for transmitting the virus in the field. On the basis of his studies, he concluded that the virus was unrelated to those described by McLean (19^1), Snyder (1942), and Yu {]Sk6) but was more likely the virus studied by Smith (192^*). Dale (1953) subsequently confirmed that the cowpea mosaic virus isolated from Trinidad was efficiently transmitted by £. ruficornis but not by aphids. Lister and Thresh (1955) isolated a virus from cowpea and identified it as a strain of tobacco mosaic virus (TMV) They observed that a purified preparation of the virus contained rod-shaped particles of varying lengths, indistinguishable from the particles of TMV, and was precipitated specifically with antiserum prepared against TMV. A cowpea strain of TMV w.as also isolated from a range of leguminous hosts at Ibadan, Nigeria (Chant, 1959). Chant (1959) also found another virus infecting cowpea in Nigeria and as its physical properties differed from other cowpea viruses, he proposed the name cowpea yellow mosaic virus (CYMV). The virus was purified and an antiserum prepared against it. Both TMV and CYMV were transmitted by the beetle Ootheca mutabi 1 is Sahib. In subsequent work, Chant (I96O) studied the influence of TMV and CYMV on growth rate and yield of cowpea, and found that infection of cowpea with the cowpea strain of TMV did not affect yield as much as infection with CYMV. Wells and Deba (I96I) tested 116 cowpea varieties and 3^2 indigenous pure lines against CYMV and observed

PAGE 17

that 6 varieties and 16 pure lines were resistant. Robertson (1965) screened 79 cowpea varieties for resistance to CYMV in a screened greenhouse. Those varieties that showed no local or systemic reactions when inoculated with the virus were classified as immune; those that developed necrotic lesions but did not become systemically infected were classified as resistant; and those that showed systemic infection were classified as susceptible. Chant (1962) found that the cowpea virus from Trinidad caused local lesions on Chenopodium ama rant i col or Coste and Reyn. Mucuna atterrina Holland, Petunia hybrida Vilm., and Pvulgaris, and that the virus was polyhedral with a mean diameter of approximately 25 nm. Doub 1 eimmunod i f f us i on tests showed that a cowpea virus from Arkansas and the Trinidad cowpea mosaic virus were closely related, but not identical serologically and that both were ant igen ical ly related to bean pod mottle virus (BPMV) (Shepherd, I963) Studying other properties of the virus. Shepherd (1964) confirmed a close similarity of the Arkansas virus with the cowpea mosaic virus from Trinidad (Dale, 19'49). Walters and Barnett (196^1), working with a cowpea mosaic virus serologically identical to the Arkansas isolate, demonstrated also that it was efficiently transmitted by the bean leaf beetle, C^. trifurcata A detailed study of three cowpea mosaic virus isolates from Surinam (South America), along with the previously reported cowpea viruses from Trinidad (Dale, \3kS) and Nigeria (chant, 1959, I960, 1962) revealed that they are strains of cowpea mosaic virus (Agrawal, 1964). Detailed descriptions of host range, biophysical, biochemical, and immunochemical properties of cowpea mosaic virus were reported, and the abbreviation CPfW was proposed to eliminate any possible confusion

PAGE 18

5 with CMV (cucumber mosaic virus). Cowpea mosaic virus (CPMV) has been extensively studied in different laboratories and was fully described by van Kammen (1971, 1972). It was selected as the type member of the comovirus group (Fenner, 1976) and reported from several other parts of the world, including Brazil (Carner et al., 1969; and Lima and Nelson, 1977), Nigeria (Williams, 1975), Venezuela (Debrot and Rojas, 1967), and Puerto Rico (Perez and Cortes-Monl lor 1970; and Alconero and Sant iago, 1973) Kuhn (I96i*b) purified and characterized a new virus isolated from cowpea in Georgia and named it cowpea chlorotic mottle virus (CCMV) which was subsequently described by Bancroft (1971). This virus belongs to the bromovirus group (Fenner, 1976) and is physically similar to brome mosaic virus (Bancroft, 1970) and broad bean mottle virus (Gibbs, 1972), neither of which produces symptoms in cowpea (Bancroft, 1971). Strains of cucumber mosaic virus (CMV) are also known to infect cowpea. Cucumber mosaic virus strains have been isolated from naturally infected cowpeas showing mosaic symptoms in southeastern United States (Anderson, 1955a; Kuhn, 196'*a; and Harrison and Gudauskas, 1968a), Italy (Vovlas and Avgelis, 1972), Morocco (Fischer and Lockhart, 1976b), and South Africa (Klesser, I96O). An aphid-transmitted, spherical virus, approximately 25 nm in diameter, was also reported from India by Chenulu et al. (1968). According to their descriptions, the virus closely resembles a strain of CMV. Shepherd and Fulton (1962) identified a seed-borne virus of cowpea as a strain of southern bean mosaic virus (SBMV) (Shepherd, 1971). Although a virus is.jlated from naturally infected cowpea in Arkansas

PAGE 19

6 had properties somewhat similar to the cowpea strain of SBMV, the two viruses were not serologically related (Shepherd, I963). A carlavirus isolated from cowpea in Ghana was described and designated as cowpea mild mottle virus (CMMV) by Brunt and Kenten (1973) and Brunt (I97'i). Cowpea mild mottle virus is seed-borne in cowpeas, is 65O nm in length and is apparently not transmitted by aphids. A virus with small isometric particles, isolated from Iranian cowpea seeds was considered as new and named cowpea ringspot virus (CpRV) on the basis of symptomatology and particle morphology, which were similar to other ringspot viruses (Phatak, 197^; and Phatak et al., 1976). According to Phatak (197^4), the virus was not transmitted by aphids, induced intracellular inclusions in cowpea, had a wide experimental host range and was serologically unrelated to hO other isometric viruses most of which commonly infect various legumes. Cowpea ringspot virus was also transmitted in 15-20% of the seeds of three cowpea cultivars (Phatak et al., 1976). McLean (19^1) studied some physical and biological properties of a cowpea virus and found that it was transmitted by the following species of aphids: Macros j£hum soJ_ajT^ Acynthos iphon pi sum (Harris) Aphis gossypi i Glover, Myzus persicae (Sulz. ) but not by the bean leaf hopper (E mpoasca fabae Le. B.), the tarnished plant bug (Lygus pratens^^ L.), the Mexican bean beetle ( Epilachra corrupt^ Mis.) and the striped cucumber beetle ( Piabrotica vittat£ Faba) Snyder (19^*2) described a mosaic disease of asparagus bean, Vigna sesgu i peda I i s Wight, and also studied some biological and physical properties of the causal agent. His positive results obtained with aphid transmission

PAGE 20

indicated that these viruses were not identical to the one described by Smith {]S2^) A cowpea virus similar to those described by McLean {]Sk]) and Snyder {\3^2) was reported from China by Yu (19^6). The virus which was transmitted by aphids was also seed-borne in cowpea. In addition to cowpea, the virus also infected lima bean and adzuki bean, Phaseol us anguiaris (Willd.) Wight (Viqna angular is (Willd.) Ohwi. and Ohshi) (Yu, ly^^S). Cowpea viruses apparently similar to those were also reported from Ceylon (Abrygunawardena and Perera, ]36k) Germany (Brandes, IS)6'), India (Nariani and Kandaswany, 1961), and New Guinea (van Velsen, I962). An aphid-borne virus isolated from cowpea in northern Italy was studied by Lovisolo and Conti (I966), and designated as cowpea aphidborne mosaic virus (CAMV). The virus was a rod, approximately 750 nm long, and was seed-borne in cowpea, but appeared to be clearly different from BICMV isolated in Florida (Anderson, 1955b). As reported by Lovisolo and Conti (1966), the virus was first recorded and described in Italy by Vidano (1959) and Rui (i960). The virus was transmitted in a non-persistent manner by M. Pers icae Aphis fabae Scop. A, medicaginis Koch, A. gossypii, and Macros i phum euphorb iae (Thomas) (Vidano and Conti, 1965). A similar virus was later isolated in East Africa and three strains of this virus were differentiated by host range and serology (Bock, 1973). It was also observed that CAMV is distantly serologically related to bean common mosaic virus (BCMV) (Lovisolo and Conti, 1966; and Bock, 1973), but no direct serological relationship was detected with the African type strain of CAMV and potato virus Y (PVY) bean yellow mosaic virus (BYMV), pea seed-borne mosaic virus (PSMV) clover yellow vein virus (CYVV) soybean mosaic virus (SoyMV) sugarcane mosaic virus (SMV)

PAGE 21

8 tobacco seviire etch virus (TEV) and iris mosaic virus (IMV) (Bock, 1973; and Bock and Conti, ]37k) A seed-transmitted virus tentatively identified as CAMV was considered to be responsible for the most important and widespr.^ad disease of cowpeas in Iran (Kaiser et al., 1968). Additional studies about various properties of the Iranian isolate of CAMV indicated its similarity to the Italian and African isolates (Kaiser and Mossahebi, 197!?). A CAMV isolate was also reported from Japan infecting adzuki bean, P. ang ularis under natural conditions (Tsuchizaki et al., 1970). Fisher and Lockhart (l976a) isolated a rod-shaped virus from severely infe.:ted cowpeas in Morocco and identified it as a strain of CAMV on the basis of its particle length, aph i d1 ransm i ss i on host range, serology, and physical properties. The Moroccan isolate differed from those CAMV isolates previously described (Lovisolo and Conti, I966; Bock, 1973; and Botk and Conti, 197'*) by failing to infect Ocimum bas i I icum L. a diagnostic species for CAMV (Bock and Conti, 1974), and other plants reported to be systemic hosts for CAMV. Padma and Summawar (1973) indicated the value of Chenopodium murale L. as a good indicator host for differentiation, screening and isolation of a rodshaped cowpea virus and the icosahedral CPMV. Cytoplasmic inclusions were observed in ph,nt cells infected with CAMV (inouye, 1973; and Nicolaeseu et al 1976) A virus isolated from cowpea in India (Khatri and Singh, ]S7h) was reported to be a strain of CPMV. However, the authors reported aphid transmission of this virus, so its identification as a strain of CPMV is questionable. A filamentous virus approximately 750 nm in length isolated from cowpeas in Ghana did not react with antisera specific for CAMV, peanut mottle virus, BCMV, and BYMV (Brunt, 197^*).

PAGE 22

9 An aphid-transmitted virus was responsible for complete loss of cowpea in irrigated areas of northern Nigeria (Raheja and Leleji, ]37k) Based on the fact that the virus was neither mechanically transmitted nor seed-borne in cowpe.i Raheja and Leleji (197'*) concluded that it was either an atypical strain of CAMV or a new virus not previously described. A virus isolated from Crota lar ia spec tabi 1 is Roth in a field at Gainesville, Florida, was studied by Anderson (l955b) and designated blackeye cowpea mosaic virus (BiCMV). Anderson (1955b, 1955c) reported that BICMV infected plants of cowpea, Crota laria and Desmodium in the field, but considered Crotalaria and Desmo d ium as secondary hosts for the virus. In a subsequent study, Anderson (1959) observed that BICMV was transmitted by M. pers icae but not by the bean leaf beetle, £• trifurcata. Coibett (1956) found that BICMV was serologically related to BYMV and identified it as a strain of BYMV. Based on Corbett's conclusion, several subsequent reports have referred to BICMV as a cowpea strain of BYMV (Brierly and Smith, 1962; Kuhn, 1 96'4a ; Kuhn et al., 1965; and Harrison and Gudauskas, 1968a). Light and electron microscopic studies of BICMV and BYMV showed marked cytological differences between these two flexuous rod-shaped viruses (Edwardson et al., 1972). According to Edwardson et al. (1972), the cytoplasmic inclusions induced by BICMV were consistently different from those induced by BYMV. In the light microscope, groups of plates were observed in cells of BYMVinfected tissues, whereas groups of tubes were seen in cells of epidermal strips obtained from Bl CMVinfected tissue. Electron microscopy of ultrathin sections indicated that BlCMV-induced cytoplasmic inclusions consisted of pinwheels with scrolls, whereas BYMV-induced cytoplasmic inclusions were made of pinwheels and

PAGE 23

10 laminated aggregates. Light and electron microscopic investigations revealed that BICMV induces nuclear inclusions in C^. spectabi 1 i s (Zettler et al., 1967; Edwardson et al., 1972; and Christie and Edwardson, 1977), while no such inclusions were observed in cells of C^. spectab i 1 i s infected with BYMV. Based on those cytological distinctions, Edwardson et al. (1972) concluded that BICMV and BYMV are distinct members of the potyvirus group Subsequently, Zettler and Evans (1972) demonstrated that BICMV and BYMV had dissimilar host ranges, providing additional evidence that they are distinct viruses. In host range studies, BICMV was shown to be very similar to BCMV, but different from v/atermelon mosaic virus-2 (WMV-2) (Uyemoto et al., 1973). Leaf-dip preparations of B I CMVinfected tissue revealed the presence of flexuous rods, 750 nm long, and double immunodiffusion tests with BCMV and WMV-2 antisera indicated that BICMV was serologically identical to BCMV and related to, but distinct from WMV-2 (Uyemoto et al 1973) • Materials and Methods Source of Virus Iso l ate The blackeye cowpea mosaic virus used in this study was isolated from infected seeds of cowpea V^. ungu iculata 'Knuckle Purple Hull' harvested from a field in Gainesville, Florida. The virus was transmitted by aphids from infected cowpea plants grown from infected seeds to non-infected 'Knuckle Purple Hull' plants. Two aphids (m. pers icae) were used per test plant and each aphid was allowed to have an acquisition period of 30 to 60 sec. A single test plant showing typical mosaic was assayed by leaf-dip electron microscopy for the presence of

PAGE 24

n rod-shaped virus particles and used as the initial source of inoculum for virus propagation. The virus was mechanically transmitted from the selected infected plant to healthy 'Knuckle Purple Hull' seedlings, where it was increased for virus and inclusion purification, and other studies. Virus and Inclusion P urificat ion Blackeye cowpea mosaic virus was propagated in either V. unguiculata or Nicotiana benthani i ana Domin, and systemically infected leaves were used for virus and inclusion purification. Either n-butanol or a combination of chloroform and carbon tetrachloride was used in the clarification process. Tlie adaxial surface of the primary leaves of 5 to 7-day old cowpea seedlings were inoculated with BICMV obtained by grinding infected leaf tissue in 0.05 M potassium phosphate (KPO^) buffer, pH 7-5 (1/2, w/v) The first trifoliolate leaves showing typical mosaic were collected 15 to I8 days later and subjected to the following purification procedures based on previous works (Hiebert et al., 1971; Hiebert and McDonald, 1973; and McDonald and Hiebert, 1975). £ Butanol clarification method. Two hundred to 'tOO g of leaf tissue were homogeni^red in a blender with two parts (w/v) of 0.5 M KPO^ buffer, pH 7-5, containing 0.5 to 1.0^ sodium sulfite (Na2S0^) The resulting extract was filtered through a double layer of cheesecloth and enough n^-butanol was added to make a final concentration of 8^ (v/v). This mixture was stirred overnight at C and the coagulated green debris obtained was removed by a low speed cent r i fugat ion at 11,700 £ in a Sorvall Centrifuge (Sorvall Superspeed RC2-B Automatic Refrigerated Centrifuge) for 10 min. Virions were precipitated from the supernantant by the addition of 6 8'^ (w/v) of polyethylene glycol

PAGE 25

12 MW 6000 (peg) followed by stirring for 60 min. The precipitated virions were collected by ceritr i fugat ion at 13,200 g for 10 niin. The resulting pellet was resuspendtd in 0.02 M KPO^, pH 8.2, containing 0.\% 2-mercapthoethanol (2-ME) (v/v) and the virus was separated from the host components by equilibrium density gradient centri fugat ion (120,000 for 16 18 hr in a beckinan SW 50.1 rotor) in 30% cesium chloride (CsCl) prepared in the same buffer. The virus zone, located at 12 to 15 mm from the bottom of i he tube, was collected dropwise through a hole punched in the bottom of the tube and diluted with 0.02 M KPO^^, pH 8.2, containing 0. U 2-ME. The virus preparation was clarified by centrifuga tion at 11,700 g for 10 min and reconcentrated by cent r i fugat i on at 85,000 cj for 90 min. The final pellet was resuspended in 0.02 M Tris buffer, pH 8.2, and i he virus concentration was determined spect rophotometrically using an extinction coefficient of 2.k mg/ml (Purcifull, 1966). The optical density (O.D.) readings for the virus at wavelengths of 260 and 280 nm were corrected for light scattering before estimating the 260/280 ratio and concentrations of virus in purified preparations. The correction for light scattering was done by plotting the log of the optical densities against the wavelengths of 320, 3^0, and 36O nm and extrapolating these values to 230 300 nm range of wavelength. C hloroform-carbon tetrachloride clarification method Th i s clarification process was selected when it was desirable to purify both the virus and inclusions from the same batch of tissue. Systemically infected tissue (200 400 g) were homogenized in a solution containing 1.30 ml of 0.5 M KPO^ (pH 7.5), 0.35 ml of chloroform, 0.35 ml of carbon tetrachloride, and 5.0 mg of Na„SO per gram of tissue.

PAGE 26

13 The homogenized mixture was centrifuged in a Sorvall Centrifuge at 5,000 rpm for 5 min and the pellet containing the organic solvents was discarded. The aqueous phase was centrifuged at 13,200 g for 15 min to precipitate the virus induced inclusions. The supernatant was treated as previously described for virus purification and the pellet containing the inclusions was resuspended in 0.05 M KPO^ pH 8.2, and 0.1^ 2-ME. The inclusion suspension was homogenized in a Sorvall Omni-mixer homogenizer for 2 min and enough Triton X-100 was added to make a final concentration of 5^ (v/v) After stirring for one hour at 4 C this mixture was subjected to a low speed cen t r i f uga t ion of 27,000 g for 15 min to precipitate the inclusions. The pellet was resuspended in 10 to 20 ml of 0.02 M KPO^, pH 8.2, containing O.U 2-ME, and homogenized for 30 sec. The inclusions were sedimented again by cen t r i f ugai i on of 2 7,000 g for 15 min. The pellet was homogenized for 30 sec and the homogenate was layered on a sucrose step gradient made up of 10 ml of 80^, 7 mi of 60% and 7 ml of 50^ (w/v) sucrose in 0.02 M KPO^, pH 8.2. The gradient was centrifuged for one hour at 27,000 rpm in a Beckman SW 25.1 rotor. The inclusions layered on top of the 80% sucrose zone and were collected by droplet from the bottom of the tube. To remove the sucrose, the inclusions were diluted in 0.02 M KPOj^, pH 8 2, and precipitated by a centr i fugat ion at 27,000 £ for 15 min. The pellet was resuspended in 0.02 M Tris, pH 8,2, and inclusion yield was estimated spect rophotomet r i ca I 1 y after being disrupted in 2'-^ sodium dodecyl sulfate (SDS) The inclusion preparations were either immediately used for immunization of rabbits or stored at -20 C by either freezing directly or by f reeze-dry ing

PAGE 27

lA Clarification with n-butanol and chloroform-carbon tetrachloride Because n-butanol resulted in virus preparations of higher purity, but chloroform-carbon tetrachloride was superior for preservation of inclusion proteins (Hiebert, unpublished), these solvent systems were combined for purification of virus and inclusions from the same batch of tissue. Infected tissue was homogenized with two parts (w/v) of 0.5 M KPO^. pH 7.5, containinc) 0 5 1 O^o Na^SO^. The homogenate was filtered through cheebecloth and subjected to cent r i fugat ion at 11,700 for 10 min. The supernatant was used for virus purification as described previously using n butanol for clarification. The pellet was resuspended in approximately 2 volumes of 0.5 M KPO^, pH 8.2, 0.5% Na^SO^, homogenized with one volume of chloroform-carbon tetrachloride (1:1, v/v) and centrifuged at 5,000 rpm for 5 min in a Sorvall Centrifuge. The aqueous phuse was subjected to a cen t r i fugat ion at 11,700 g for 15 min. The supernatant was collected for additional virus purification using PEG, equilibrium density-gradient centr i fugat ion and differential cent r i fuqat ion The pellet was resuspended in 0.05 M KPO^, pH 8.2, containing 0.]% 2-ME and treated with 5t Triton X-100. The inclusions were then purified by sucrose step gradient centrifugation as described above. Virus Particle Size Determination Crude leaf extracts from systemically infected cowpea plants and purified virus preparations were negatively stained in 2% potassium phosphotungstate (PTA) pH 6.5, containing 0.]% bovine serum albumin (BSA) prior to photography in an electron microscope. The procedure used was similar to tf,ose previously described (Edwardson et al., I968 and Purcifull et al., 1970). Small pieces of 8 1 CMVi nf ec ted leaf were

PAGE 28

15 chopped with a razor blade in 1% PTA, pH 6 5, containing O.R BSA on a glass slide and a small quantity of the resulting cell extract was deposited on a carbon coated Formvar film supported by 75 x 300 mesh copper grids. Excess liquid was then removed by touching momentarily the edge of the grid with a filter paper and the specimen was allowed to air-dry. The purified virus was stained directly on the grid. A small drop of virus solution was deposited on the grid. After 1 2 min, the virus solution was partially blotted with a piece of filter paper and a small drop of 2% PTA solution was added. The grid was blotted and allowed to air-dry. The grids were then examined in a Philips Model 200 electron microscope. The virus particles were observed, photog raph>:'d and their sizes were estimated by comparing projected micrographs to micrographs of a diffraction grating (2l60 lines/mm). Twenty-five virus particles from leaf extracts and 190 particles from a purified preparation were measured and classified according to their length at intervals of 50 and 20 nm. Blackeye cowpea mosaic virus-grids prepared according to the serologically specific electron microscopic technique (SSEM) developed by Derrick and Brlansky (1976) were also used for virus particle measurements. Parlodion film grids sensitized with 81 CMV-ant i serum (BICMV-As) were treated with cowpea leaf extract containing BICMV and positively stained with \% uranyl acetate in 50% ethanol The SSEM technique will be described in more detail in Chapter II. Stability of Virus in Sap Thermal inactivation point (TIP), 1 ongev i ty W t r£ (LIV) and dilution end point (OEP) were determined for BICMV using C. amarant icolor as an assay plant. The TIP was determined by heating crude sap of

PAGE 29

16 BICMVinfected cowpea leaves to ^45, 50. 55, 60, 65, 70, and 75 C for 10 min. All treated saps as well as unheated sap of B 1 CMVinfected tissue were rubbed on the test plants, which were maintained in greenhouse conditions for at least three weeks for observation of symptoms. Crude sap of infected leaves obtained in deionized water was placed in test tubeb and assayed for infect ivity after storage at room temperature for 0, 8, 16, 2^4, ^8, and 72 hr. For the DEP determination, crude Juice was extracted from B 1 CMVinfected leaves, and the extract was diluted to lo"', lo"^ ]0-\ lo'^ and lo'^ with deionized water prior to assay. Pojj^acryjamide Gel L lectrophores is of Viral and Inclusion Proteins The polyacryl amide gel electrophoresis studies were performed according to the meihod of Weber and Osborn (I969) as modified by Hiebert and McDonald (1973). Running gels of approximately 75 mm in height were prepared with 61 acrylamide (7.5 ml sodium phosphate buffer, pH 7.2; 15.0 ml water; 0.15 ml lO^o SDS; 6.0 ml of 30Z acrylamide; 0.045 ml N, N, N', N'-tetramethylenediamine (TEMED) and 1.2 ml ammonium persulphate 15 mg/ml;, and a well-forming gel of 8t acrylamide with onefifth the electrophoresis buffer concentration (1.2 ml buffer; 7.2 ml H^O; 0.2 ml ]Q% SDS; 3.0 ml of 30Z acrylamide; 0.0^* ml TEMED, and 0.3 ml ammonium persulphate 15 mg/ml) was cast on top of them. Disassociated protein solutions, 20 50 yl samples in approximately 20^ sucrose and one-fifth the electrophoresis buffer concentration, were placed into the formed wells. The top of the samples were covered with a cap gel of composition similar to the well-forming gel. The electrophoresis was performed in a vertical slab electrophoresis apparatus, Ortec. Model kO]0/kOU Ortec, I ncorporated, Oak Ridge, Tenn.,

PAGE 30

17 for \.b to ^4.0 hr at 160 V with a pulsed constant power supply at 300 pulses per second and about 90 mA current. Prior to being used for electrophoresis, the protein was disassociated by mixing 0.2 ml of protein solution with 0.1 ml of ]Q% SDS and 10 20 pi 2-ME and heating tliis mixture in boiling water for 1 to 2 min. Samples of 20 50 yl of disassociated proteins were added to 0.1 ml of one-fifth of the electrophoresis buffer concentration, containing 30% sucrose and 0.15% SDS. Serum albumin (MW 67,000), glutamate dehydrogenase (MW 53,000), ovalbumin (MW ^3,000), carbonic anhydrase (MW 29,000), and TMV coat protein (MW 17,500) were used as protein markers to estimate the molecular weight values for inclusions and virus coat protein subunits. After electrophoresis, the gel slabs were stained and fixed overnight in a staining solution containing 50% methanol, 10% glacial acetic acid, and 0.\t Coomassie brilliant blue R250. Before photography, the gels were destained by soaking them for 8 hr in a solution made up of 10% methanol and 7-5% acetic acid followed by several changes in the solution over a period of several days. The distances migrated by the protein subunits into the running gels were measured from the photographs of the stained gels. Sedimentation Coefficient Determinat i on The sedimentation rates of fresh and stored purified BICMV in either 0.02 M Tris buffer, pH 8.2 or 0.05 M borate buffer, pH 8.2 were measured with a Beckman Model E analytical ul tracentrifuge according to the method of Markham (i960). After the rotor reached a speed of 27,690 rpm photographs were taken at k min intervals using

PAGE 31

18 Schlieren optics. The datg were corrected for standard water viscosity conditions at 20 C, but not for the effect of virus concentration. The virus concentrations used varied from 0.5 to 1.0 mg/ml Sero 1 ogy Antiserum prod i i ction for virus a nd cytoplasmic i nclus ions A n t i s e r a were obtained by injecting a New Zealand white rabbit with untreated virions and a second rabbit with pyrrol id ine-degraded virus protein. All rabbits selected for immunization were first bled to produce normal sera. The concentrations of untreated BICMV in 0.02 M Tris buffer, pH 8.2, used in the immunization process varied from 1.0 to 2.0 mg of nucleoprotein per ml of purified solution. BICMV used for pyrrolidine degradation was suspended in 0.005 M borate buffer, pH 8.2. The virus protein was degraded according to the method used by Shepard (1972). A virus solution was mixed with an equal volume of ^% pyrrolidine in distilled water (v/v) The mixture was then immediately dialyzed against two liters of 0.05 M borate buffer, pH 8.2, containing 0.37^ actual formaldehyde for approximately k% hr at ^ C to remove the pyrrolidine and fix the protein subunits. A series of ^ to 5 intramuscular injections was given to each rabbit with an interval of 10 to 15 days between the injections. Each Injection consisted of 1.0 to 2.0 ml preparations of virus or degraded viral protein vigorously emulsified with equal volume of Freund's complete or incomplete adjuvants (Difco). Booster injections were given at intervals of about 2 months. The immunized animals were bled every week, starting 10 to 15 days after the last injection of the initial series of 4 5 injections.

PAGE 32

19 The rabbits were farted for k \2 hr prior to each bleeding and 30 50 ml of blood were collected into glass tubes according to the procedure described by Purcifull and Batchelor (1977). Blood samples were allowed to clot for approximately min at 37 C in a waterbath. The clotted blood was subjected to a cen t r i f uga t i on of 2,000 rpm in a Sorvall table centrifuge for 10 min. The antisera were transferred with a Pasteur piperte to con ical -bottomed tubes and clarified by a second cent r if ugat ion at 5,000 rpm for 10 min. Antiserum specificity and titer were deteimined by Ouchterlony (I962) double-diffusion tests In SDS-agar plates. The antisera were stored at -20 C by either freezing directly 01 after f reeze-dry i ng The BlCMV-induced cytoplasmic inclusions (BlCMV-l) used for antiserum production were purified from N, ben t ham i ana Freshly purified cylindrical inclusiuns, which were unreactive with antiviral sera, were used for immunization and the foot pad route of immunization (Zlemiecki and Wood, 1975) was used. The rabbit received three injections into the foot pad, each >:ontaining 0.1 ml of purified Inclusions (O.l 0.2 O.D. units/ml at 28o nm) in 0.02 M Tris, pH 8.2, emulsified with an equal volume of eitlier Freund's complete or incomplete adjuvants. S erological tests Both double and single Immunodiffusion tests In agar gel were used in the present study. Most double immunodiffusion tests were performed in agar medium containing 0.8% Noble agar (Difco); 0.5^ SDS (Sigma) and 1.0?; NaN^ (Sigma) in deionlzed water (Purcifull and Batchelor, 1977), or 0.05 M Tris-HCl buffer pH 7-2. Reactant wells were punched in the solidified agar medium with an adjustable gel cutting device made by Grafar Corp., Detroit, Mich. Routinely the wells (7 mm in dlam.iter) were punched in an hexagonal arrangement

PAGE 33

20 consisting of a cenier well with six peripheral wells spaced ^ S ™^ from the center well as measured from the edges of the wells. Different gel patterns were also used in certain tests. Antigens used as reactants were prepared either in deionized water or in ] .5% SDS solution, according to Purcifull and Batchelor (1977). In the first case, fresh tissue was ground with a mortar and pestle in deionized water (1/2, w/v) and expressed through cheesecloth. The second method which was more commonly used, consisted of grinding fresh tissue in 1.0 ml of water per gram of tissue and adding 1.0 ml of 3.0% SDS per gram of tissue prior to expressing the sap through cheesecloth. The antigens and undiluted antibi.;ra were pipetted directly into the appropriate wells, and the plates were incubated in a moist chamber at 2k C for 2*4 48 hr. The development of precipitation patterns was observed by looking at the plates, which were illuminated from the bottom with indirect lighting. The reactants were removed and 15^ charcoal (Norit a) in water (w/v) was added into the wells before photographs were taken Single radial immunodiffusion tests were conducted in agar media containing O.Bt Noble agar, 1.0^ NaN^ 0.3 or 0.5^ SDS, and 10, 15, or 20% BICMV antiserum. Media were prepared either with antiserum obtained for untreated BICMV and antiserum for pyrrolidine degraded BICMVprotein. Each SDS concentration in the media was tested with antigens prepared in distilled water or in ] .5% SDS. During medium preparation, care was taken to avoid heating the antisera over 50 C and while exposed to SDS, the antisera were maintained at 50 C for less than 2 min. Single radial diffusion plates were also prepared with a mixture of antisera to BlChV and CPMV. The CPMV-an t i serum was prepared by

PAGE 34

21 immunizing a rabbit with CPMV degraded by SDS according to a procedure described by Purcifull and Batchelor (1977). A lyophilized, purified preparation containing approximately 3 mg of CPMV was resuspended in 1 ml of 1.0^ SDS solution containing 2.01 2-ME, and boiled for approximately 5 min before emu I s i f i ca t ion with Freund's adjuvant and intramuscular injeci ion into a rabbit. Three similar injections were given into the same rabbit with 7-day intervals between injections. Serological rel ationship between BICMV and other potyviruses Reciprocal double immunodiffusion tests with BlCMV and the following potyviruses were conducted in SDS-conta in ing media: bean yellow mosaic virus (BYMV), bean c:ommon mosaic virus (BCMV-BV-l), bean common mosaic virus-siratro isolate (BCMV-S) bidens mottle virus (BiMV), dasheen mosaic virus (DMV) lettuce mosaic virus (LMV) pepper mottle virus (PeMV), potato viru. Y (PVY) soybean mosaic virus (SoyMV) tobacco etch virus (TEV) turnip mosaic virus (TuMV) watermelon mosaic virus-l (WMV-1), and waternu-lon mosaic virus-2 (WMV-2) The source of each antiserum was as follows: BYMV (jones and Diachun, 1977); BCMV-BV-l (J. K. Uyemoto, New York State Agricultural Experiment Station); BCMV-S (Lima et al., 1977); DMV (Abo El-Nil et al., 1977); BiMV, LMV, PeMV, PVY, SoyMV. TEV, TuMV, WMV-1, and WMV-2 (d. E. Purcifull, University of Florida, Gainesvi lie). Using BICMV-As, the serological relationship of BICMV with commelina mosaic virus (CoMV) (Morales and Zettler, 1977), a Moroccan isolate of CAMV (Fischer and Lockhart, 1976a), pepper veinal mottle virus (PVMV) and pokeweed mosaic virus (PWMV) were also studied in double diffusion tests with SDS-treated antigens. In all serological tests, the reactants were arranged so that BICMV was always placed in

PAGE 35

22 a well adjacent to the other virus-well. Sap extracts from appropriate healthy host tissues were included as controls in all serological tests, and all antigens were also tested against normal serum. The intragel c ross -absorpt ion technique described by van Regenmortel (1966) was also used to study the serological relationships of BICMV with BCMV-S and CAMV. Purified preparations of heterologous antigens (BCMV-S or CAMV) were placed in the center well and allowed to diffuse for approximately 2'^ hr. The excess of the antigen preparations were then removed and the BICMV antiserum was added in the same well. At the same time, the homologous and the heterologous antigens were positioned in the outer wells. Light and Electron Microscopy of Virus Induced Pinwheel inclusions Epidermal leaf strips obtained from systemically infected cowpea V. ungu i cu 1 ata were floated on a b% solution of Triton X-100 for 5 to 10 min and subsequently stained with a combination of calcomine orange and "luxol" brilliant green as described by Christie (1967). The stained leaf strips were mounted in euparal on glass slides and examined with a light microscope for the presence of cytoplasmic inclusions. Similarly, strips from non inocu 1 ated V. unguiculata were also stained and examined in the light microscope as controls. Cylindrical inclusions were examined in situ in ultrathin sections with an electron microscope. Small pieces were taken from symptomatic areas of systemically infected cowpea leaves and fixed for 2 to 3 hr at room temperature in Karnovsky's formaldehydeg 1 utara 1 dehyde fixative prepared in 0.1 M cacodylate buffer, pH 7.2 (Karnovsky, 1965). After washing with 0,1 M cacodylate buffer, the small leaf pieces were postfixed for 1 to 2 hr at room temperature

PAGE 36

23 in 2% osmium tetroxide and progressively dehydrated in an increasing ethanol solution series. The leaf pieces were maintdined for 5 to 15 min in each ethanol solution at room temperature. The pieces were stained overnight at k C In a solution of 7S% ethanol containing 2% uranyl acetate and subsequently dehydrated in a second series of ethanol solutions (75 100.^) followed by 100^ acetone or propylene oxide. They were then embedded in plastic containing Epon 8|2, Araldite 502, and dodeceny 1 succ in ic anhydride. Ultrathin sections were cut with a diamond knife in a Sorvall MT-2 ul t ramie rotome and mounted on copper grids with carbon-coated Formvar film. The specimens mounted on the grids were poststained with 3% potassium permanganate (2 min), \Z uranyl acetate (2 min), and lead citrate (2 min). These sections as well as those obtained from non inocu lated cowpea plants were examined with a Philips Model 200 electron microscope. Purified BICMV-I preparations were mounted on carbon-coated Formvar film supported by copper grids and stained with either ]% ammonium molybdate or 2% uranyl acetate, before examination by electron microscopy. Host Range and Screening Cowpea Varieties for Resista n c e Test plants were inoculated with crude sap from 'Knuckle Purple Hull' systemically infected with BICMV. The inoculum was prepared by grinding leaf tissue in 0.05 M KPO^, pH 7-5 ( 1 /2 w/v) The inoculations were done by rubbing the inoculum on carborundum-dusted leaves of the test plants which were maintained in greenhouse conditions for at least one month for observation of symptoms. All inoculated plants, including those that did not show any symptoms were checked serologically for the presence of BICMV.

PAGE 37

2k The cowpea varieties were also inoculated with CPMV, CAMV and BCMV-S. Crude sap from all inoculated cowpea plants were also tested in double immunodiffusion against antisera specific for CPMV, BICMV, and BCMV-S, respectively. Since CAMV was shown to be serologically related to BICMV, the serological tests to detect its presence in the inoculated plants were done with BICMV antiserum. Resul ts Purification and Pro perties of Blackeye Cowpea Mosa i c Virus Purified preparations of BICMV were obtained from systemically infected leaves of either V. un cjuiculata 'Knuckle Purple Hull' (Fig. 1 -A) Of fibenthamiana using the purification procedures diagrammed in Figures 2, 3, and k. The best yield with the highest degree of purity was obtained using the first method of virus purification (Fig. 2) and infected cowpea leaves (Fig. I-A) as a source of virus. The first trifoliolate cowpea leaves collected 15 to 18 days after inoculations gave the highest yield of virus (8 10 mg) per 100 g (fresh weight) of infected tissue and n^-butanol proved to be the best clarifying agent for cowpea tissue. An opa lescent, sharp virus-band was usually obtained after equilibrium density gradient cent r i fugat ion in 30^ CsCl. The virus zone was located at 12 to 15 nm from the bottom of the tube while most of the green host components stayed at the top portion of the gradient. The clear pellet obtained after a high speed centrifugation of virus removed from CsCl gradients confirmed the absence of colored host components. The combination of chloroform and carbon tetrachloride, although necessary for inclusion purification, was an inferior method of clarification for obtaining virus from cowpea

PAGE 38

Figure 1 Systemic and localized symptoms induced by blackeye cowpea mosaic virus (BICMV) in cowpea, V^. ungu i cu 1 ata 'Knuckle Purple Hull' and C. a marant {color A) Typical mosaic on secondary trifol folate leaf of cowpea plant inoculated with BICMV (l), and primary trifoliate leaf showing vein clearing (2). B) Local lesions on leaf of C. amarant i col or inoculated with BICMV. ~

PAGE 40

Figure 2 Flow diagram outlining the procedure of purification of BICMV using n-butanol as clarifying agent, polyethylene glycol (peg) for virus concentration, CsCl gradient cent ri fugat ion for separation of virus from host components, and differential centr i fugat ion for further virus purification. For details, see description in materials and methods section.

PAGE 41

28 PELLET — (Discard) SUPERNATANT(Discard) PELLET— (Discard) SYSTEMICALLY INFECTED TISSUE 0.5M KPO4 pH 7.5 + 0.5-1.0% Na2S02 GRiriD I FILTER 8% nUTANOL STIR: OVERNIGHT CENTRIFUGATION: n700g lOmin SUP 8% [RNATANT 'EG STIR : 60min CENTRIFUGATION: 11700 g lOmin PELLET CO^M KPO^ pH 8.2 + O.n 2-ME CsCl GRADIENT CENTRIFUGATION: d=1.28g/cc 120000g 18 hr COLLECT VIRUS ZONE CENTRIFUGATION: n700g lOmin SUPFRNATANT CENTRIFUGATION: 85000g 90niin SUPERNATANT(Discard) PEL ET 0.02M TRIS pH 8.2 VIRUS

PAGE 42

Figure 3 Flow diagram outlining the procedure for purification of BICMV and its cytoplasmic inclusions, using chloroform and carbon tetrachloride as clarifying agents. The procedure is described in the text.

PAGE 43

30 SYSTEIICALLY INFECTED TISSUE 0.5M KPO^ pH 7.5 + CHCI3+ CCI4+ i: Na^SOj CENTRIFUGATION: 4,000g Smin PELLET (Discard) SUPERNATANT CENTRIFUGATION: ll,700g IBmin SUPERNATANT(Discard) PELLET — (Discard) SUPERNATANT^ (Discard) SUPERNATANT— (Virus) 8% |eG ST IP : 60min CEN RIFUGATION: ll,700g lOmin PELpT O.Oj'M KPO4 pH 8.2 + 0.1% 2-ME CsCl GRADIENT CENTRIFUGATION: d=lj28g/cc-120000g 18hr COL|-ECT VIRUS ZONE CENTRIFUGATION: ll,700g lOmin SUPERNATANT CEN RIFUGATION: 85,000g 90niin PELLET 0.02M TRIS pH 8.2 VIRUS PELLET ( Inclusions) 0.05M KPO4 pH 8.2 + 0.1". 2-ME homAgenization 5% IRITON-X CENTRIFUGATION: 27,000g ISmin -SUPERNATANT (Discard) PELLET SUCROSE STEP GRADIENT CENTRIFUGATION: 45,^00g eOmin COLLECT INCLUSION ZONE CEN RIFUGATION: 27,000g ISmin -SUPERNATANT (Discard) PELLET 0.02M TRIS pH 8.2 INCLUSIONS

PAGE 44

Figure ^ Flow diagram outlining the steps carried out during the purification of BICMV and its cytoplasmic inclusions by a combination of the first (Fig. 2) and second (Fig. 3) methods for purification of virus and inclusions.

PAGE 45

32 INFECTED TISSUE 0.5M KPO4 pH 7.5 + 0.5-1.0% Na2S03 GRIND FILTER VIRUS ONLY PELLET — (Discard) SUPERNATANT( Discard) PELLET — (Discard) SUPERNATANT(Discard) CENTRIFUGATION: lUOOg-lOmin SUPERNATANT( Virus) I -8% BUTANOL STIB : OVERNIGHT CENTRIFUGATION: lUOOg-lOmin SUPpNATANT 8% PEG-^ STIP eOmin CENTRIFUGATION: 11700g-10min PELLET 0.02M KPO4 pH 8.2 + 0.1% 2-ME CsCl GRADIENT CENTRIFUGATION: d=1.28g/cc 120000g IShr COLLECT VIRUS ZONE CENTRIFUGATION: 1 1 ZOOg-lOmin SUPERNATANT I CENTRIFUGATION: 85000g-90min PELLET 0.02M TRIS pH 8.2 VIRUS PELLET (Inclujions + Some Virus) 0.5M KPO4 pH 8.2 + Na2S0,+ CHCl, + CC^. I CENTRIFUGATION: 4000g-5min PELLE"F — (Discard) -SUPERNATANT(Virus) SUPERNATANT(Discard SUPERNATANT( Discard) AQUEOUS PHASE CENTRIFUGATION: 11700g-15niin PELjET HOMjGENIZATION 5% TRITON-X CENTRIFUGATION: 27000g-15min PELLET I 0.05M KPO4 pH 8.2 + O.li 2-ME SUCraSE STEP GRADIENT CEN|RIFUGATION: 45000g-60niin COL ECT INCLUSION ZONE CENTRIFUGATION: 27000q-l 5min i PELl ET 0.02M TRIS pH 8.2 INCLUSIONS

PAGE 46

33 tissues. With this inerhod, a clear sap was obtained after the first low speed cent r i fuqa r ion but the virus zone in the CsCI gradient was not very well separated from the host components. Plants of C, am.uant icolp r and V. unguiculcita mechanically inoculated with purified preparations of BICMV showed the first symptoms of local lesions and systemic mosaic (fig. I) ^ and 7 days after inoculation, respectively. The ultraviolet absorption curve (Fig, 5) obtained for the purified preparations of BICMV had a maximum between 260 and 262 nm, and a riiinimum at 2kh to 2^5 nm. The ratio between the absorption at wavelengths of 260 and 280 nm was approximately 1,2 after correction for light scattering, as would be expected for a member of the PVY group. This value is consistent and agrees with those of other long flexuous rod-shaped viruses (Shepherd and Purcifull, 1971; Tosic et al., 197'^; and Barnett and Alper, 1977), The virus solutions showed strong stream birefringence and electron microscopic examinations indicated that 73% of the 190 virus particles examined were between 700 and 800 nm (Figs. 6, 7, 8). The rods observed in the purified preparations (Fig. 6) indicated a low percentage of virus fragmentation during the purification processes. As the result of end-to-end virus aggregation, a few particles with 1^00 to 1500 nm were also observed. Purified virus preparations usually were relatively free of normal plant constituents when examined with the electron microscope and in the spectrophotometer. Sedimentation coefficients determined for the virus at 20 C either In 0,02 M Tris buffer, pH 8.2, or in 0.05 M borate buffer, pH 8.2, indicated that BICMV sedimented as a single species with the S2Q values of 157 159 S. On the other hand, the Schlieren pattern (pig9)

PAGE 47

Figure 5 Absorption spectra of purified preparations of BlCMV in 0.02 M Tris buffer, pH 8.2, and B1CMV cytoplasmic inclusions in the same buffer.

PAGE 48

35

PAGE 49

Figure 6 Electron microscopy of BlCMV in a purified preparation and in cowpea leaf extracts. A) Purifieii preparation of BlCMV negatively stained with 2% phosphotungst ic acid, pH 6.5, containing Q.n BSA; B) Serologically specific electron microscopy (SSEM) of leaf extract from cowpea plants systemically infected with BlCMV. Antiserum for BlCMV diluted 1/1000 in 0.05 M Tris buffer, pH 7.2, was used to sensitize the grid and the virus particles were positively stained with \% urany] acetate. Note the considerable increase in virus concentration compared with the normal leaf-dip preparation (C) ; C) Leaf-dip preparation of cowpea leaf tissue systemically infected with BlCMV, negatively stained with 2% phosphotungstate

PAGE 50

37

PAGE 51

Figure 7 Histograms of lengths of BICMV particles from purified preparation negatively stained with phosphotungstate (a), and cowpea leaf extract using the serologically specific electron microscopy and uranyl acetate as a positive stain (B) Class interval for both histograms 50 nm.

PAGE 52

39 120 100 30 60 40 20 cc. o EC 100 80 60 40 20 [SI El 200 400 T 600 800 1000 PARTICLE LENGTH (nm) 1200 1400 1600

PAGE 53

Figure 8 Histograms of BICMV particle lengths from two different electron microscopic preparations to show particle length distribution from 600 to 900 nm. Class interval = 20 nm. a) Particle length distribution of BICMV from purified preparation negatively stained with phosphotungs tate ; B) Particle length distribution of BICMV from cowpea leaf extract prepared on grids sensitized with BICMV antiserum and positively stained with uranyl acetate.

PAGE 54

41 600 700 800 PARTICLE LENGTH (nm) 900

PAGE 55

42 revealed a difference in S values between BICMV in fresh preparations and BICMV in purified preparations stored at C for more than 30 days. Both virus preparations showed a single sedimenting peak, but the s^Q values for BICMV in fresh preparations and at a concentration varyiny from 0.5 to 1.0 mg/ml ranged from 157 to 159 S while the s^q values for the virus in the stored preparations and at the same concentrations ranged from 1^0 to 1^42 S (Fig. 9). The lower sedimentation coefficients obtained for the stored virus suggested that a change in virus mass (mW) had occurred. Hiebert and McDonald (1976) observed some possible enzymatic degradation of caps id protein of purified turnip mosaic virus. Proteolytic degradation of capsid protein of stored purified preparations of BICMV was also observed by polyacry lamide gel electrophoresis (PAGE) studies. Polyacry lamide gel electrophoresis analysis of SDS-degraded virus of a freshly purified preparation of BICMV revealed a main protein component with an estimated molecular weight (MW) of 3^,000 daltons and two smaller ones with MWs of 29,000 and 27,000 daltons (Fig. 10). These smaller components may have arisen by degradation of the slow moving component during storage (Hiebert and McDonald, 1976). Stored BICMV preparations contained only the faster moving protein components with MWs of 29,000 and 27,000 daltons (Fig. 10), presumably derived from 3^,000 daltons component Purified Inclusion Preparations Using either of the methods outlined in Figs. 3 and ^, purified cylindrical inclusions induced by BICMV were obtained from the same batches of systemically infected leaf tissue of V. unguiculata or

PAGE 56

Figure 9 Schlieren patterns from sedimentation velocity experiment with stored (A), and fresh (B) purified preparations of BICMV. Photograph was taken 8 minutes after the rotor reached a speed of 27,690 rpm. Sedimentation is from left to right.

PAGE 58

0) > -t-* CO (0 o <+o o CO 3 1 0) 0) l/l •o (D 1L. ro c — 10) Q c m >1 — -i: 0) 0) o > > VOl ^ OJ 03 o — X) o E OJ 1 o u X) > -a CO -Q XJ ro >~ — C x; • <-> E 0) 0) c — 3 o > 0) X) — CO .z: Dl 0) — I/) o — O c — o u/ 1/1 t/1 OQ — [ — m 1_ c O tH" 4-1 ro o O <-> 4—* "D l/l o 3 • — D O CL o Wl LX O Ol "O •— 4-* d ro c c u — 0) C71 o — (D o CL (D u 1 — E o 0) 1Q. c lA C i_ o ro O l/l 1E -C o c/1 01 Q. XI O -Q 0) >+1— U tJ (0 CO — o ro ro c > — E ro O l/l to • — (/I c: u >, 01 3 "O — CN x: I(11 a)
  • , O >4• — CO O OJ ~o C "O D i/i r* c > c o L. 4-1 o O ro s: — 0) _c 14MXI O >. Mn. (D 4-' o — 1-14o Mro CQ U D L, O > L. XI c (tJ J3 4J z: u. — O o c o XI O • — (U E 1O 0) CO • • ro I/) Q. (D 01 4-1 01 U1 o 0) -C o X) -C 3 o. I0) 1O ^ >^ vO I/) 0) Q. — 1C71 • — o cn 4u m m f 4-# — ro c a 01 X) O 3 ~ XI 0) 4) O > 1if} XI — -M (/) iC c ro O O u > — XI > -C 1-o m 01 OJ 4-> 4-" 4-* O OJ — ^ o o c ^ 1T3 o a 4t-J •— ^-v CL ^ CL a in — O (/I t/1 (U Q. Q — mm ui o — < O i_ XI 4-> c o 9) *< t/1 0) c 1 0> E 0) Q. c l/l O 4-1 ro 01 0) o; ro i_ c o o i_ 4-' 14c L. X) ro ro XI 0 Ul a X) 01 L. E > l/l l/l c c Ol ro ro -C 0) ro l/l o 4-1 ro X) c 4-1 c o 3 4-* ro 01 c 3 ro c XI o XI ro ro 0) O ro o 4-> l/l cn o +j c c X) > E l/l 4-> l/l o > CO ro x; — 01 C71 >~ j: — 14-1 0) o S ro cri >C 1o o CL — o ro OJ c o iQX) 0) ro 3 o l/l 0) — 0) O — — o — CO CO ro i_ ro Q0) L. Q. > <_> CO xi 0) c o ro c E 10) 4-* 0) XI Ol 01 2 i_ ro 3 O 0) o C 0) 4- o i. Q. 01 XI -d 0) 4-1 101 -C XI 4-1 E — 3 2 c C in 3 C 01 0) ^ 4-i K O L. D. in U C OJ — _^ 0) L. 4-1 ro 0 E IQ. cn c I— 01 2 o I. — ro — E O O) c 01 — _c l/l 4-> 3 4-* in c — 0) l/l l/l OJ 01 U CO Q. x: OJ Q. lc — o l/l XI C IO ro 4-1 o ro X) ^ l/l o c o o O 4-" ro vO X) o c o — o E' 3 ro XI -Jro c E — 3 E I3 0) XI l/l — ro 0) > c o > o J3 l/l c o .. <-( E — O ro +-• XI 4-< o o -Q O O o 4-" (V-> Lf\. Q. o 4-> 01 l/l E ro o c Mcn O T3 4-' u OJ in x: o Ia. x: Q ro 1cn O OJ -o in >~ OJ ^ -C OJ 4-1 -o — O ro x: • o Q. o c o — LA X) r-^ OJ — i_ — 3 in c ro — OJ OJ E 4J O in IOJ Dl O C X) ro ._ 4-. in in Q— ro XI o OJ > x: x *-> hO X) 4-1 c ro X) c o ^ Q. in in c OJ O i4-> l— O ro o X) O Qo o O 4-1 > 4-1 o z: o o o — CD -3XI OJ ^ X) eg ro — c_ a, OJ I > in X c t-> o — 4-> CO — — ^ ro c X) — in OJ C O 4-> O O O O 1— Qro cn X) csi in 3 O ^ lO rr, — O — > o -a r-, OJ C XI — — ro — OJ >4-1 CJ) O OJ 1XJ .. Q.^ 3 O OJ o E XI 01 4-t ro E OJ o — <4> ro z: X) in o ro — o CO o OJ o IXI ro OJ XI CM in ro c I^ — cn-3OJ OJ --^ 4-1 XI O C TO >3 C Q. — ro I o 3 CTl

    PAGE 59

    46

    PAGE 60

    Hj. benthamian a used for virus purification. Electron microscopy of purified BICMV-I negatively stained with molybdate revealed the presence of tubular inclusions with only trace amounts of host components (Fig. 11). At high magnification, striations of protein subunits were observed on individual tubes (Fig. 1 1 -D) These regularly spaced striations were estimated to have a periodicity of approximately 5 nm. Striations with similar periodicity have been observed in cytoplasmic inclusions induced by several other potyviruses (Edwardson et al., 1968; Hiebert and McDonald, 1973; and Morales and Zettler, 1977). Few virus particles were observed in the purified preparations of BlCMV-l which were not reactive to BICMV-As (Fig. 12). Purified preparations of B1CM\/-I with the highest degree of purity were obtained from N^. benthamiana with yields of 5 to 20 l^2^Q units were usually obtained from 100 g of fresh weight of N. benthamiana or V. unguiculata tissues. The ultraviolet absorption spectrum obtained for SDS disassociated BICMV-I was typical of proteins, with a maximum at 277 nm and a minimum at Ihd 2^48 nm (Fig. 5). Polyacrylamide gel electrophoresis of SDS-di srupted inclusion proteins revealed a single subunit component estimated to have a MW of 70,000 daltons (Fig. 10). Virus Particle Size and Stability in Sap Electron microscopic examinations of purified preparations of BICMV negatively stained with PTA indicated that IZt of 190 virus particles measured were between 700 and 800 nm with a modal length of 753 nm. Particle measurements of several leaf-dip preparations negatively stained with PTA and of grids prepared for SSEM with infected cowpea leaf tissue gave modal lengths of 758 and 780 nm,

    PAGE 61

    Figure II Electron micrographs of purified preparations of BiCMV cytoplai.mic inclusions stained with molybdate. All purified preparations consisted of tubes, most of which were fragmented during the purification process. Note striations (St) on high magnification (d)

    PAGE 63

    U1 C T3 o 1 0) c XJ 2 4-" in 1 to (U re us CO um ro — — — i-C — ro 0 0) 0) in c H a o > G (0 i_ •— > 0 2 i_ 0) ro >• z • D. — 2: • — L. 0 in Q) 0) ro > ro 0 ro 1+0 3 4-" t— — 3 0) I/) If) in (U — E C 0) Q. o > — c l_) x: ca u in c 0) c z 3 • O ^— 0 4-" •— > 0 • ro 0 — CO <414c u E 0) OJ 0 E i/i 0 0 ro 0 0) 3 XI x: •* C 3 14X) •lu — x: u 4-1 >1o — 0 4-1 0) c 4-1 0 ro 0) x: m in in CO XL 4-> Ol in l/l l/l in ro z ro 0) 0 4-' (0 •— c i_ • — 2 E • — — ro •M — o 0 4-1 0 X) (U ro 5 OJ 0) C o • — X B in in (U z c 1E E jr — c c 4-1 0) 3 — 4- 0 0 u 0 X) • — 0 \OJ O CTl u 01 (U 0) 0 E c 44-) L. z o • — ro X) l/l u L. 0 3 u OJ — 4-' a 3 0 E 14u 0) • — ~in OJ CL — a c Q) I' — >3 1 z in 4-" ctCL CO Ul (0 i_ L. l/l > c z 0 c 3 • CL E 2 0) Q ro E ^ — ro 1 o U. — cn ^ — ^ i_ in 1/1 0 0) 0 X O Q. c X) 0 z — — X3 XJ 4-' in Ol o — — ^ c 4-1 x: CO Mc X OJ XJ c > 3 ro c 4-1 14ro •— > o 14X) 4-> ro M0 01 ro c ro c — o — •— c in XI 3 4J X) OJ o — 1_ ro c c > c ro E 0) (0 — XI c 3 ro X) (U — — 3 4-> c +-> 4CL ro 0 0) 4-1 ro 0 1ro ro ro c *— o ro cn > — 0. ro \in 01 0) OJ OJ o U (U ^ — ^ (U ro CQ L. — 4-1 •— in Q. XI u 0) X) jr Q. 0 ro 0 in 4-' 2 D c 4-1 — 3 X) — X in '> 4-1 1 0 > E 1/1 0 (U ca u ro z: c CO 0 3 x: 0 4-1 x: 0 ro Q 4-1 (U > > in c 4-1 ^ — c • — CO X) 4-1 XI (U (U — 1>0) 1/1 z ro 0) (D 0) 4-1 0) 3 0) z 0) 1 -C OJ B l. — 4-' 2 2 — 2 0) > 4-' X) x: l-J X) ca 0 4- x> 4-1 ^ z: c 1. l/l 0) in (U (U in 0) 4-> 0 3 ro (0 C +-* X) 14— XI — — ro XI CO u cn o l/l — 0) c ro — — OJ E C D• — OJ Q. Q. c — ^ ix: 0 ro x: CO u cn • • > in O) 0) — c x: 4-1 0 4-1 z: OJ z: 0) c l/l > UOJ ro l/l Q. 0 ro 4-1 u 0 > f-) i/i — 0 CTi < — 0) >— CO 0) CO z: i) 0) 4-C OJ 3 3 c M 4-1 (U l/l 4-* 0) (U C 4-1 Lx: o XI 2 4-1 c 3 CL 0 -a in 4-" m — o CO ro — 1 ro — L0) — (/) 0 ro ro L. 01 > 0) TO 4-> cn 3 43 O E ro I/) i_ u (U 2: X ro I— 0) M3 14-1 4-> 10 i_ ro 3 X) Mcn u (U X < cn c Ol x: OJ 0) O (U X LA (U 0 0) CD n: 0 in 0 4-" 4-1 XI l/l Q. 0 ro ro O "o 0 0 E 0) u OJ C u i-l 1'0' ro i_ OJ in X) u 0 u OJ 43 (U c
    PAGE 64

    51

    PAGE 65

    52 respectively, with 90?; of the particle lengths ranging from 700 to 800 nm (Figs. 7 and 8). Some variation was observed with the particle size of purified virus stained with PTA and virus particles in leaf extracts prepared by SSEM and stained with uranyl acetate (Figs. 7 and 8). On the other hand, grids with less plant debris and higher concentrations of virus particles were obtained with SSEM than with the conventional leaf-dip preparation (Fig. 6). Using normal leaf-dip prepaiations at least four grids were prepared and 10 electron micrographs were taken to measure a maximum of 25 virus particles. On the other hand, 132 virus particles were measured by examining two micrographs obtained by SSEM. In cowpea leaf extracts, BICMV had a TIP of 65 C, LIV of k8 hr, and DEP of 10 Biackeye cowpea mosaic virus was still infectious after 10 min at 60 i: but not at 65 C and lost its infectivity after ^8 hr at room temperature, but not at 2h hr. Sap of cowpea leaves systemically infected with BICMV lost infectivity when diluted more than 10 ^ with distilled water. Serology Antisera specific for BICMV were obtained against untreated virions and pyrrolidine degraded viral protein. Both antisera reacted with SDSor pyrrol idine-treated BICMV in purified preparations or in plant sap in double and single radial diffusion tests (Figs. 12, 13, 1^). Most bleedings were specific for viral antigens; however, some bleedings also reacted with extracts from healthy plants, suggesting the presence of antibodies specific for normal plant components. To remove these antibodies the antiserum was absorbed with plant

    PAGE 66

    Figure 13 Single radial diffusion tests in agar media containing different concentrations of SDS and antisera for blackeye cowpea niDsaic virus (BICMV-As) and cowpea mosaic virus (CPMV-As), The media in (A, B, C) contain 0.8% Noble agar, 1.0^ NaN 0.3^0 SDS, and lO;^ BICMV-As (a) \5l BlCMV-As (B), and^20^n BICMV-As (C) The media in (D. E, F) contain 0.8t Noble agar, 1.0?; NaN3, O.^l SDS, and 10^ BICMV-As (D), \5t BICMV-As (E) and 201 BlCMV-As (F) The wells in (A, B, C, D, E, F) were charged with: (l) extracts from B I CMVinfected cowpea prepared in 1.5^ SDS 1/2 (w/v), (2) solution used in "I" diluted 1/2 with SDS, (^4) solution used in "1" diluted ]/^ with 1.5V, SDS, (8) solution used in "1" diluted 1/8 with ].S-o SDS, and (H) extract from healthy cowpea prepared in 1.5^ SDS. The media in (G, H) contain 0.8^ Noble agar, ].0% NaN 0.5^ SDS, and 15!^ BlCMV-As + ]5% CPMV-As (G), and^lO?, B1Cmv-As + 10% CPMV-As (H) The media in (I, J) contain 0.8% Noble aqar, ] .0% HaH-^ 0.3Z : SDS, and ]0% BlCMV-As + \0t CPMV-As (l), and 8% BICMV-As + 8'/o CPMV-As (j) The wells in (G. H, I J) were charged with SDS-treated extracts from: BICMVY infected cowpea (row no. 1), CPMVi nf ected cowpea (row no. 2), cowpea leaf tissue containing both BICMV and CPMV (row no. 3), and healthy cowpea (row no. k)

    PAGE 67

    54

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    Figure ]k Single radio! diffusion tests with SDS and pyrrolidine degraded capsid protein of blackeye cowpea mosaic virus (BICMV) and cowpea mosaic virus (CPMV) The media in (A, B, C) contain 0.8% Noble agar, ].0l NaN ().5>o SDS, and 1 5>o BlCMV-As (A) 15% CPMV-As (B), and^lOX BICMV-As + 10% CPMV-As (C) The wells were charged with SDS-treated extracts from: BICMVinfected cowpea (row l), CPMVinfected cowpea (row 2), BICMV and CPMV in cowpea (row 3), and healthy cowpea (row A) The media in (D, E, F) contain 0.8% Noble agar, CPMV-As (E), and 10% BICMV-As + 10% CPMV-As (F) prepared in 0.05 M Tris-HCl buffer, pH 7.2. The wells were (barged with py r ro 1 i d i ne-t reated extract from: Bl CMVinfected cowpea (row 1), CPMVinfected cowpea (row 2), BICMV and CPMV in cowpea (row 3), and healthy cowpea leaves (row k) BICMV-As (D), 15%

    PAGE 69

    56 €> • • 9 • • &

    PAGE 70

    57 components purified from V^. ungu icu I ata by high speed cent r i fugat ion according to the method used by Purcifull et al. (1973). The high specificity of most of the antisera obtained against purified BICMV preparations confirmed the efficiency of the methods used for its purification. The titers of antiserum varied depending on the bleeding date and on the rabbits, but 32 was the highest antiserum titer estimated by SDS-gel double immunodiffusion tests with a series of dilutions (1/2, 1/4, 1/B, 1/16, and 1/32) of B 1 CMVi nf ected cowpea tissue prepared in 1.5^ SDS Antiserum specific for cytoplasmic inclusions induced by BICMV was obtained from a rabbit injected with preparations of BICMV-I purified from infecred tissue of N^. b enthamiana The BlCMV-l antiserum reacted specifically with purified preparations of BICMV-I and crude sap of Bl CMVinfect ed cowpea, but not with either purified preparations of BICMV or crude sdp of non inocu lated plants (Fig. 12-B). The positive reactions with BICMV-I were more evident after 48 hr of incubation. The results obtained with BICMV antiserum also indicated that BICMV was not serologically related to its cytoplasmic inclusions (Fig. 12-A). Attempts to obtain specific antiserum by injecting rabbits with BICMV-I purified from infected cowpea tissue were unsuccessful. All three rabbits injected with BICMV-I purified from infected cowpea developed high titers of antibodies specific for normal plant tissue antigens. Single radial immunodiffusion studies in SDS-agar medium impregnated with the virus antiserum indicated that appropriate SDS and antiserum concentrations need to be previously selected for highest sensitivity and to avoid spurious reactions. The best results were

    PAGE 71

    58 observed when the antigens were prepared in 1.5^ SDS and the medium used had 0.3^^ SDS and lO;^ antiserum (Fig. 13-A) or 0.5^ SDS and ]5Z antiserum (Fig. 13-E). The same results were consistently observed with different batches of plates with the same medium compositions. Similar results were also observed with CPMV using antiserum obtained for SDS-treated virus. On the other hand, different results were observed in SDS medium containing a mixture of BlCMV and CPMV antisera. All media containing 0.3^ SDS were cloudy with all combinations of BlCMV and CPMV antisera used (Fig. 13-1, J ) i nd i ca t i ng some type of interaction between SDS and antiserum proteins. However, even with the cloudy appearance, some virus-specific reactions were still observed (Fig. 13-1, -J). Clearer media were obtained with 0.5^ SDS and 10 or ]5l of each antiserum. The best reactions, however, were observed when both BlCMV and CPMV antisera were used at concentrations of ]0% in media containing O.SZ SDS (Fig. 13-H). Strong precipitin rings were observed around the wells containing BlCMV or a combination of BlCMV and CPMV whereas weaker reactions were observed around the wells containing only CPMV (Figs. 13-H, \h'C) Unexpectedly, no reactions were observed around the wells containing only CPMV in a medium containing \S% of each antiserum and 0.5% of SDS (Fig. 13-G). Virus-specific reactions using BlCMV and CPMV antisera were also obtained with the single radial diffusion method described by Shepard (1972). Precipitin rings around the virus-wells were observed when the antigens were prepared in 1.5 or 2.5% pyrrolidine and the medium used contained 0.8"^ Nuble agar, 0.2^ NaN^ and 10 to 1 5^o virus antisera prepared in 0.05 M Tris-HCl buffer, pH 7.2, containing 0.85^ NaCl. Sharp, white precipitin rings were formed close to edges of

    PAGE 72

    59 the wells containing BICMV in agar medium prepared with BlCMV antiserum, whereas whitish halos with greater diameters were formed around the wells containing CPMV in agar medium impregnated with CPMV antiserum (Fig. lA-D, -E, -F) The same distinction between these two types of precipitin rings was observed when both antisera were added into the same medium, so that two concentric rings were formed around the wells containing both viruses (Fig. I^i-F). The inner ring was the result of BlCMV-ant ibody specific reactions and the larger halos resulted from CPMV-spe.;if ic reactions. This difference in types of precipitin rings could be related to the concentration of the antigens placed in the wells and to the reciprocal of antibody concentration (Shepard, 1972). Stronger and more compact rings were observed with CPMV when the antigens were diluted or the antiserum concentration was increased. Reciprocal double immunodiffusion tests with SDS-treated antigens showed that BICMV is serologically related to, but distinct from, the following potyvi ruses; BCMV-BV-1, BCMV-S, BYMV DMV LMV, PVY, SoyMV TEV, and WMV-2 (Fig. 15). No reactions were detected, however, with certain potyvi ruses, including BiMV, PeMV TuMV WMV-1, CoMV PVMV, and PWMV. Antiserum for BICMV also reacted specifically with CAMV forming a distinct spur which extended past the heterologous reaction (Fig. 16-A). In all positive serological relationships observed in the reciprocal serological tests, spurs were formed in both directions (Fig. 15). The serological distinctions observed between BICMV and BCMV-S, and CAMV by spur fonnation were demonstrated by the intragel cross absorption technique (Fig. 16-B, -D) The heterologous antigens.

    PAGE 73

    Figure 15 Reciprocal double immunodiffusion tests with BICMV and other potyviruses in medium containing 0.8^ Noble agar 1.0^ NaN3, and 0.5^ SDS prepared in 0.05 M Tris-HCl buffer, pH 7.2. All the antigenic solutions were prepared in 1.5'-^ SPS. The center wells were charged with: (1) BICMV antiserum, (2) PVY antiserum, (3) TEV antiserum, (k) WMV-2 antiserum, (5) DMV antiserum, (6) BCMV-BV-l antiserum, (7) BCMV-S antiserum, (8) SoyMV antiserum, (9) BYMV antiserum, (lO) BiMV antiserum, and (ll) I MV antiserum. The top rows of wells in all cases were charged with SDS-treated extracts from: (a) BICMVinfected cowpea, and (c) healthy cowpea. The bottom rows of wells were charged with SDS-treated extracts from: A) PVYinfected tobacco (b) and healthy tobacco (d) ; B) TEVinfected tobacco (b) and healthy tobacco (d) ; C) WMV-2infected pumpkin (b) and healthy pumpkin (d) ; D) DMVinfected dasheen (b) and healthy dasheen (d) ; E) BCMV-DV-l infected bean (t)),and healthy bean (d) ; F) BCMV-Sinfected bean (b) and healthy bean (d) ; G) SoyMV-infected N. bent hamiana (b) and healthy N. bent hamia na (d) ; HFBYMVinfected pea (b) and healthy pea Td) ; l) B iMVi n f ect ed Nicot iana hybrid (b) and healthy N i c ot iana hybrid (d) ; anJTPLMVinfected pea (b) and healthy pea (d)

    PAGE 74

    61 ^oH^n o 0| m OMO ^ 0 1r O El D E MSk ]E8r in mm Msm'^^ro o o o i # G o o o m ^
    PAGE 75

    Figure 16 Immunodiffusion tests with BlCMV, Moroccan isolate of CAMV and siratro strain of bean common mosaic virus (BCMV-S) in agar medium containing 0.8^^ Noble agar, 1.0% NdN,, cjnd 0.5% SDS prepared in 0.05 M Tris-HCl buffer, pH 7.2. A) Serological tests with BlCMV and CAMV. The center wells were charged with: BlCMV antiserum (Bis), and normal serum (Ns) The peripheral wells were filled with SOS-treated extracts from: (BI) B1 CMVinfected cowpea, (Ca) CAMVinfected cowpea and (H) healthy cowpea B) Intragel cross-absorption test with BlCMV and CAMV. The center wells were charged with: (l) BlCMV antiserum, (2) purified CAMV and 20 hr later BlCMV antiserum. The peripheral wells were filled with SDStreated extracts from: (Bl) BI CMVinfected cowpea, (Ca) CAMVinfected cowpea, and (H) healthy cowpea. C) Serological tests with BlCMV and BCMV-S. The center wells wp.re charged with: (Bis) BlCMV antiserum, (Ss) BCMV-S antiserum. The peripheral wells were filled with SOS-treated extracts from: (Bl) Bl CMVinfected cowpea, (S) BCMV-Sinfected bean, (Hb) healthy bean, and (He) healthy cowpea. The arrows point to spurs. D) Intragel cross-absorption test with BlCMV and BCMV-S. The center wells were filled with: (l) BlCMV antiserum, end (2) purified BCMV-S and 20 hr later BlCMV antiserum. The peripheral wells were charged with SDb-treated extracts from: (Bl) B 1 CMVi nf ec ted cowpea, (S) BCMV-Sinfected bean, (Hb) healthy bean, and (Hc) healthy cowpea. E) Serological tests with BCMV-S and CAMV using two different antisera for BCMV-S. The center wells were charged with: (l) BCMV-S antiserum from a rabbit inoculated with freshly purified preparations of BCMV-S, and (2) BCMV-S antiserum obtained from the same rabbit after a booster injection with purified BCMV-S stored at '4 C for 30 days. The peripheral wells were charged with SOS-treated extracts from: (S) BCMV-Sinfected bean, (Ca) CAMVinfected cowpea, (Hc) healthy cowpea, and (Hb) healthy bean. F) Serological test with fresh and stored purified preparations of BlCMV. The center well was charged with BH.MV antiserum, and the peripheral wells with SDS-treated new purified preparation of BlCMV (Np) old purified BlCMV (Op), and healthy cowpea extracts (H).

    PAGE 76

    63

    PAGE 77

    64 which were placed in the center well prior to the antiserum, crossreacted with and fully precipitated the cross-reacting antibodies at the region of optimal proportions close to the center well. Serological distinction was also observed between a freshly purified preparation of BICMV and purified BICMV stored at ^ C for more than 30 days (Fig. .16-F). This suggested some enzymatic degradation of certain BICMV antigenic determinants during the storage period Serological relationship studies between CAMV and BCMV-S using BCMV-S antisera obtained by different bleedings of the same rabbit indicated that the antiserum specificity varied according to the Immunization program and the conditions of the antigenic solution used A highly specific antiserum for BCMV-S was obtained from a rabbit injected with approximately 8 mg of freshly purified preparations of BCMV-S. Using this specific antiserum it was possible to show a complete serological distinction between BCMV-S and CAMV (Fig. 16-E). About three months after the initial immunization, a booster injection with a purified preparation of BCMV-S stored at 4 C for more than one month was given to the same rabbit. All antisera obtained 15 days or more after the booster injection reacted with CAMV, forming a spur between CAMV and BCMV-S when they were placed into adjacent antigen wells around the an t i serum we 1 1 (Fig. 16-E). Light and Electron Microscopy Light microscopic observations of epidermal leaf strip preparations from plants systemically infected with BICMV revealed the presence of tubular cytoplasmic inclusions similar to those described by Edwardson et al. (1972) and Edwardson (197'*) for BICMV. Side views of groups of tubular inclusions were easily observed in B 1 CMVInfected

    PAGE 78

    65 leaves (Fiq. 17), and at high maqn i f i ca t ion end views of them could be seen as small dots by changing the microscope focus. In ultrathin sections of B 1 CMVinfected tissue, these inclusions consisted of tubes attached to a central core, forming pinwheels (Fig. 18), similar to those induced by the potyviruses from Edwardson's subdivis ion-l (Edwatdson, 197'*)As reported by Edwardson (1974), the pinwheels contained arms with pronounced curvatures and tight scroll-like tubular inclusions. Only tubes were observed in purified preparations of cytoplasmic inclusions induced by BlCMV (Fig. 11). Host Range and Resistant Cowpea Varieties Blackeye cowpe.j mosaic virus was readily transmitted mechanically from cowpea 'Knuckle Purple Hull' to the following plants in which it was detected serologically and caused the following symptoms: ^'"ilta Uria spectabi I is (mosaic); Glycine max (l.) Mer. (mild mottle and chlorotic spots); Macropt i 1 i um at ropurpureum (DC.) Urb. (mosaic); Macroptilium bracteatum (L.) Urb. (mosaic); N icotiana bent ham i ana (mottle); Ocimum basi 1 icum (local lesions); Phaseolus vulgaris 'Black Turtle-2' (epinasty, necror.is, yellowing) and 'Bountiful' (chlorotic spots on inoculated leaves); V igna unguiculata 'Black Local' (mosaic), 'Early Ramshorn' (mottle), 'Knuckle Purple Hull' (mosaic), and 12 Brazilian cowpea cultivars in which the reactions varied from symptomless to mosaic (Table I). Small chlorotic lesions were found on the leaves of C henopodiu m amaran t i co lor inoculated with purified preparations of BlCMV or cowpea sap containing BlCMV (Fig. 1-B). Based on failure to induce symptoms and on negative serological results, BlCMV did not infect Arachis hypogaea L. 'Florunner', Capsicum

    PAGE 79

    Figure 17 Photomicrographs (A, B, D) of cytoplasmic inclusions in epidermal strips of cowpea leaves systemically infected with BICMV, stained with a combination of calcomine orange and luxol brilliant green. A) cells with masses of cytoplasmic inclusions, B) details of mass of inclusions seen in "A", c) general view of the inclusion distribution in epidermal cells, and n) phase contrast micrograph of the area photographed in "A". (Ci) cytoplasmic inclusions, (CW) plant cell wall, (G) guard cells, and (Nu) nucleus.

    PAGE 80

    67

    PAGE 81

    Figure 18 Electron micrographs of ultrathin sections of cowpea leaf cells infected with BlCMV showing c ross -sect i ons (A, B, C) and longitudinal sections (O) of pinwheel inclusions.

    PAGE 82

    69

    PAGE 83

    70 > (0 *J > 3 O ^— > < XI 3 ~ — CD >. C cn 0 (D — 0 LO C u 1 cn 0) > — m 1) a. 5 O > o z: CJ 14. > 0 cn cu t/l 0 (U *— X) •M c 0) > • s: > > 2: 0 < c 0 10 >• 1 (0 > ;/) 1/1 0 CO < 1/1 0) > E 0 X. 0 0 J-cn Q. 0 a:> E CO I "o 4-) X 0) ej in i 4T3 0 (U 4-> 1/1 (D M — > 3 z: 3 0 in 1 — C 1. • 0 >c — ro I I + I I > I I I I > I I I + • + + + + + + + + + + + + I I + + 1 2: z: I z: I I -J 0) (U Q Q E oi ro 10) ro 1(U to — ro Q< CL Q. 0) ro -M -ctn c (u I I -2:^CLCLi/ii/)co>> c 10 0 1-C ro ro 3 I/) 0) u 0 E CL 0 0 ro _i de LA LA a: er -T cr> 1 E >-a 0 ro 00 ro ro ro 1 1 1 1 1 0) u 0 "o LU UJ LU UJ LU lEa L oa 00 <_) CJ 0 CJ 0 CJ 3 — CL — 0) > Xi — 0) ro u 0) L. — j3 nj o c ro -iii 3 E "o ro 3 o — o — 0) C7, 3 +-I 4-1 L. CL ro c — o 0) II z II z: n in O E c O *^ l/l •u c 4— Q) 1/1 -0 0 4-> Wl <+(/I (U m 3 4-' 0) H4c 0 11 X> XI 01 _l a) 3 MXI 4X 3 0 X) ro 1/1 X) a 3 c E c 0 ro >0 XJ 1/1 a c 0 0 II c ro di (U c Q II L. 0 1 4-J in ro X) 0 ro {r> c 0 ro Ol i_ u 0 0 1/1 "o ro X 1_ 0 0 1/1 (U 0 (/I cn 0 i_ 0 0 E > 0 1/1 E 0 0 4) Q. c II 4-> 1/1 II II -C >CJ l/l + 1 -o 0) t/i
    PAGE 84

    71 annuuni L. Ear ly Ca I wonder ; C ucumis s at i vus L. ; Cucurb i ta pepo L. •Small Sugar'; Lupinus a ngust i fol ius L. 'Bitter Blue'; Lupinus luteus L. 'Sweet Yellow'; P haseolus vulgaris 'Black Turtle-1', 'Green Northern 11'40', 'Improved Tendergreen 'Lake Shasta', 'Michel ite 62', 'Pink Rosa', 'Pink Viva', 'Puregold Wax', "Red Mexican U-3A', 'Red Mexican U-35', 'Top-crop' and 'VC 1822'; Pisum sativum L. 'Alaska', 'Bonneville' and 'Ranger'; and V^ici^ faba L. The reactions uf cowpea varieties to mechanical inoculations of BICMV, BCMV-S, CAMV, and CPMV are indicated in Table I. All inoculated plants were assayed serologically for the presence of the viruses (Table I). D i scuss io n Blackeye cowped mosaic virus and its cytoplasmic inclusions were successfully purified from systemically infected cowpea or N. benthamiana leaf tissues with the procedures outlined herein. The first method of virus purification (Fig. 2) gave good yields of highly purified BICMV, and the combination of n butanol and chloroform-carbon tetrachloride (Fig. 4) was the better procedure for purification of BICMV and its cytoplasmic inclusions from the same batch of tissue. The high degree of purity of the BICMV preparations indicated by spectrophotometry, analytical cen t r i f uga r ion and PAGE analyses, as well as serological studies and electron microscopic observations, confirmed the efficiency of the pur i f i cat ion procedures. Aggregation of virus particles and virus and host components during purification appears to be a limiting factor for obtaining high

    PAGE 85

    72 yields of viruses in the PVY group (Shepherd and Pound, I960; van Oosten, 1972; Hiebert and McDonald, 1973; Uyeda et al., 1975; and Barnett and Alper, 1977). Hiebert and McDonald (1973) reported aggregation of virus particles after PEG precipitation. The losses of BICMV by low speed centr ifugat ion due to aggregation of virus particles were reduced by maintaining the virus in KPO^, buffer, pH 8.2, after precipitation with PEG. Another critical aspect on purification of potyviruses for obtaining maximum virus yield is the host used for virus increase. In order to obtain a good yield of BICMV from the 'Knuckle Purple Hull' variety of cowpea the virus was inoculated into the source plants at the age of 3 to days after emergence and the systemically infected leaf tissues were harvested 15 to 18 days after inoculation. Attempts to purify the virus from plants inoculated later than that or from tissue harvested more than 30 days after inoculation resulted in very poor yields of virus and cytoplasmic inclusions. Electron microscopic examinations of purified preparations of BICMV indicated a low percentage of virus fragmentation during the purification processes (Fig. 6-A) Particle measurements of purified BICMV and of BICMV particles on grids prepared for SSEM with infected cowpea leaf tissue gave two modal lengths (Figs. 7, 8) which differed by approximately 30 nm. Variations in lengths of virus particles have been extensively observed (Edwardson, 197'*). As reviewed by Edwardson (197'*), virus length variations may be attributed to several factors, including sample preparation, host influence, virus strain differences, and normal fluctuations in the electron microscope magnification. Increase of 50 to 100 nm in certain potyvirus particle lengths induced

    PAGE 86

    73 by magnesium ions were reported by Govier and Woods (1971). They indicated that in the presence of Mg ions the particles were straight, contrasting with tht: flexuous particles observed in the absence of Mg ions. On ant i serum-coated grids several antibodies combine with a single virus particle and, possibly, increase its length. Because of the specific antigen-antibody reaction the BICMV particles were so strongly attached to the surface of the ant i serum-coated grids that they could not be rt;moved by repeated washing. On the other hand, positive staining of BICMV particles with ethanol ic uranyl acetate may have induced some changes in their lengths. Measurements of 25 BICMV particles on ijrids prepared by conventional leaf-dip preparation with PTA gave a modcil length similar to that estimated for purified BICMV negatively st.ilned with PTA. Milne and Luisoni (1977) emphasized that negative staining gives better preservation and better resolution of viral capsids than positive staining. Using SSEM with uranyl acetate as a negative stain, Milne and Luisoni (1977), observed no change in the normal lengths of TMV and a potexvirus. However, leafdip preparations often contain too few particles to photograph conveniently for virus particle measurements, whereas relatively large numbers of particles can be photographed by using the serologically specific electron microscopic technique. As indicated by Derrick and Brlansky (1976), the addition of sucrose in the extracting and washing buffers greatly reduced the amount of plant debris on the SSEM grids. High amounts of plant debris in electron microscopic preparations are frequent problems in establishing the dimensions of a virus.

    PAGE 87

    7^ Polyacryl amide gel electrophoresis of SDS dissociated cytoplasmic inclusions and viral coat proteins clearly indicated that ths viral coat protein subunit was smaller than the inclusion subunit (Fig. 10). The PAGE results revealed that the inclusions were made of a single kind of protein with an estimated molecular weight of 70,000 daltons. Polyacrylamide gel electrophoresis of cytoplasmic inclusion preparations conducted by Hiebert and McDonald (1973) showed one protein component with molecular weight of 67,000 daltons for PVY; 67,000 for PeMV; 69,300 for BiMV; 69,600 for TEV; and 70,300 for TuMV. The PAGE studies also indicated that freshly purified BlCMV consisted of a main protein component with a molecular weight around 3^*,000 daltons. Two smaller protein components were also revealed by PAGE analysis of SDS denatured viral coat protein (Fig. 10). Since only traces of the faster moving proteins were observed with fresh purified BlCMV, and greater amounts of these prote inaceous components were revealed by PAGE analyses of stored purified BlCMV preparations (Fig. 10), it is assumed that the smaller components are due to the degradation of the slow moving protein during purification and storage. Hiebert and McDonald (1976) observed that some possible enzymatic degradation of TuMV capsid protein occurred during storage of purified virus preparations. The lower sedimentation coefficient estimated for stored purified BlCMV preparations (Fig. 9) is further evidence of proteolytic degradation of viral coat protein during storage at C. According to Hiebert and McDonald (1976), it is likely that "s^q values reported for potyviruses that are near ]kO S represent virus with partially degraded capsid protein, whereas those near 160 S represent virus with intact capsid protein." This proteolytic degradation also changes

    PAGE 88

    75 the antigenic properties of viral coat protein (Hiebert and McDonald, 1976; and Purcifull and Batchelor, 1977). Using antiserum obtained for freshly purified BICMV, serological distinction was observed between freshly purified preparations of BICMV and purified BICMV stored at ^ C for more than 30 days (Fig, 16-F). The serological distinctions between different antigen preparations of the same virus observed herein are of great significance for serological identification and characterization of potyviruses as pointed out by Hiebert and McDonald (1976) and Purcifull and Batchelor (1977). It IS important to keep in mind that purification, storage of either purified virus preparations or crude sap containing virus, and mailing of virusInfected fresh plant tissues may all result in modifications in the antigenic properties of viral coat protein. To solve this problem, the preservation of plant virus antigens by 1 yoph i 1 i zat ion of crude extracts from infected plants (Purcifull et al., 1975) or purified virus preparations is recommended. Blackeye cowpea mosaic virus has been maintained in lyophilized condition either in crude sap or purified preparation over two years during the course of this study without any perceptive change in its antigenic properties. Another factor that should be considered during serological relationship studies between viruses in the PVY group is the specificity of antisera. Variations in the degree of cross reactivity exhibited by different antisera obtained against the same virus have been attributed to differences between individual animals (van Regenmortel and von Wechmar, 1970), route and number of injections used in the immunization program (Hoi lings and Stone, 1965) and time of bleeding

    PAGE 89

    76 (Tremaine and Wright, 1967; and Koenig and Givord, \37^) The results of the present study indicated that the immunization program and the conditions of the antigenic solution used for rabbit immunization may also affect the antiserum specificity. A highly specific antiserum for BCMV-S was obtained from a rabbit immunized with freshly purified BCMV-S, whereas antiserum with a broader cross-reactive spectrum was obtained from the same rabbit after a booster injection with a purified preparation of BCMV-S stored at 4 C for more than one month. The use of such antisera would make it difficult to distinguish between certain plant viruses in SDS immunodiffusion tests. The serological distinction between B I CMV and BCMV-S was impossible to detect in SDS doubleimmunodiffusion when the BCMV-S antiserum with a wider cross-reactivity was used. On the other hand, an antiserum with a wide spectrum of activity should be useful for identification of vi rusinfected tissue used for plant propagation and possibly for identification of virus at the group level. As any v i rusinfected plant organ is undesirable for plant propagation the specific virus identification may not be necessary in such cases. For example, the BCMV-S antiserum was successfully used to identify cowpea seeds infected with BICMV. Unilateral serological relationships observed between BlCMV and SoyMV (Fig. 15-G) and with BICMV and BYMV (Fig. 15-H) showed the nenessity of reciprocal tests for demonstrating the absence of serological relationship between two viruses. According to Matthews (1970) "to demonstrate that two viruses are serologically unrelated, reactive antisera must be prepared against each of the viruses under test." Reciprocal tests are also important to show distinction between two closely related viruses. It was more difficult to observe a spur

    PAGE 90

    77 between BlCMV and WMV-2 when both viruses were tested against antiserum to WMV-2 than when they were tested against BlCMV antiserum (Fig. 15-C) Similar results wete observed with BCMV isolates and BlCMV (Fig. I5-E, -F) which may explain the identical reaction reported by ilyemoto et al. (1973). It is noteworthy that BlCMV and BYMV are serologically distinct, though related. This supports the contention of Edwardson et al. (1972) and Zettler and Evans (1972) that BlCMV and BYMV should be considered distinct viruses. Serological differences between closely related viruses are better detected with antisera of fairly low titer (Matthews, 1970). On the other hand, he also stated that a high titer antiserum is preferable for demonstrating distant serological relationship. This can be illustrated by the serological tests carried out with BlCMV and CAMV isolates using a BlCMV antiserum with a titer of 32. By diluting the antiserum to 1/4, no reaction was observed with the heterologous virus (CAMV) whereas a fairly good reaction was still detected with the homologous antigen. The absence of reaction between BlCMV and LMV-ant i serum (Fig. I5-J) may be a result of the low titer of the antiserum. The intragel cross-absorption test was effective for demonstrating distinctions between two closely related viruses (Fig. I6-B, -D) This is additional evidence that serological distinctions that are undetectable in conventional doub I eimmunod i f f us ion tests may be clearly revealed by intragel cross-absorption. Using this test, Matthews (1970) revealed a serological difference between type TMV and a nitrous acid induced mutant which showed a reaction of identity when tested against

    PAGE 91

    78 unabsorbed TMV antiserum. For a full precipitation of the crossreact ing antibodies close to the center well, a fairly high concentration of the heterologous antigen should be used to fill the antiserum well. This is illustrated by the intragel cross-absorption tests with BICMV antiserum shown in Figure 16. A precipitin ring was formed very close to the center well when a highly concentrated purified preparation of BCMV-S (0.5 1.0 mg/ml) was used to absorb BICMV antiserum (Fig. 16-D) whereas the ring formed approximately 2 mm away from edge of the well when BICMV antiserum was absorbed with a less concentrated preparation of CAMV (0.01 0.05 mg/ml) (Fig. 16-B). In both cases, though, the intragel cross-absorption test showed serological distinction between the viruses. The intensity of the reaction between the homologous antigen and the cross absorbed antiserum may give some information about the degree of relationship between the viruses. Weaker homologous reaction indicates closer serological relationship. Based on this, the results of the present study clearly indicate that BICMV is more closely related serologically to BCMV-S than to CAMV (Fig. 16-B, -D) The different degrees of serological relationships are also indicated by the intensity of the precipitin lines spurring over the heterologous virus reactions in straight diffusion tests (Fig. 16-A, -C) Serological relationship between different potyvi ruses has been commonly observed (Bercks, I960; Purcifull and Shepherd, 1964; Purcifull and Gooding, 1970; Uyemoto et al., 1972; and Shepard et al., 197^*), and the cross absorption of an antiserum with heterologous viruses has also been used to study serological relationship between plant viruses in tube precipitin tests (Wetter, 1967; and Alba and Oliveira, 1977), and in combination with gel diffusion tests (van Regenmortel 1966; Wetter,

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    79 1967; Nelson and Knuhtsen, 1973; Shepard et al., 197^4; and Jones and Diachun, 1977). The intragel cross-absorption technique was also observed to be useful in demonstrating cross-protection between serologically distinct strains of plant viruses (Lima and Nelson, 1975). The fact that most of the antisera obtained against purified BICMV preparations did not react with extracts of noninfected cowpea tissue can be added to confirm the efficiency of the virus purification procedures described herein. On the other hand, the high population of antibodies for normal plant antigens developed by the rabbits injected with BICMV-I purified from infected cowpea was an indication that v i rusinfected cowpea tissue may have a high concentration of host antigens, which were difficult to separate from the BICMV cytoplasmic inclusions. However, using N, bent hamiana as a source plant for BlCMV-l purification, antiserum specific for BlCMV-l was obtained. This is an additional indication of the useful application of N. benthamiana in plant virus research. Nicotiana benthamiana has been artificially infected with more than 50 plant viruses (Quacquarel 1 i and Avgelis, 1975; and Christie and Crawford, in press), showing its great potential for cytologlcal, serological, and physiological studies of different viruses in the same biological system. The foot-pad route of rabbit immunization (Ziemiecki and Wood, 1975) used to obtain the antiserum specific for BlCMV-l was an efficient procedure. The high yield of antibodies obtained for BCMV-S using the same route of immunization (Lima et al., unpublished) is additional evidence that a high titer antiserum can be obtained at the expense of very little ant igen

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    80 Reciprocal immunodiffusion tests with antisera specific for BICMV and BICMV-I (Fig. 12-A, -B) confirmed the findings of Hiebert et a1. (1971), Purcifull et a1. (1973), Batchelor (197'*), and McDonald and Hiebert (1975) that the inclusion body proteins are immunochemically distinct from viral coat protein and host proteins. The results of single radial immunodiffusion tests indicated that agar-media impregnated with mono-specific antiserum or with a mixture of antisera can be used for serodiagnos is of two morphologically distinct legume viruses. Single radial immunodiffusion tests were first used in plant virology by Shepard (1969) for serod iagnos i s of potato virus X in potato tuber sprouts. Subsequently the same method was successfully used to identify plant tissue infected with carlaviruses (Shepard, 1970; and Shepard et al. 1971) potyviruses (Uyemoto et al., 1972; and Casper, 197^*), a cucumovirus (Richter et al., 1975), a hordeivirus (Slack and Shepherd, 1975) and tobamovirus (Granett and Shalla, 1970; and Clifford, 1977). Rad ia 1 immunod i f fus ion plat es containing a mixture of antisera to two or three filamentous viruses have been used for detection of potato viruses X, S, and M (Shepard, 1972). This, however, appears to be the first report of a mu 1 t i p le-ant i sera medium for detection of both an isometric and a rod-shaped plant viruses. Shepard (1969) observed that single radial immunodiffusion was more sensitive than double immunodiffusion for detection of PVX in infected plant tissue, but Richter et al. (1975) obtained better results with double diffusion tests than with single diffusion for serological detection of CMV in naturally infected herbaceous plants. No attempts to compare these two serological techniques were made in the present study. Some observations, however, indicated that single radial

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    8] immunodiffusion requires fairly large amounts of antiserum and that the proper antiserum concentration needs to be previously determined for highest sensitivity and to avoid spurious reactions in SDS-agar media. Better results in mu 1 t i -ant i sera media were obtained when pyrrolidine was used as denaturant of virus coat protein. The three serologically related but distinct legume viruses, BICMV, BCMV-S, and CAMV can also be differentiated by some biological properties. The CAMV isolate was well adapted to cowpea infecting and causing symptoms in all 20 inoculated cowpea varieties. On the other hand, five cowpea varieties showed immunity to BICMV, and only two were infected with BCMV-S, which caused very mild symptoms (Table I). The different symptoma to 1 og i ca 1 reactions induced by CAMV and BICMV in some of the varieties (Table I) clearly indicate that they can be used to distinguish these two potyviruses. It was observed, however, that some of the symptoms induced by the viruses varied with temperature, light conditions, and age at which the plants were inoculated, but no variation was observed with the immunity of any cowpea variety. The cowpea varieties that showed immunity to BICMV (Table l) should be included in a cowpea breeding program or in a control program for this virus in the southeastern United States. Cowpea lines with resistance to other viruses have been selected in different parts of the world (Williams, 1977a; and Beier et al., 1977). Virus-resistant lines with resistance to other plant pathogens have also been identified (Williams, 1977a and 1977b). Attempts to compare BICMV with the East African type of CAMV were impossible because all samples of vi rusinfected leaf-tissue arrived in high degree of decomposition with the virus already inactivated.

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    82 Serological studies with such decomposed leaf tissue and BICMV antiserum gave results similar to those obtained with the CAMV isolate (Fig. 16-A) obtained originally from Morocco. As the inactivation of the vi rus in the decomposed tissue may have destroyed some of its antigenic determinants, no conclusive results about its serological relationship with BICMV can be derived from these tests. Light and electron microscopy of cowpea and other host cells infected with any one of these three legume potyvi ruses revealed that their cytoplasmic inclusions are morphologically similar. In ultrathin sections, their inclusions consisted of pinwheels similar to those induced by the potyviruses from Edwardson's subd i v i s i onI (Edwardson, 197^). The cytoplasmic inclusions induced either by BCMV-S or CAMV, however, failed to react with antiserum for BICMV induced inclusions. The low titer of the inclusion antiserum, however, may be one of the reasons for the absence of reactions. Despite the great similarity in the ul trastructures of pinwheel inclusions induced by BICMV and CAMV, they showed some difference at the light microscope level. Whereas BICMV induced big masses of cytoplasmic inclusions in 'Knuckle Purple Hull' (Fig. 17), only scattered small bundles of inclusions were observed in the cells of this host infected with CAMV. This is also an indication of no direct correlation between the severity of the symptoms and abundance of cytoplasmic inclusions induced by these potyviruses, since 'Knuckle Purple Hull' is more susceptible to CAMV than to BICMV (Table I). A similar phenomenon was observed with these two viruses in C. spect abi lis In addition to this, the nuclear inclusions readily observed in cells of C. spec tabi I is systemical ly

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    83 infected with BICMV (Christie and Edwardson, 1977) were not seen in leaf tissue of this host infected with CAMV. In summary, BlCMV is a potyvirus that belongs to Edwardson's subdivision-! (Edwaidson, \37^) and has a modal length of approximately 750 nni. The BICMV particles have a single sedimenting peak with s^q = 157 159 S and have a main protein component with a MW of 3^,000 daltons. Its cytoplasmic inclusions are made of tubes which show striations with periodicities of approximately 5 nm and consist of a single type of prott-in estimated to have a MW of 70,000 daltons. The virus also induces nuclear inclusions in certain hosts including C. spectab ills Blacktye cowpea mosaic virus is serologically unrelated to seven potyviruseb and serologically related to, but distinct from eight other potyviruses in SOSimmunod i f fus ion The virus has a narrow host range outside leguminosae, is seed-borne in at least two cowpea varieties and is transmitted by aphids in a nonpers i s ten t manner. Based (jn its physicdl, biological, cytological and immunochemical properties, BICMV cjn be differentiated from any other virus that infects cowpea. The antisera prepared for BICMV and its cytoplasmic inclusions were essential tools for the development of the serological techniques for detection of v i rusinfected seeds described in Chapter II.

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    CHAPTER I I IMMUNOCHEMICAL AND CYTOLOGICAL TECHNIQUES FOR DETECTION OF LEGUME VIRUSES IN INFECTED SEEDS Introduct ion The transmission of plant viruses through seed of infected host plants was first demonstrated by Reddick and Stewart (1919), who showed that bean common mosaic virus (BCMV) was transmitted by approximately SOI of seeds from infected P haseolus vulgaris. Since then, the phenomenon of seed transmission of plant viruses has received considerable attention and an appreciable number of viruses have been demonstrated to be seed-borne to some extent (Fulton, 1964; Bennett, 1966; Shepherd, 1972; and Phatak, 197^). Virus can be introduced into a crop at an early stage of plant development through infected seeds. Thus, the production of virus-free seeds, or seed lots with very low virus content may provide a very effective control of seed-borne plant viruses. Seed certification programs have been developed to test seed lots for the presence of viruses and to select virus-free seeds.. Barley stripe mosaic virus, which is responsible for a serious disease in Montana (Afanasiev, 1956), and lettuce mosaic virus(LMV), the causal agent of an important disease of lettuce (Grogan et al 1952), are good examples of virus diseases against which seed certification programs have been successful (Zink et al., 1956; Hamilton, 1965; Phatak, 197't; and Slack and Shepherd, I975). 84

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    85 Barley stripe mosaic virus has no l
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    86 The main purpose of the present investigation was to develop efficient and rapid serod iagnost ic techniques to assay legume seed lots for the presence of virus-infected seeds. Seeds of cowpea, V. ungu iculata 'Knuckle Purple Hull' infected with BICMV were used as a model hostvirus combination. Immunochemical techniques for detection of BICMV, BCMV, and SoyMV in hypocotyls of germinated v i rusinfected seeds of V^. unguiculata P^. v ulgaris and G^. Max, respectively, are described in this chapter. Abstracts of portions of this research have already been published (Lima and Purcifull, 1977a, 1977b), Literature Review The phenomenon of seed transmission of plant viruses was first demonstrated by Reddick and Stewart (1919), who presented strong evidence of seed transmission of BCMV in Phaseolus vulgaris. Since then, a large body of information has been accumulated about the transmission of numerous plant viruses through the seeds of infected host plants (Fulton, 196^; Bennett, 1966; Shepherd, 1972; Baker, 1972; and Phatak, 197^). Among the 183 plant viruses described in the Commonwealth Mycological Institute up to September, 1977, (Doi et al., 1977), 51 viruses have been experimentally demonstrated to be seedborne to some extent. Several plant viruses are known to be seed-borne in many leguminous host plants, but this review will cover only those viruses transmitted through seeds of Vigna spp.. Glycine max. and Phaseol us vulgaris.

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    87 Seed-Borne Viruses in V igna spp. Gardner (1927) apparently was the first to report the transmission of a cowpea virus through seeds of cowpea. Since then, many viruses which naturally infect cowpea have been demonstrated to be seed-borne in this host. A virus isolated from cowpea in Trinidad was demonstrated to be transmitted through 8^ of seeds of asparagus-bean (V igna sesquipeda l is) obtained from vi rusinfected plants (Dale, 19^9). The virus is believed to be a representative strain of cowpea mosaic virus (Agrawal, ]SS^; and van Kammen, 1971, 1972). It seems that the seed transmissibility of CPMV is erratic and depends on the type of virus isolate and the cowpea variety involved. A cowpea mosaic virus isolated from cowpea grown in Arkansas was seed-borne in this host (Shepherd, 196^*). Approximately 620 'Blackeye' cowpea plants grown from seeds harvested from plants artificially inoculated with CPMV failed to develop mosaic symptoms (Perez and Cortes-Mon 1 1 or 1970). On the other hand, Haque and Persad (1975) observed that the rate of seed transmission of CPMV varied from zero to 5.8^ depending on the cowpea varieties and select ions Anderson (1957) reported the seed transmission of three cowpea viruses, including a strain of CMV, which was transmitted through 4 28% of cowpea seeds from artificially infected plants, A virus closely resembling a strain of CMV was seed-borne in cowpea with a transmission rate of 5 to 16^ (Chenulu et al., 1968). On the basis of symptoms observed on cowpea plants grown from commercial seeds, Gay and Winstead (1970) reported seed transmission of CMV, a cowpea

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    88 strain of southern bean mosaic virus and a virus referred to as a cowpea strain of bean yellow mosaic virus. A strain of CMV isolated from cowpeas in Morocco was transmitted through approximately 1^% of the seeds harvested from artificially inoculated cowpeas (Fischer and Lockhart, 1976b). The seed transmissibi 1 i ty of the cowpea strain of SBHV was first demonstrated by Shepherd and Fulton (1962). McLean (I9'l) observed that a cowpea virus was transmitted through approximately 5% of seeds of highly susceptible cowpeavar iet ies but slightly susceptible or somewhat resistant varieties produced lower percentages of v i rusinfected seeds. Snyder (19'*2) observed only 3 to k% transmission of a cowpea virus through commercial seeds and 37 to k\X transmission of the same virus through seeds obtained from virus-infected plants. Yu (19^6), however, found no difference in the percentages of v i rusinfected cowpea plants grown from seeds obtained from artificially or naturally infected plants. These three viruses were also transmitted by aphids (McLean, \^k\; Snyder, 19'<2; and Yu, I9'*6). Similar aphid-transmitted viruses reported from India were also demonstrated to be seed-borne (Nariani and Kandaswamy, 1961; and Verma, 1971). A rod-shaped virus isolated from cowpea in northern Italy was designated cowpea aphid-borne mosaic virus (CAMV) and studied by Lovisolo and Conti (1966) who demonstrated its seed transmissibil ity in cowpea. The t ransmi ss ib i I i ty of CAMV through seeds of cowpea has been confirmed by several studies involving different strains of the virus and varieties of the host (Kaiser et al., 1968; Tsuchizaki et a 1., 1970; Bock, 1973; Bock and Conti, 197^4; Phatak, 197^*; Khatri and Singh, 1974; and Fischer and Lockhart, 1976a).

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    89 The mosr important cowpea virus in the southeastern United States was first isolated in Florida by Anderson (1955a) who later designated it blackeye cowpea mosaic virus (BlCMV) (Anderson, 1955b) and demonstrated its seed transmissibil ity in cowpea (Anderson, 1957). The seed transmiss ibi 1 i ty of BlCMV was confirmed by subsequent studies at the University of Florida, Gainesville. Zettler and Evans (1972) found as much as 18;^. of B 1 CMVinfected seeds in lots of certified cowpea seeds, and Uyemoto ei ai. (1973) reported 28^ of seed transmission for this virus in cowpea 'Knuckle Purple Hull'. Cowpea mild mottle virus, a carlavirus isolated from cowpea in Ghana, was demon strafed to be transmitted through seeds of cowpea, bean, and soybean in variable but sometimes large proportions (Brunt and Kenten, 1973, 197'*). Cowpea banding mosaic virus, which was identified as a member of the cucumovirus group in India, was transmitted through high percentages of seeds from infected cowpea varieties (Sharma and Varma, 1975). A new isometric cowpea virus isolated from cowpea seeds from Iran and designated cowpea ringspot virus had a seed transmission rate of 15 to 20% in three cowpea cultivars (Phatak, 197^; and Phatak et al. 1976) Thus, among the four filamentous and the six isometric viruses known to naturally infect cowpea, only TMV (f i 1 amen tous) and cowpea chlorotic mottle virus (isometric) have not been demonstrated to be seed-borne in cowpea. Kuhn (196^b) found no evidence of seed transmission of cowpea chlorotic mottle virus (CCMV) in more than 2,100 seeds harvested from infected cowpea, and Gay (1969) observed no virus symptoms in 3,000 cowpea plants grown from seeds harvested

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    90 from CCMV-infected plants. According to Gay (I969), CCMV was not transmitted through seeds of cowpea because of virus inactivation during seed maturat ion Seed-Borne Viruses in Glycine max. Soybean mosaic virus (SoyMV) which is probably common wherever soybean is cultivated (Bos, 1972) was first demonstrated to be seedborne in soybean by Gardner and Kendrick (I92l). Subsequently, they observed that the virus could overwinter in the v i rusi nf ected seeds lying in the field (Kendrick and Gardner, 192^). Although Kendrick and Gardner (192^) hcd also observed that mottled seeds were produced by both healthy and v i rusinfected plants and that diseased seedlings were produced by mottled as well as clean seeds, the association of seedcoat mottling and virus infection has been reported (Koshimizu and lizuka, 1957; Ross, 1963, 1968, 1969; and Kennedy and Cooper, 1967). A mechanical selection of nonmottled soybean seeds has been suggested as a measure for controlling SoyMV in Brazil (Lima-Neto and Costa, 1976). However, Ross (1970) observed that SoyMV was equally transmitted through mottled and nonmottled seeds from virus-infected soybean plants, and concluded that the percentage of seed transmission for SoyMV in soybean could not be estimated from the amount of mottled seeds. Working with a Brazilian isolate of SoyMV, Porto and Hagedorn (1975) reported the production of mottled seeds by supposedly noninfected soybean cultivars and observed that SoyMV was not transmitted through seeds of v i rusinfected soybean cv, 'Bienville', which produced seeds with a high percentage of mottling. Bean pod mottle virus.

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    which is not seed-borne in soybean, also increased the percentage of mottled seeds (Ross, 1963). Ghanekar and Schwenk (197^) showed a rate of seed-transmission for tobacco streak virus varying from 2.6 to 30.6^ according to the soybean cul t i var Among the nine viruses known to infect and cause diseases in soybean in Japan, SoyMV, soybean stunt virus, soybean mild mosaic virus, peanut stunt virus, southern bean mosaic virus, and a strain of alfalfa mosaic virus were reported to be transmitted to some extent through seeds of soybean varieties (Koshimizu and lizuka, 1963; I izuka and Yunoki, 1974; Takahdshi et al., 197'*; lizuka, 197'; and Tamada, 1977). The transmission of nepoviruses through seeds of their host is well documented. Tobacco ringspot virus (TRSV), a nepovirus (Fenner, 1976) responsible for a disease commonly called bud blight of soybean, was first reported to be transmitted through seeds of artificially inoculated soybean plants in 195^* (Desjardins et al., 195^). These results were confirmed by subsequent studies (Kahn, 1956; Owusu et al., 1968; Athow and Bancroft, 1959; and Demski and Harris, 197^)). Kahn (1956) also demonstrated the seed-t ransmi ss ib i I i ty of tomato ringspot virus in soybean and observed that soybean seeds from plants infected with either tobacco or tomato ringspot virus had a lower percentage of emergence than seeds from virus-free plants. Three other nepoviruses: arabis mosaic, raspberry ringspot and tomato black ring viruses were experimentally demonstrated to be transmitted through seeds of soybean plants artificially inoculated with these viruses (Lister, I960; and Lister and Murant, 1967).

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    92 Seed-Borne Viruses in Phaseol us vulgaris A large body of evidence (Bos, 1971) has confirmed the first demonstration of seed transmission of BCHV in P. vulgaris by Reddick and Stewart (1919). Burkholder and Muller (1926) reported that seeds from bean plants showing mosaic gave rise to 50% diseased plants. The virus remained viable within bean seeds for a period of at least 30 years (Pierce did Hungerford, 1929). Fajardo (1930) observed that both infected and noninfected bean seeds germinated with equal readiness and vigor, and concluded that the viability of the seeds was not directly affected by the presence of BCMV. Fajardo (1930) and others (Crowley, 1957, 1959; and Schippers, 1963) found that plants infected at early vegetative growth produced more virus-infected seeds than those infected at later stages. It has been demonstrated that BCMV is transmitted irregularly through seeds harvested from vi rusinfected plants. Fajardo (1930) observed that some of the seeds in a single pod from bean plants infected with BCMV were v i rusi nf ected and some were noninfected. By crossing vi rusinfected and healthy bean varieties. Nelson and Down (1933) provided evidence that BCMV was equally transmitted by ovule or pollen from infected parent plants. Similarly, Medina and Grogan (1961) obtained high percentage of seed transmission of BCMV through either the pollens or ovules of infected plants, but also observed that the pollens usually transmitted the virus to a largernumber of progeny plants than the ovules. Cross-pollination experiments performed by Schippers (1963) revealed that the embryo infection with BCMV and consequently the seed-transmission might originate from

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    93 an infected egg-cell or an infected pollen grain. Studies to determine the distribution of BCMV in developing reproductive tissues suggested that successful seed transmission was insured by the presence of infective virus within the embryo itself (Ekpo and Saettler, \S7k) A correlation between symptom severity and percentage of seed transmission of BCMV has been observed in different bean varieties (Smith and Hewitt, 1938). Medina and Grogan (I96I) suggested that the percentage of seed transmission of BCMV was greatly affected by the differences in varietal susceptibility of the beans. However, Zettler (1966) found similar percentages of seed transmission of BCMV in 5 different bean varieties. Using the bean cultivar VC1822 as an indicator host for BCMV, Provvidenti and Cobb (1975) demonstrated a rate of seed transmission from 7 to 22% for BCMV in tepary bean ( Phaseolus acutifol ius Gray var. latifol ius Freem.), and observed that the virus was carried in the embryo but not in the testa. Two BCMV isolates obtained from bean grown in the Netherlands were observed to be transmitted through approximately 20 to 80:^ of seeds harvested from infected bean plants (Drijfhout and Bos, 1977). A strain of CMV isolated from bean plants grown in Spain was also demonstrated to be seed-borne in one of twelve bean varieties studied by Bos and Maat (1974). As they tested only a few seeds from virusinfected plants of each bean variety, they suggested that a low percentage of seed transmission could possibly occur in other varieties. The transmissibi 1 i ty of SBMV through seeds of bean was first demonstrated by Zaumeyer and Harter {]3k3) Cheo (1955) found a very

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    9^ high content of SBMV in the embryos of immature seeds from virusinfected bean plants, but observed that the virus concentration dropped to a low level or disappeared as the seed matured and dehydrated. Cheo's finding was confirmed by Crowley (1959), who found that approximately 100^ embryo infection occurred when bean plants were inoculated with SBMV at any time prior to flowering, but no seed transmission of thib virus occurred when samples of mature dry seeds were sown. These findings were supported by the results of McDonald and Hamilton (1972), which confirmed that in mature bean seeds, infectious SBMV is confined to the seed coat. An ilarvirus was also reported to be seed-borne in bean. Tobacco streak virus, the causal agent of a severe disease outbreak of pinto bean in State of Colorado, U.S.A., in 19^7, was demonstrated to be transmitted by approximately 26% of seeds from plants grown from virus-infected seeds (Thomas and Graham, 1951). In summary, seed-transmission is an important factor in the perpetuation and dissemination of numerous viruses that cause diseases in cowpea, soybean, and bean. The following methods for detecting selected seedborne viruses in legumes may be useful in virus disease control programs and as research tools. Materials and Methods Source of Seed and Seed Germination Seeds of cowpea. V. unguiculata 'Knuckle Purple Hull' and 'Early Ramshorn', harvested from BlCMV-infected plants were used in the present tests. All seed lots showing B 1 CMVt ransmi ss ion were obtained either

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    95 in fields of 'Knuckle Purple Hull' cowpeas in Gainesville, Florida, or from Gilvan Pio Kibeiro, University of Georgia, Athens. As controls, virus-free 'Knuckle Purple Hull' seeds produced in Texas were obtained from a local commercial source. Seeds were surface sterilized in 0.5^ sodium hypochlorite for 10 min, rinsed thoroughly with sterile deionized water and placed in moistened paper towels to germinate. The towels were rolled up, placed upright in 30 ml beakers and the seeds were allowed to germinate for 3 to 5 days at 25 27 C in an incubator. Prepar at ion of Ant i gens for Serology Hypocotyls from germinated seeds, singly or in groups of 5 or 10 were tested in double or single radial immunodiffusion tests. If information about the percentage of infected seeds was wanted, I 2 mm thick di scs from individual hypocotyls were cut with a razor blade which was rinsed with 35Z ethanol and deionized water after cutting each hypocoty 1 These hypocoty] discs were tested individually in double or single immunodiffusion tests by embedding them directly into the agar. Groups of 5 or 10 hypocotyls were ground in water or 1.5^ SDS (l/l, w/v) with a mortar and pestle as described previously (Purcifull and Batchelor, 1977) and tested against BICMV and BICMV-I antisera in double and single radial diffusion. Single or bulked hypocotyls were checked by SSEM for the presence of BICMV particles. Double Immunodiffusion Tests Individual hypocotyls or groups of hypocotyls from germinated cowpea seeds were tested against BICMV antiserum in double diffusion

    PAGE 109

    96 tests in agar medium containing 0.8?; Noble agar, 0.5% SDS, and 1.0^ NaN^ prepared either in deionized water or in 0.05 M TrIs-HCl buffer, pH 7.2. Eight to tv/elve discs of individual hypocotyls were embedded in the agar medium, to 5 mm away from each antiserum well. Undiluted antiserum for BICMV was routinely used in these tests, but dilutions of 1/2, I/'*, and 1/B of the antiserum with either normal serum or 0.05 M Tris buffer, pH 7.2 were also tested against hypocotyl discs and extracts of hypocotyl tissue. As controls each hypocotyl was also tested against normal serum, and hypocotyls from noninfected seeds were also included in each test. Extracts from groups of hypocotyls were tested by double immunodiffusion against BlCMV antiserum. Hypocotyl extracts were pipetted into the antigen wells distributed in an hexagonal arrangement around the antiserum well. All plates were incubated in a moist chamber at 2h C for 2^ to ^8 hr. The sensitivity of double immunodiffusion for detection of BICMV in extracts of bulked hypocotyls of germinated 'Knuckle Purple Hull' seeds was tested by mixing l.O g of infected hypocotyl tissue with different amounts of noninfected hypocotyls. Single Radial Immunod if fusion Tests Discs and extracts of hypocotyls were tested by single radial immunodiffusion in medium containing 0.8^ Noble agar, 0.5^ SDS, ].0% NaN^ and 15^ antiserum for BICMV buffered with 0.05 M Tris-HCl, pH 7.2. Hypocotyl discs were embedded directly into the solidified agar medium with the aid of forceps and the plates were incubated in a moist chamber at 2k C for 2k to 72 hr. Precipitin reactions were detected by direct observation of the plates on a darkfield

    PAGE 110

    97 light box or with a binocular microscope (6 to 15 x) in which the plates were illuminated from the bottom. Extracts of groups of 5 hypocotyls were placed into wells (3 nim in diameter) punched in the agar medium in a row arrangement. The wells were spaced 3 to ^ mm from each other as measured from the edges of the wells and as many as 230 germinated seeds could be tested in a 90 x 15 mm plastic petri dish. Precipitin rings around the wells charged with extracts from infected tissue could be detected anywhere between one to 2** hr. Serolog ical iy Specific Electron Microscop y Hypocotyl extracts prepared in 0.05 M Tris buffer, pH 7.2, containing 0.15 M NaCI and 0.^ M sucrose were examined for the presence of BICMV by SSEM as described previously (Derrick and Brlansky, 1976). Copper grids with P.jrlodion film coated with carbon were treated with BICMV antiserum diluted to 1/1000 in 0.05 M Tris buffer, pH 7.2. The grids were washed with 0.05 M Tris buffer and floated on drops of hypocotyl extracts tor 3 to i hr at room temperature. After washing with approximately 2 ml of the extracting buffer and then with approximately 1 ml of deionized water, the grids were positively stained with 1.0^ uranyl acetate in 50^ ethanol. The grids were washed again with 50^ ethanol, dried and examined in the Philips Model 200 electron microscope. The sensitivity of SSEM to detect the presence of BICMV particles in extracts from a mixture of hypocotyls was determined by diluting infected 'Knuckle Purple Hull' hypocotyl tissue with noninfected tissues up to a dilution of 1/50 (w/w) The different mixtures of infected and noninfected hypocotyl tissues were ground in

    PAGE 111

    98 extracting buffer ()/l, w/v) with mortars and pestles and small drops from these extracts were used to treat the ant 1 serum-sens i t i zed grids. Double Immunodiffusion Tests and SSEM for Detection of Other Viruses in Germinat ed Legume S eeds Double immunodiffusion tests using discs or extracts of hypocotyls, and SSEM using hypocotyl extracts were used to detect the presence of BCMV and SoyMV in germinated seeds. A seed lot of bean, P. vulgaris 'Black Turtle' containing BCMVinfected seeds was obtained from Dr. Rosario Provvidenti, New York State Agricultural Experiment Station, Geneva. Infected seeds were detected by double immunodiffusion and SSEM using antiserum obtained for a severe strain of BCMV (BCMV-S) isolated from siratro> Macropt i 1 ium at ropurpureum in Florida (Lima et al.,1977). Extracts of these germinated bean seeds prepared in 0.05 M potassium phosphate buffer, pH 7.5, were also inoculated in a very sensitive local lesion bean line VC-1822 (Provvidenti and Cobb, 1975). Antiserum for SoyMV was also used to detect SoyMVinfected seeds of soybean, G lycine max. Serology and Microscopy of Cytoplasmic Inclusions Induced by BICMV and SoyMV in Hypocotyls of Germinated Seeds Germinated seeds of cowpea 'Knuckle Purple Hull' infected with BICMV and germinated seeds of soybean 'Midwest' infected with SoyMV were serologically identified by double immunodiffusion tests using antiserum for BICMV and SoyMV, respectively, and hypocotyl discs as assay antigens. Hypocotyl extracts from the v i rusinfected 4-5-dayold seedlings were tested by SDS double immunodiffusion tests against BICMV and SoyMV inclusion antisera.

    PAGE 112

    99 Cytoplasmic irn lusions were examined in epidermal strips of the hypocotyls of germinated cowpea seeds infected with BICMV, and of SoyMVinfected hypoi:otyls of germinated soybean seeds, after staining with a combination of calcomine orange and "luxol" brilliant green (Christie. 196?). Small pieces of B 1 CMVinfected and non infected cowpea hypocotyls were prepared for ultrathin sectioning as described previously in Chapter I. Similarly, healthy and SoyMVinfected soybean hypocotyls were prepared for ultrathin sectioning. Sections were cut with a diamond knife and stained with potassium permanganate, uranyl acetate, and lead citrate. All specimens were examined with a Philips Model 200 electron microscope. Resul ts Preparation of Antigens for Serological Tests Antigens prepared from hypocotyls of germinated seeds proved to be very satisfactory for indexing seeds in double and single immunodiffusion tests. Neither small discs nor extracts from hypocotyl tissues showed any kind of nonspecific reaction that could interfere with the virus-specific reaction in double or in single inmunod i f f us ion tests. On the other hand, extracts obtained from whole seedlings, including roots, cotyledons and primary leaves showed several types of precipitation patterns when tested against any serum in double diffusion tests (Fig. 19). Such nonspecific precipitations were also observed with extracts obtained from cotyledons, primary leaves or root tissues. These nonspecific reactions were reduced when the agar

    PAGE 113

    Figure 19 Double immunodiffusion tests witli extracts from different portions of B] CMVinfected and healthy, ^J-S-day-old cowpea seedlings. A) medium containing 0.8^ Noble agar, 1.0^ NaNj, and 0.5% SDS prepared in deionized water. B) medium with tbe same composition except that it was prepared in 0.05 M Tris-HCI buffer, pH 7.2 instead of water. Center wells were charged with: (As) BICMV antiserum, and (Ns) normal serum. The peripheral wells were filled with extracts from: (a) hypocotyl from Bl CMVinfected cowpea seedlings, (b) hypocotyl from healthy cowpea seedlings, (c) cotyledons and primary leaves of BICMVinfected seedlings, and (d) cotyledons and primary leaves of healthy cowpea. Note the nonspecific precipitate formed with cotyledons and primary leaves of virusinfected (c) and healthy (d) seedlings and normal serum (Ns) and BICMV antiserum (As). The virus-specific precipitin lines clearly shown with v i rusi nf ected hypocotyls (a) and the antiserum (As) was masked by the nonspecific precipitates when extracts from cotyledons and primary leaves (c) of infected seedlings were used.

    PAGE 114

    101 e o o o e e o o ^ ^ o

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    102 medium was prepared in 0.05 M Tris buffer, pH 1.1 (Fig. 19-B). Based on these results a diagram showing several procedures for preparation of antigens from leijume seeds is suggested (Fig. 20). Double Immunodiffus i on Test s Blackeye cowpea mosaic virus was detected in hypocotyl extracts from bulked seedl inijs and in small discs of individual hypocotyls of cowpea 'Knuckle Purple Hull' and 'Early Ramshorn' by double immunodiffusion tests in SDS-gel plates. Virus specific precipitin lines were observed with extracts of mixtures of B 1 CMVi nfected and noninfected cowpea 'Knuckle Purple Hull' hypocotyls up to a dilution of 1/30 (w/w) respectively (Fig. 21-B). This indicated that germinated seeds can be divided into groups of up to 30 seedlings to be tested in double immunodiffusion. Approximately equal amounts of hypocotyl tissue (O.l ^.h g) were cut from each seedling and the extracts obtained from each hypocotyl group were deposited into individual antigen wells. Precipitin reactions were observed with those groups in which at least one seedling was infected with BICMV. On the other hand, no precipitin reactions were observed with extracts obtained from noninfected hypocotyl tissue, nor with any hypocotyl extract and normal serum (Figs. 19, 21 ) When information about the infection percentage was wanted, discs of individual hypocotyls were used in double immunodiffusion. Virusspecific precipitin lines formed between B 1 CMVi nfected hypocotyl discs and antiserum wells, whereas no reactions were observed with noninfected hypocotyls (Fig. 21-C, -D, -F). Good results were observed with undiluted antiserum and with antiserum diluted 1/2 and \lk with

    PAGE 116

    Figure 20 Diagram showing methods for assaying legume seeds by single and double radial immunodiffusion: I) seeds are placed on moistened paper towels, 2) the towels are rolled up and placed upright in beakers, 3) germinated seeds after four to five days, individual seedlings are divided into three parts: roots (r) hypocotyl (hy) and epicotyl (ep) consisting of cotyledons .and primary leaves, 6) hypocotyl tissue is ground with mortar and pestle, 7) hypocotyl extracts are rested in double radial immunodiffusion (DRD) 8) hypocotyl extracts are also tested in single radial immunodiffusion (SRD) 9) small discs are cut from each hypocotyl, 10) discs from different hypocotyls are tested by DRD, and 11) discs from different hypocotyls are tested by SRD.

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    Figure 21 Double Immunodiffusion tests with hypocotyl from healthy and Bl CMVinfected '-5-day-old cowpea seedlings in medium containing 0.8^ Noble agar, I Oi^ NaN and 0.5^ SDS, prepared in water. A B) Serological tests with hypocotyl extracts prepared in water. The center wells were charged with: (As) BlCMV antiserum, and (Ns) normal serum. The peripheral wells were charged with extracts from: (1) BlCMVinfected hypocolyls, (H) healthy hypocotyls, (5) one gram of B 1 CMVi nf ec ted hypocotyl mixed with g of healthy hypocotyls, (lO) one gram of B 1 CMVinfected hypocotyl and 9 g of healthy hypocotyls, (15) one gram of infected hypocotyl and 14 g of healthy hypocotyls, (20) one gram of infected hypocotyl and 19 g of healthy hypocotyls, (25) one gram of infected hypocotyl and m 9 of healthy hypocotyls, and (30) one gram of infected hypocotyl and 29 g of healthy hypocol y 1 s C D) Serological tests with saml 1 discs of different hypocotyls embedded into the agar medium ^ S mm away from the antiserum well. Ten (c) or twelve (D) hypocotyl discs are embedded around the center wells and the hypocotyl discs are numbered from the top in a clockwise direction (arrows). Wells were charged with: (As) BlCMV antiserum, and (Ns) normal serum. Note v i rus-ant i serum specific reactions with hypocotyls; 1, 2, 8, 11, ]k, l6, 18, 19 (C), and 6, 8, 9. 16, 20, and 23 (D) E F) Serological tests with hypocotyl extracts (E) and hypocotyl discs (F) from healthy and BICMVinfected cowpea seedlings. The center wells were charged with: (A) undiluted BlCMV antiserum, (A:2) BlCMV antiserum diluted 1/2 with normal serum, (A:'4) BlCMV antiserum diluted \/k with normal serum, and (A:8) BlCMV antiserum diluted 1/8 with normal serum. The peripheral wells were charged with extracts from: (5) one gram of B 1 CMVi nf ected hypocotyl s and k of healthy liypocotyls, (lO) one gram of BlCMV-infected hypocotyls and 9 g of healthy hypocotyls, and (H) healthy hypocotyls. Hypocotyl discs from healthy (h) and BlCMV-infected (i) seedlings were alternated around the antiserum wells in F.

    PAGE 119

    106 ((i) Q ^ o

    PAGE 120

    107 normal serum (Fig. 21-F). The percentages of BICMVinfected seeds in four different lots of cowpea 'Knuckle Purple Hull' and one 'Early Ramshorn' seed lot were estimated by this simplified double immunodiffusion test and by the growing-on test (Table II). Over a hundred seedlings tested by this serological technique were randomly checked by SSEM for the presence of BlCMV particles and in all cases the resu corresponded Single Radial Immun od if fusion Tests The presence of BICMV in cowpea hypocotyl extracts was detected by single radial immunodiffusion tests in agar medium impregnated with BICMV antiserum. Precipitin rings were observed around the wells charged with extracts from groups of 5 individual hypocotyls in which at least one was infected with BICMV, but not around those wells charged with extracts of noninfected hypocotyls (Fig. 22). The virus-specific reactions started to appear at approximately one hour after the hypocotyl extracts had been added into the wells and became very clear and evident 3 to 23 hr later. The reactions were still very distinct 2^4 hr after the hypocotyl extracts had been added into the we 1 1 s When small discs of individual hypocotyls were directly embedded into the agar medium containing BICMV antiserum, the virus-specific reactions took longer to appear. These specific reactions were recognized as opalescent precipitates around infected hypocotyl discs that could be better detected under a binocular microscope. The opalescent precipitates which usually were located at the ends of the hypocotyl discs, started to appear in 2^ hr, but were more evident

    PAGE 121

    108 O 1) d) tU lO *— • d) o C -M o •M 1 CTl (/I £c o o d) d) o C LA c O CD — > t-i d) *o ^ M 0 O > cu C o Q_ — o If) i CO 4T3 o dJ d) 4-1 C c/o t/) (A O dJ 0) U C (/I o u o a dJ — X) O Mw 0) D 4- a. — 1/1 (1) 1in — a> 0) u Io I/) X) > > > > 2: X 0 <_) <_) ca CO CO CO CO > X >o 0) X) (U 0 CA LA LA *-> -3CA in LA ^ 00 te rA OA CO OA CM rA OA 00 fA CTl o CO 3 o C 3 (U Q. 2 O o 3 3: Q, L. O C < CO 0 Q •M M M 0 0 0 0 _l _l _l _J c in > 0 1_ u 0 X 01 >*-C XI 1 > 0) E 0 M D c Z D. u n3 Q. (D (D 0) 1 UJ -Q -> C CO > <0 0 0) J; to CQ

    PAGE 122

    Figure 22 Single radidl immunodiffusion tests with hypocotyl extracts from healthy (H) and B 1 CMVinfected (I), i*-5-day-old cowpea seedlings in media containing 0.8% Noble agar, 1.0% NaN^, 0.5% SDS, and 15% BICMV antiserum (A, C) and 15% normal serum (B). The top row and the two bottom rows of wells in C were charged with extracts from healthy hypocotyls and the others were randomly filled with extracts from groups of 5 hypocotyls containing 1 or 2 B 1 CMVi nf ecied hypocotyl per group.

    PAGE 123

    110 o ooooo ooooo o ooooo

    PAGE 124

    ) 1 1 at A8 to 72 hr after the test had been set up. Usually after 72 hr, a nonspecific precipitate also started to appear throughout the agar med i um. Serolog ica 1 1 ^ Specific Electron Microscopy The SSEM techn ique developed by Derrick and Brlansky (1976) was adapted with great success to identify seed lots of cowpea infected with BICMV (Fig. 23). Virus particles were still observed in extracts from hypocotyl tissue which was mixed 1/bO (w/w) with noninfected hypocotyls (Fig. 2^4-6) This indicates that to test several seed lots by SSEM for the presence of BlCMV, germinated seeds can be divided Into groups of up to 50 equal pieces of individual hypocotyls. Each group of hypocotyl -pieces is ground in the extracting buffer and a drop of the extract is then examined by SSEM using antiserum sensitized grids. Virus particles will be observed in extracts obtained from those hypocotyl groups in which at least one hypocotyl is infected with BICMV. To illustrate the sensitivity of SSEM for detection of virus particles in hypocotyl extracts, the same Bl CMV-infected hypocotyl was used for four different electron microscopic preparations: a) SSEM; b) normal dip preparation on a carbon coated Formvar film supported in copper grids and negatively stained with 2% PTA; c) preparation similar to SSEM using normal serum instead of B 1 CMV-an t i serum ; and d) preparation similar to SSEM using grids with Parlodion film coated with carbon but treated with 0.05 M Tris buffer instead of antiserum (Fig. 23). The results showed a great increase of virus concentration on the grids sensitized with BICMV antiserum when compared with the

    PAGE 125

    Figure 23 Electron micrographs of BICMV in hypocotyl extracts from the same cowpea seedlings using different preparations. (a) serologically specific electron microscopy (SSEM) with BICMV antiserum diluted 1/1000 in 0.05 M Tris-HCl buffer, pH 7.2, and positively stained with uranyl acetate, (B) dip preparation with 1% phosphotungst ic acid pH 6.5, containing O.i;^ BSA, (C) preparation similar to SSEM using normal serum instead of Bl CMV-ant i serum, and (d) preparation similar to SSEM using Tris buffer instead of antiserum. The micrograph A represents a typical view of the entire grid whereas micrographs B, C, and D are selected areas of the grids showing virus part icles (arrows)

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    Figure 2k Electron micrographs of serologically specific electron microscopy vnth BICMV, BCMV-S, and CPMV. A) BICMV antiserum grid and extracts from a BICMVinfected cowpea hypocotyl ; B) BICMV antiserum grid and extracts from one gram of B I CMVinfected hypocotyl and ^3 g of healthy cowpea hypocotyls. Arrows point to virus particles; C) BCMV-S antiserum grid and leaf extract from BCMV-S infected bean; D) CPMV antiserum grid and leaf extract from CPMVinfected cowpea.

    PAGE 129

    116 other grid preparations (Fiq. 23). As the antiserum sensitized grids were washed several times during their preparation, a great reduction in the amount of pliinr debris on the grids was observed. On the other hand, when grids not previously treated with the antiserum were washed, the virus particles were also removed (Fig. 23-C, -D) The SSEM seemed to be also adequate to assay seeds for polyhedral viruses since it was successfully used to observe CPMV particles in cowpea leaf extracts using grids treated with antiserum specific for CPMV (Fig, 2h-D). Double immunodiffus ion Tests and SSEM for Detection of Other Viruses in Germinat ed Legu me Seeds The double immunodiffusion tests and SSEM used to detect BICMV in germinated cowpea seeds were also useful for detecting BCMV in infected germinated bean seeds and SoyMV in hypocotyls of germinated soybean seeds infected with SoyMV. Both legume viruses were detected by double immunodiffusion tests using hypocotyl extracts from groups of 5 seedlings and small discs of individual hypocotyls as assay antigens (Fig. 25). The results obtained in the simplified double immunodiffusion tests with hypocotyl discs were confirmed by SSEM, which also proved to be a very good technique to assay bean and soybean seed lots for the presence of BCMV (Fig. 26-A) and SoyMV (Fig. 26-B) respectively. The results obtained from the inoculation of the hypersensitive bean line VC-1822 with extracts from germinated bean seeds were also in agreement with those obtained by the immunochemical tests for BCMV. The percentages of BCMVinfected bean 'Black Turtle' seed lot and of

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    Figure 25 Double immunodiffusion tests with hypocotyls of ^-^-dayold bean and soybean seedlings using antiserum for BCMV-S and for SoyMV. A B) Serological tests with hypocotyl discs (A) and hypocotyl extracts (B) from healthy and BCMVBVI infected bean seedlings. The center wells were charged with: (As) BCMV-S antiserum, and (Ns) normal serum. The peripheral wells were filled with extracts from: (i) BCMV-BV-1inft'Cted hypocotyls, and (H) healthy bean hypocotyls. The hypocotyl discs were embedded directly into the agar medium and numbered from the top in a clockwise direction (arrows). Note a vi rus-ant iserum specific reaction with hypocotyl "1" in A. C D) Serological tests with hypocotyl discs (C) and hypocotyl extracts (D) from healthy and SoyMVinfected soybean seedlings. The center wells were charged with: (As) SoyMV antiserum, and (Ns) normal serum. The peripheral wells were filled with extracts from: (l) SoyMVinfected hypocotyls, (2) SoyMVinfected Nicotiana bentha miana leaves, (3) healthy N^. be nt ham i ana leaves, and (4) healthy soybean hypocotyls. Note a positive reaction with hypocotyl "l4" in C

    PAGE 131

    118

    PAGE 132

    f Figure 26 Electron micrographs of serologically specific electron microscopy with extracts from BCMVand SoyMVinfected hypocoty 1 s a) BCMV-S cjnt i serum-sens i t ized grid and extract from bean hypocoty 1 infected with BCMV-BV-l; B) SoyMV a(it iserum-sensitized grid and extract from SoyMVi iifec ted soybean hypocotyl

    PAGE 133

    120

    PAGE 134

    121 SoyMVinfected soybean 'Jupiter' seed lot were also estimated by the simplified double iivimunod i f f us ion test using hypocotyl discs (Table ll). Serology and Microsc o py of Cytoplasmic Inclusions Induced by BICMV and SoyMV in Hypocot yl s of Germinated Seeds Cytoplasmic inclusions induced by BICMV in cowpea and by SoyMV in soybean were detected by serology, light microscopy and electron microscopy in hypocotyls of A-5-day-old seedlings. In double immunodiffusion tests, specific precipitin lines were observed with the inclusion antisera and extracts of v i rusinfected hypocotyls but not with extracts from liealthy hypocotyls (Fig. 27). Light microscopic observations of epidermal strips of hypocotyls from BICMVinfected cowpea and SoyMVi nfected soybean seedlings readily revealed the presence of cytoplasmic inclusions (Figs. 28, 29). Groups of inclusion^) induced by BICMV and by SoyMV were abundant in cells of virus-infected hypocotyls (Figs. 28, 29), but were not observed in cells of noninfected hypocotyls. Pinwheels with scrolls were observed in ultrathin sections of hypocotyl tissues infected either with BICMV or SoyMV (Figs. 30, 31), A large number of longitudinal sections of pinwheel inclusions in hypocotyl tissues showed that they were abutted to the cell wall close to the p 1 asmodesmata (Fig. 31-C, -D) Similarly, tubes were observed in molybdate-treated extracts from hypocotyls infected with either virus.

    PAGE 135

    Figure 27 Double immunodiffusion tests with hypocotyl extracts from ^-5-day-old seedlings of cowpea (A) and soybean (B) using antisera for BICMV, SoyMV and their cytoplasmic inclusions. A) Serological test for detecting BICMV cytoplasmic inclusions (BlCMV-l) in hypocotyls of ^-5-day-old cov\/pea st-.edlings grown from B 1 CMVinfected seeds. The center wells were charged with: (Is) BlCMV-l antiserum, (Vs) BICMV antiserum, and (Ns) normal serum. The peripheral wells were charged with SDS-treaied extracts from: (l) Bl CMVinfected hypocotyls, and (H) healthy hypocotyls. B) Serological test for detection of SoyMV cytoplasmic inclusions (SoyMV-l) in hypocotyls of '4-5-day-old soybean seedlings grown from SoyMVinfected seeds. The center wells were charged with: (is) SoyMV-l antiserum, (Vs) SoyMV antiserum, and (Ns) normal serum. The peripheral wells were charged with SDS-treated extracts from: (1) SoyMVinfected hypocotyls, and (H) healthy soybean hypocotyls.

    PAGE 137

    Figure 28 Photomicrographs showing different views (A, B, C, D) of cytoplasmic inclusions (arrows) induced by BlCMV in epidermal strips of cowpea hypocotyl tissue stained with a combination of calcomine orange and luxol brilliant green. (Cl) cytoplasmic inclusions, (CW) plant cell wall, and (Nu) nucleus.

    PAGE 138

    125

    PAGE 139

    Figure 29 Photoinicrogrdphs showing different views (A, B, C, D) of epidermal cells of hypocotyls from 4-5-day-old soybean seedlings containing cytoplasmic inclusions (arrows) induced by SoyMV. The hypocotyl epidermal strips were stained with a combination of calcomine orange and luxol brilliant green. (Cl) cytoplasmic inclusions, (CW) plant cell wall, and (Nu) nucleus.

    PAGE 140

    127

    PAGE 141

    Figure 30 Electron mictagraphs of ultrathin sections of cells from hypocotyls ot '4-5-day-ol d cowpea seedlings inft;cted with BICMV. Note cross-sections (A, B, C) and longitudinal sections (D) of pinwheel inclusions induced by BICMV. (CW) plant cell wall, (IS) intercellular space, (m) mitochondrion, and (pw) pinwheel inclusions.

    PAGE 142

    129

    PAGE 143

    Figure 31 Electron micrographs of ultrathin sections of hypocotyl cells of 'l-S-day-ol d soybean seedlings grown from SoyMVinfected seeds. Note cross-sections (A, B) of the pinwheels and longitudinal views of the inclusions abutted to the plant cell wall at the p 1 asmodesmata (C, D) (CW) plant cell wall, (pi) plasmodesma, and (pw) pinwheel i nc 1 us ions

    PAGE 144

    131

    PAGE 145

    132 D i scuss io n Seed-transmiss if)n of plant viruses is of agricultural importance because efficient virus transmission in space and time can be provided through seeds of inff^cted host plants. The international exchange of several legume seeds is probably partly responsible for the worldwide distribution of important virus diseases of legumes. The occurrence of SoyMV in soybean cind BCMV in bean in most countries where these crops have been tested for viruses (Bos, 1971, 1972) are good examples to illustrate this point. According to Phatak (197^), several legumes are propagated in association with their seed-borne viruses on a wide scale in certain subiropical and tropical regions that lack organized seed certification and breeding programs. Brunt and Kenten (1973) explained the prevalence of cowpea mild mottle virus in cowpea in Ghana by the high rate of seed transmission in cowpeas and by the traditional practice of sowing st:eds from crops previously grown in the same region. The control of virus diseases of legumes through the production of virus-free seeds or seed lots with very low percentage of infected seeds has been advocated frequently (Fajardo, 1930; Thomas and Graham, 1951; Zettler and Evans, 1972; Baker, 1972; Phatak, 197^; Sinclair and Shurtleff, 1975; and Williams, 1975). The increase in percentage of infected seeds produced in crops with low initial infection and the efficient secondary spread of certain viruses into the growing crop from a small amount of primary inoculum randomly located throughout the planting, emphasize the importance of maintaining a low tolerance level in seed certification programs.

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    133 Several techniqiies have been developed for diagnosing seed-borne viruses in routine seed healtti testifig laboratories. Phatak (197'*) grouped those techniques into dry examination (visual inspection of dry seeds), biologicjl tests (growing-on and infectivity tests), biochemical tests (color imetric, h i stochemica 1 and serological tests), and biophysical tests (electron microscopy). According to Phatak {]S7h) the highly specific serological tests are the best among the tests available to a;.say seed lots for the presence of v i rusinfected seeds. Several serological tests have been used for detecting virusinfected seeds (Scott, 196l; Hamilton, 1965; Phatak, \S7^; Slack and Shepherd, 1975; Lund^gaard, 1976; and Lister, 1977). However, the present investigation contains the first successful results with double immunodiffusion tests for detecting potyviruses in germinated seeds. The use of hypocotyl tissue of germinated legume seeds eliminated the problem of nonspecific reaction commonly observed with extracts from seeds per se or germinated seeds, including roots, cotyledons, and primary leaves in immunodiffusion tests (Fig. 19). Cockbain et al. (1976) reported that when embryos of Vi cia faba L. minor seeds were tested by double immunodiffusion, the agar became clouded, obscuring the virus-specific precipitation lines. The nonspecific precipitates that interfered with the virus-specific reactions (Fig. 19) may be related to the high concentration of haemagg lut in ins {lectins) reported to be present in legume seeds (Toms and Turner, 1965; Moreira and Perrone, 1977; and Fountain and Yang, 1977). It has been demonstrated that lectins bind specifically to mono and polysaccharides as

    PAGE 147

    13^ well as to globulins of normal serum and to virus coat protein. Marshall and Nor ins (I965) showed that extracts of P. vulgaris seeds containing lectins precipitated a and 3 globulins of normal rabbit serum. Gumpf and Shannon (1977) demonstrated that a barley lectin interacted with purified BSMV i n^ v i n;o and formed insoluble aggregates that greatly reduced the virus inPectivity. Phatak (197^) assumed that the high content of lectin in soybean seeds was responsible for the unsatisfactory results of the passive haemagg I ut i na t ion test with SoyMV in seed extracts. Thus, the absence of nonspecific reactions with extracts of hypocotyl tissue in contrast to the nonspecific precipitates observed with extracts of rooi or cotyledons and primary leaves (Fig. 19) of A-5-day-old legume s.-edlings may be an indication that a very low content of lectin is present in the hypocotyl. In support of the use of germinated seed for serology, instead of seed £er se, is the fact that if any seed certification program or seed producers will assay for the presence of v i rusinfected seeds, they will also test some other seed properties such as percentage of seed germination. Consequently, the germinated seeds could be used in a concomitant serological indexing program. The sensitivity of the double immunodiffusion test indicates that germinated seeds can be divided into groups of 5 to 30 seedlings and precipitin reactions will be observed with those groups containing at least one infected seedling (Fig. 21). Immunochemical tests with hfgher sensitivity have been also used for indexing v i rusinfected seeds. The extremely sensitive enzymeI i nked innunosorbent assay (ELISA) (Voller et aL, 1976; and Clark and Adams, 1977) was used to

    PAGE 148

    135 detect TRSV and SoyMV in individual soybean seeds (Lister, 1977). The high sensitivity of SSEM for detection of particles of BICMV, BCMV, and SoyMV in infected hypocotyl extracts as well as of other seed-borne viruses in seed extracts (Brlansl
    PAGE 149

    136 infected with alfalfa mosaic virus could be detected only U days after germination and symptom expression was not reliable enough for the estimation of percentage of v i rusi nf ected seeds. Phatak et al. (1976) used the ind icatorinocu lat ion test in association with the growing-on test to estimate the percentage of cowpea seeds infected with cowpea ringspot virus because infected seedlings were either symptomless or the symptoms were very mild. In assessing the proportion of V. faba i^inor seeds infected with broad bean stain virus, Cockbain et al. (1976) found that under high temperature the symptoms were often mild and evanescent, and v i rusinfected seedlings remained without obvious symptoms for more thjn 6 weeks. Thus, the quantitative estimation of vi rusinfected seeds of leguminous crops by the growing-on test may often be difficult due to symptomless infections. On the other hand, the reliability and :.implicity of the hypocotyl -disc-double immunodiffusion test make it highly suitable for commercial certification programs. Observations during the course of this research indicated that a trained person can set up approximately 50-60 hypocotyl discs per hour in an agar plate to be tested by this technique. This simplified double immunodiffusion technique does not require tissue grinding, antigen wells, and chemical treatment of the antigen prior to incorporation into the agar matrix. The single radial immunodiffusion test also proved to be satisfactory for detection of BICMV in cowpea hypocotyls. These results provide another option for a routine seed health testing program. Single radial immunodiffusion in mu I t ipl e-ant i sera media could be used for indexing legume seeds for more than one virus. As shown in the

    PAGE 150

    first part of this research, an agar medium impregnated with a mixture of antisera was used for serod iagnos i s of two morphologically distinct viruses in cowpea (F i
    PAGE 151

    LITERATURE CITED Abo El-Nil, M. M., F. W Zettler. and E. Hiebert. 1977. Purification, serology, and some physical properties of dasheen mosaic virus Phytopathology 67: \^^S--]^50. Abrygunawardena, D. V. W. and S M. D. Perera. 196^ Virus diseases foI^?oI"^ Trop. Agric. Mag. Ceylon Agric. Soc. 120: I b 1 -204 Afanasiev, M. M. 1956. Occurrence of barley stripe mosaic in Montana. Plant Dis Rep. 'tO: ]k2. Agrawal, H. 0. 196't Identification of cowpea mosaic virus isolates Mededel. van de Landbouth Wageningen, Nederland 6^4: 1-53. Alba, A. P. C, and A. R. Oliveira. 1977, Serological studies on viruses of the potato virus Y group occurring in Sao Paulo. Summa Phytopathologica 2:178-186. Alconero, R. and A. Santiago. 1973. Phaseolus lathyroides as a reservoir of cowpea virus in Puerto Rico. Phy topaThoT^gy 63:120Anderson C. W. 1955a. Vigna and Crotalaria viruses in Florida I Preliminary report on a strain of cucumber mosaic virus obtained from cowpea plants. Plant Ois. Rep. 39 : 3't6-3i8 Anderson, C. W. 1955b. Vigna and Crotalaria viruses in Florida II Notations concerning cowpea mosaic virus (Marmor Vignae) Plant Dis. Rep, 39:3'9-352. Anderson, C. W. 1955c. Vigna and Crotalaria viruses in Florida III Notations concerning identification difficulties, indicator plants possible vector relationships, and virus maintenance. Plant Dis. Rep. 36:354-357 Anderson C. W. 1957^ Seed transmission of three viruses in cowpea. Phytopathology ^7:515 (Abstr ). Anderson. C. W. 1959. Vigna and Crotalaria viruses in Florida V Comparative transmission tests with aphids and beetles. Phytopathology itg; 117-1 18 ^ Andrews J. h. and T. A, Shalla 197^, The origin, development, and conformation of amorphous inclusion body components in tobacco etch VMus-infected cells. Phytopathology 6/( : 1 23^)1 2^3 138

    PAGE 152

    139 Athow, K. L., and J. B. Bancroft. 1959Development and transmission of tobacco ringspot virus in soybean. Phytopathology '9:697-701. Baker, K. F. 1972. Seed pathology. Pages 317'-'l6 in^T. T, Kozlowski, ed. Seed Biology Vol. II. Academic Press, New York, ''7. Bancroft, J, B, 1970. Brome mosaic virus. No, 3 in Descriptions of plant viruses. Commonw. Mycol Inst., Assoc. Appl. Biol, Kew Surrey, England. k p. Bancroft, J. B. 1971. Cowpea chlorotic mottle virus. No. kS in Descriptions of plane viruses. Commonw. Mycol, Inst., Assoc. Appl. Biol., Kew, Surrey, England, k p. Barnett, 0. W. and M. Alper. 1977. Characterization of Iris fulva virus. Phytopathology 67;'t'*8-'45'4. Batchelor, D. L. 197'*Immunogen ic i ty of sodium dodecyl sulfate denatured plant viruses and paint viral inclusions. Ph.D. Dissertation, University of Floridc), Gainesville, 8| p, Beier, H. D J, Siler, M. L, Russell, and G, Bruening. 1977. Survey of susceptibility to cowpea mosaic virus among protoplasts and intact plants from Vign a s inens i s lines. Phytopathology 67:917-921. Bennett, C. W. I966 Seed transmission of plant virus. Pages 221261 J_n K. M. Smith and M, A Lauffer, eds. Advances in virus research, Vol. I'*, Academic Press, New York. 350 p. Bercks, R. i960. Serological relationships between beet mosaic virus, potato virus Y, and bean yellow mosaic virus. Virology 12:311-313, Bock, K. R. 1973East African strains of cowpea aphid-borne mosaic virus. Ann. Appl. Biol. 7'<;75-83. Bock, K, R. and M. Conti. 197'^. Cowpea aphid-borne mosaic virus. No. h]^ Descr ipt ions of plant viruses. Commonw, Mycol, Inst., Assoc. Appl. Biol,, Kew, Surrey, England, k p. Bos, L 1971. Bean common mosaic virus. No. 73 in Descriptions of plant viruses. Commonw. Mycol, Inst,, Assoc. Appl. Biol., Kew, Surrey, England, k p. Bos, L. 1972. Soybean mosaic virus. No. 93 j_n Descr i pt ions of plant viruses. Commonw. Mycol. Inst., Assoc, Appl. Biol., Kew, Surrey England, k p. Bos, L. and D. Z. Maat. 197'i. A strain of cucumber mosaic virus, seed-transmitted in beans. Neth, J. PI. Pathol. 80:113-123. Brandes, J. 1964. I den t i f i z ierung von gestreckten pf 1 anzenpa thogenen viren auf morpholog i scher Grundlage. Mitt. biol. BundAnst, Ld u. Forstw. 110:1-130.

    PAGE 153

    Brantley, B. B. C. W. Kuhn, and G. Sowell. 1965. Effect of cucumber mosaic virus on southern pea (Vigna^ s i nens i s). Proc. Am. Soc. Hortic. Sci. 87:355-358. Brierley, P., and F. F. Smith. 1962. Three cowpea mosaic viruses from gladiolus. Plant Dis. Rep. '6:335-337. Brlansky, R. H. and K. S. Derrick. 1976. Detection of seed-borne plant viruses using serologically specific electron microscopy. Proc. Am. Phytopafhol. Sor,. 3;33'4-335 (Absrr.). Brunt, A. A. 197^Tropical leguminous crops. Glasshouse Crop Research Institute. Annual Report. 197** (Abstr.). Brunt, A. A., and R. H. Kenten. 1973. Cowpea mild mottle, a newly recognized virus infecting cowpeas (Vigna unguiculata ) in Ghana Ann. Appl. Biol. 7'<:67-7'. Brunt, A. A., and R. H. Kenten. 197^Cowpea mild mottle virus. No. 1^0 jji^ Descr ipt ions of plant viruses. Comnionw. Mycol. Inst., Assoc. Appl. Biol., Kew, Surrey, England, k p. Burkholder, W. H. and A. S. Muller. 1926. Hereditary abnormalities resembling certain infectious diseases of beans. Phytopathology 16:731-737. Camargo, I. J. B. E. W. Kitajima, and A. S. Costa, 1968. Estudo ao microscdpio electronico de tecidos de planras infetadas pelo virus do mosaico comum e mosaico amarelo do feijoeiro. Bragantia 27:'*09-'15. Garner, J., K. S i 1 berschmidt and E. Flores. 1969. Ocorroncia do virus do mosaico da Vigna no Estado de Sio Paulo. 0 Blologico 35: 13-16. Casper, R. 197'*. Serodiagnos is of plum pox virus. Acta Hort icul turae i*'*: 171-172. Chant, S. R. 1959. Virus of cowpea, V igna ungu icu lat a L. (Walp.) in Nigeria. Ann. Appl. Biol. 47:5iB5-572. ~ Chant, S. R. I960. The effect of infection with tobacco mosaic and yellow mosaic viruses on the growth rate and yield of cowpea in Nigeria. Empire J. Exp. Agric. 28:ll'*-120. Chant, S. R. 1962. Further studies on the host range and properties of Trinidad cowpea mosaic virus. Ann. Appl. Biol. 50:159-162. Chenulu, V. V., J. Sachch i dananda and S. C. Mehta. 1968. Studies on a mosaic disease of cowpea from India. Phytopathol Z. 63:38l387.

    PAGE 154

    Cheo, P. C. 1955Effect of seed maturation on inhibition of southern bean mosaic virus in bean. Phytopathology ^45: 17-21, Christie, R G. 1967. Rapid staining procedures for differentiating plant virus inclusions in epidermal strips. Virology 31:268-271. Christie, R G. and J R. Edwardson. 1977. Light and electron microscopy of plant virus inclusions. Florida Agric. Exp. Stn. Monogr. Ser. 9, 150 p Christie, S, R. and W. E. Crawford. 1977Plant virus range of N icot iana benthamiana. Plant Ois, Rep. (in press). Clark, M. F. and A N. Adams. 1977. Characteristics of the microplate method of enzyme1 inked immunosorbent assay for the detection of plant viruses. J. Gen. Virol. 3^:^7S-h83. Clifford, H. T. 1977Immunodiffusion techniques for the detection of the Cymbidium mosaic and Odontog lossum ringspot viruses of orchids. M. S. Thesis, University of Florida, Gainesville, 63 p. Cockbain, A. J., R. Bowen and S. Vorra-Urai. 1976. Seed transmission of broad bean stain virus and Echtes ackerbohnenmosa i k-v i rus in field bean (V ic ia faba) Ann. Appl. Biol 8^4:321-332. Corbett, M. K. 1956. Serological and morphological relationships of plant viruses. Florida Univ. Agric. Exp. Stn. Annu. Rep., p. 1171 1 8 Crowley, N. C. 1957. Studies on the seed transmission of plant virus diseases. Aust. J. Biol. Sc i ]0 :kkS-^\(>k Crowley, N. C. 1959. Studies on the time of embryo infection by seedtransmitted viruses. Virology 8:116-123. Dale, W. T. 19^9. Observations on a virus disease of cowpea in Trinidad. Ann. App. Biol. 36:327-333. Dale, W. T. 1953. The transmission of plant viruses by biting insects, with particular reference to cowpea mosaic. Ann. Appl. Biol. hO: 385-392. Debrot, A. E. and C. E. Benitez de Rojas. 1967. I dent i f i cac ion del virus del mosaico de la soya en Venezuela. Agron. Trop. 17:75-86. Demski, J. W. and H. B. Harris. 197'*. Seed transmission of viruses in soybean. Crop Sci. 1^:888-890. Derrick, K. S., and R. H. Briansky. 1976. Assay for virus and mycoplasmas using serologically specific electron microscopy. Phytopathology 66:815-820.

    PAGE 155

    142 Desjardins, F. K. R. L. Latterell, and J. E. Mitchell. \S5^. Seed transmibsion of tobacco-r ingspot virus in Lincoln variety of soybean Phytopathology h^:8S, Doi, Y., M. U. Chanc), and K. Yora. 1977Orchid fleck virus. No. 183 in Descriptions of plant viruses. Conimonw. Mycol. inst., Assoc. Appl. Biol., Kew, Surrey, England. 3 p. Drijfhout, E., and L. Bos. 1977. The identification of two new strains of bean common mosaic virus. Neth. J. PI. Pathol. 83:13-25. Edwardson, J. R. 197^. Some properties of the potato virus-Y group. Fla. Agric. Exp Stn, Monogr. Ser, ^. 398 p. Edwardson, J. R. D, E. Purcifull, and R. G. Christie. 1968. Structure of cytoplasmic inclusions in plants infected with rod-shaped viruses. Virology 3^:250-263. Edwardson, J. R. F, W. Zettler, R. G, Christie, and I. R. Evans. 1972. A cytological comparison of inclusions as a basis for distinguishing two filamentous legume viruses. J. Gen. Virol. 15:113-118. Ekpo, E. I. A., and A W Saettler. 197^4 Distribution pattern of bean common mosaic virus in developing bean seed. Phytopathology 6'4:269270. Elliot, J. A. 1921 A mosaic of sweet and red clovers. Phytopatholoov ll:U6-li(8. ^ Fajardo, T. G 1930. Studies on the mosaic disease of the bean. Phytopathology 20 : '469-'49A Fenner, F. 1976. Classification and nomenclature of viruses. InterV i rology 7:^-115. Fischer, U and B, E. Lockhart. 1976a. A strain of cowpea aphidborne mosaic virus isolated from cowpeas in Morocco. Phytopathol Z. 85:A3-i*8. Fischer, H. U., and B. E. Lockhart. 1976b. A strain of cucumber mosaic virus isolated from cowpeas in Morocco. Phytopathol. Z. 85:132-13C. Fountain, D, W. and W Yang. 1977. Isolectins from soybean ( Glycin e max) Biochhm. Biophys. Acta ^92:176-185. Fulton, R. W 196*4. Transmission of plant viruses by grafting, dodder, seed, and mechanical inoculation. Pages 39-67 in M. K. Corbett, and H. D. Sisler, eds. Plant Virology. Univ.. PTorida Press, Gainesville, 527 p,

    PAGE 156

    1^3 Gardner, W. 1927 Seed transmission of cowpea mosaic Proc Ind Acad. Sci. A3. Gardner, M. W. and j. B. Kendrick. 1921. Soybean mosaic. J. Aqric Res. 22: m-l 1/4 ^ Gay, J. D. 1969. Effect of seed maturation on the infectivity of cowpea chlorotic mottle virus. Phytopathology 59:802-80'i. Gay, J. D., and E. E. Winstead. 1970, Seed-borne viruses and fungi from southern pea seed grown in eight states. PKint Dis Reo 5A:2/*3-2it5. Ghanekar. A. M. and F. W. Schwenk. 197^. Seed transmission and distribution of tobacco streak virus in six cultivars of soybeans Phytopathology G^:]]2-]]h. Gibbs, A. J. 1972. Broad bean mottle vi rus. No. 101 in Description of plant viruses Commonw. Mycol. Inst., Assoc. Appl. Biol Kew, Surrey, England. ^ p. Govier, D. A., and R. D, Woods, 1971. Changes induced by magnesium ions in the morphology of some plant viruses with filamentous particles. J. Gen. Viol. 13:127-132. Granett, A. L. and T. A. Shalla. 1970. The relation of tobacco mosaic virus X-protein to amorphous cellular inclusions (X-bodies) Phytopai hology GO : h]S-^2k Grogan, R. G., J. E. Welch, and R. Bardin. 1952. Common lettuce mosaic ?2 573!57r"'' ^ mosaic-free seed. Phytopathology Gumpf, D.J., and L M. Shannon. 1977. Interaction of barley stripe mosaic virus and barley lectin. Proc. Am. Phytopathol Soc k (in press) (Abstr ) Hamilton, R. I. 1965. An embryo test for detecting seed-borne barley stripe mosaic virus in barley. Phytopathology 55:798-799. Hampton, R. 0., S. Phillips, J. E. Knesek, and G. I. Mink 1973 Ultrastructural cytology of pea leaves and roots infected by pea seed-borne mosaic virus. Arch. Ges. Virusforsch. lil :2k2-2S3. Haque, S. Q. and G. C, Persad 1975, Some observations on the seedtransmission of beetle-transmitted cowpea mosaic virus. Pages iiy-i^l in J. Bird, and K. Maramorosh eds. Tropical Diseases of Legumes. Academic Press, New York, I71 p. Harrison, A. N.. and R. T. Gudauskas.. 1968a, Identification of viruses isolated from cowpeas in Alabama. Plant Dis, Rep. 52:

    PAGE 157

    Harrison, A. N., and R. T. Gudauskas. 1968b. Effects of some viruses on growth and seed production of two cowpea cultivars Plant Dis. Rep. 52:509-511. Hiebert, E., and J. G. McDonald. 1973. Characterization of some proteins associated with viruses in the potato Y group. Viroloqv 56:3')9-36l. Hiebert, E. and J. G. McDonald. 1976. Capsid protein heterogeneity in turnip mosaic virus. Virology 70:1'4^-150. Hiebert, E. D. E. Purcifull, R.. G. Christie, and S. R. Christie. 1971. Partial purification of inclusions induced by tobacco etch virus and potato virus Y. Virology '(3 : 638-6'*6 Rollings, M. and 0. M. Stone. 1965Studies of Pelargonium leaf curl virus. II. Relationships to tomato bushy stunt and other viruses. Ann. Appl. Biol, 56:87-98. lizuka, N. 1973. Seed transmission of viruses in soybean. Tohoku Natl. Aqric. Exp. Stn. Bull. ^6 : 1 3 1 1 I lizuka, N. lS7k. Southern bean mosaic virus in soybean. Plant Protection 28:471-'t7'4 lizuka, N., and T. Yunoki. 197'*. Peanut stunt virus isolated from soybean, Glycine max Merr. Tohoku Agric. Exp. Stn Bull kl 1-12. Inouye, T. 1973. Characteristics of cytoplasmic inclusions induced by bean yellow mosaic virus. Nogaku Kenkyu 5': 155-171. Jones, R.T., and S. Diachun. 1977. Serologically and biologically 83^838^ ^^^'^ yt^I low mosaic virus strains. Phytopathology 67: Kahn, R. P. 1956. Seed transmission of the tomato-ring spot virus in the Lincoln variety of soybean. Phytopathology ^6:295. Kaiser, W. J., D. Danesh, M. Okhovat, and G. H. Mossahebi. I968. Diseases of pulse crops (edible legumes) in Iran. Plant Dis Rep. 52:687-691. Kaiser, W. J. and G. H. Mossahebi. 197^. Natural infection of mungbean by bean common mosaic virus. Phytopathology 6^* : I 2091 2 1 Kaiser, W. J., and G. H. Mossahebi. 1975. Studies with cowpea aphidborne mosaic virus and its effect on cowpea in Iran. FAO Plant Protection Bui 1 23:33-39.

    PAGE 158

    1^5 Karnovsky, M. J. I965. A formaldehyde-glutaraldehyde fixative of high osmolality for the use in electron microscopy. J. Cell Biol. 27:137A. Kendrick, J. B. and M. W. Gardner. 192'*. Soybean mosaic: seed transmission and effect on yield. J. Agr. Res. 27:91-98. Kennedy, B. W. and R. L. Cooper. 1967. Association of virus infection with mottling of soybean seed coats. Phytopathology 57:35-37. Khatri, H. L. and L. Singh. 197^. Studies on a mosaic disease of cowpea. J. Res. 11:289-29'*. Klesser, P. J. i960. Viruses diseases of cowpea. Bothalia 7:233-251. Koenig, R. and L. Givord. 197^4. Serological interrelationships in the turnip yellow mosaic virus group. Virology 58:119-125. Koshimizu, S. and F. lizuka. 1957Relationship between the brown speck of soybean seed and soybean mosaic. Ann. Phytopathol Soc. Japan 22 : 18 (Abstr ) Koshimizu, Y., and H. lizuka. I963. Studies on soybean virus diseases in Japan. Tohoku Natl. Agric. Expt. Stn. Bull. 27:1-103. Kuhn, C. W. 196'4a. Separation of cowpea virus mixtures. Phytopathology 5'<:739-7'40. Kuhn, C. W. \'-jGkh. Purification, serology, and properties of a new cowpea virus. Phytopathology 5^4:853-857. Kuhn, C. W. B. B. Brantley, and G. Sowel 1 I965. Immunity to bean yellow mosaic virus in cowpea. Plant Dis. Rep. ^49:879-881. Kuhn, C. W. B. B. Brantley, and G. Sowel 1. 1966. Southern pea viruses: identification, symptomatology, and sources of resistance. Univ. Georgia Agric. Exp. Stn. Bull, I57. 22 p. Lima, J. A. A., and M. R. Nelson. 1975Squash mosaic virus variability: nonreciprocal cross-protection between strains. Phytopathology 65:837-8/40. Lima, J. A. A., and M. R. Nelson. 1977. Etiology and epidemiology of mosaic of cowpea in Ceara, Brazil. Plant Dis. Rep. 61:86^4-867. Lima, J. A. A. and D. E. Purcifull. 1977a. Immunochemical tests for detection of blackeye cowpea mosaic virus in infected seeds. Proc. Am. Phytopathol. Soc. 4 (in press) (Abstr.). Lima, J. A. A., and D. E. Purcifull. 1977b. Serology and microscopy of cylindrical inclusions induced by blackeye cowpea mosaic and soybean mosaic viruses in hypocotyls of germinated seeds. Proc. Am. Phytopathol. Soc. ^4 (in press) (Abstr.).

    PAGE 159

    Lima, J. A. A., D. E. Purcifull, and E. Hieberr. 1976. Purification and serology of blackeye cowpea mosaic virus. Proc. Am. Phytopathol Soc. 3:2^8 (Absrr.). Lima, J. A. A., D. E. Purcifull, and R. Sonoda. 1977. Some properties of a potyvirus isolated from siratro in Florida Proc. Am. Phytopathol. Soc. ^4 (in press) (Abstr.). Lima-Neto, V. C, and A. S. Costa. 1976. Transmissao comparative do vfrus do mosaico comun da soja por sementes com manchas-cafe e nao manchddas. Fi topatolog ia 11;20 (Abstr.). Lister, R. M. I960. Transmission of soil-borne viruses through seed Virology 10:5^7-5'*9. Lister, R. M. 1977. Detection of viruses in soybean seed by enzymelinked immunosoi bant assay. Proc. Am. Phytopathol. Soc. ^ (in press) (Abstr.) Lister, R. M., and A F. Murani. 1967. Seed-transmission of nematodeborne viruses. Ann. Appl Biol. 53:^3-62. Lister, R. M., and J M. Thresh. 1955. A mosaic disease of leguminous plants caused by a strain of tobacco mosaic virus. Nature 17510'47-10'i8. Lovisolo, 0., and M, Conti. 1966. Identification of an aphidtransmitted cowpea mosaic virus. Neth. J. PI. Pathol. 72:265-269. Lundsgaard, T. 1976 Routine seed health testing for barley stripe mosaic virus in barley seeds using the latex-test. J. Plant Dis Protect ion 83:2/8-283. Markham, R. I960. A graphical method for rapid determination of sedimentation coefficients. Biochem. J. 77:516-519. Marshall, W. H and L. C. Norins. 1965. Antigenic properties of extract of Phast-.olus vu lgaris seeds (phytohaemaggi ut in in) routinely used in leucocyte cultures. Aust. J. Exp. Biol. Med. Sci ^43213-228. Martelli, G. P., and M. Russo. 1977. Plant virus inclusion bodies Pages 175-266 hi M. A. Lauffer, F. B. Bang, K. Maramorosch, and K. M. Smith, eds. Advances in virus research, Vol. 21. Academic Press, New York. ^03 p. Matthews, R. E. F. 1970. Plant Virology. Academic Press, New York. 77o p. McDonald. J. G. and R. I. Hamilton. 1972. Distribution of southern bean mosaic virus in the seed of Phaseolus vulgaris Phvtopathology 62:387-389. ^ ^

    PAGE 160

    1^7 McDonald, J. G. and E. Hiebert. 1975. Characterization of the capsid and cylindrical inclusion proteins of three strains of turnip mosaic virus. Virology 63:295-303. McLean, D. M. 1941. Studies on mosaic of cowpea Vigna sinensis. Phytopathology 3 1 : 420-'30. Medina, A. C, and R. G. Grogan. 1961. Seed transmission of bean mosaic viruses. Phytopathology 51:452-'456. Milne, R. G., and E. Luisoni. 1977. Rapid immune electron microscopy of virus prepar.Jtions. Pages 265-281 |n K. Maramorosch, and H. Kiprowski, eds. Methods in Virology, Vol. VI. Academic Press New York. 5^2 p. Morales, F. J., and F. W. Zettler. 1977. Characterization and electron microscopy of a potyvirus infecting Commel ina diffusa. Phytopathology 67:839-8i)3. Moreira, R. de A., and J. C. Perrone. 1977. Purification and partial characterization of a lectin from Phaseolus vulgaris. Plant Physiol. 59; 783-787. Nariani, T. K. and T. K. Kandaswany. 1961. Studies on a mosaic disease of cowpea (Vigna sinensis Savi). Indian Phytopathol 1077-82. Nelson, R. and E. E Down. 1933. Influence of pollen and ovule infection in seed transmission of bean mosaic. Phytopathology 2325 (Abstr.). Nelson, M. R. and H. K. Knuhtsen. 1973. Squash mosaic virus variability: review and serological comparison of six biotypes. Phytopathology 63:920-926. Nicolaescu, M. H. Titu, and M. Paraschiv. 1976. Electron-microscopical investigations on the pinwheel structures detected in soybean mosaicvirus infected plants. Rev. Roum. Biol. Biol. Veg. 21:67-69. Ouchterlony, 0. 1962. Di f fus ionin-gel methods for immunological analysis II. Prog. Allergy 6:30-15'). Owusu, G. K. N. C. Crowley, and R. I. B. Francki. I968. Studies of the seed-transmission of tobacco ringspot virus. Ann. Appl. Biol 61:195-202. Padma, R. and A. S. Summanwar. 1973. Che nopodium murale a differential host for cowpea mosaic virus. Curr. Sci. i2 -620. Perez J. E. and A. Cortes-Monl lor. 1970. A mosaic virus of cowpea from Puerto Rico. Plant Dis. Rep. 5'<:212-2l6.

    PAGE 161

    1A8 Phatak, H. C. 197'*. Seed-borne plant virus identification and diagnosis in seed health testing. Seed Sci. Techno). 2:3-155. Phatak, H. C, J. R. Diaz-Ruiz, and R. Hull. 1976. Cowpea ringspot virus: a seed transmitted cucumov i rus Phytopathol. Z. 87:132Pierce, W. H., and C. W. Hungerford. 1929. A note on the longevity of the bean mosaic virus. Phytopathology 19:605-606. Porto, M. D. M., and D. 1. Hagedorn. 1975. Seed transmission of a Brazilian isolate of soybean mosaic virus. Phytopathology 65: 713-716. Provvidenti, R. and E. D. Cobb. 1975. Seed transmission of bean common mosaic virus in tepary bean. Plant Dis. Rep. 59:966-969. Purcifull, D. E. 1966. Some properties of tobacco etch virus and its alkaline degradation products. Virology 29:8-1'*. Purcifull, D. E., and D. L. Batchelor. 1977. Immunodiffusion tests with sodium dodecyl sulfate (SDS) -treated plant viruses and plant viral inclusions. Univ. Florida Agric. Exp. Stn. Bull. No. 788 (Tech.). 39 p. Purcifull, D. E., S, R. Christie, and D. L. Batchelor. 1975. Preservation of plant virus antigens by f reeze-dry ing Phytopathology 65:1202-1205. Purcifull, D. E., J. R. Edwardson, and S. R. Christie. 1970. A morphological comparison of inclusions induced by tobacco etch and potato Y viruses. Phytopathology 60:779-803. Purcifull, D. E., and G. V. Gooding. 1970. Immunodiffusion tests l^^^potato Y and tobacco etch viruses. Phytopathology 60:1036Purcifull, D. E., E. Hiebert, and J. G. McDonald. 1973Immunochemical specificity of cytoplasmic inclusions induced by viruses in the potato Y group. Virology 55:275-279. Purcifull, D. E., and R. J. Shepherd. 196i. Preparation of the protein fragments of several rod-shaped plant viruses and their use in agar-gel diffusion tests. Phytopathology 5^4:1102-1108. Quacquarelli, A., and A. Avgelis. 1975Nicotiana bent ham i ana Domin. as host for plant viruses. Phytopathol. Medit. 1^:36-39. Raheja, A. K. and 0. I. Leleji. 1974. An aphid-borne virus disease of irrigated cowpea in Northern Nigeria. Plant Dis. Rep. 58:1080I OoH

    PAGE 162

    Reddick, D. and V. B. Stewart. 1919. Transmission of virus of bean mosaic in seed and observations on tiiermal death-point of seed and virus. Phytopathology 9:^1^)5-^50. Richter, J., J. Polak, and E. Proll. 1975. Ausarbeitung von Verfahren zum Serolog ischtn sche 1 1 nachwe i s des Gu rkenmosa i k-v i rus in naturlich infizierten krautigen Pflanzen. Arch. Phytopathol. v. Pf anzenschutz 1 1 :307-3l8. Robertson, D. G. 1965. The local lesion reaction for recognizing cowpea varieties immune from and resistant to cowpea yellow mosaic virus. Phytopathology 55:923-925. Ross, J. P. 1963. Interaction of the soybean mosaic and bean pod mottle viruses infecting soybeans. Phytopathology 53:887 (Abstr.). Ross, J. P. 1968. Effect of single and double infections of soybeans with soybean mosaic and bean pod mottle viruses on yields and seed characters. Plant Dis. Rep. 52:3kk-}k8. Ross, J. P. 1969. Effect on time and sequence of inoculation of soybeans with soybean mosaic and bean pod mottle viruses on yields and seed characters. Phytopathology 59 : 1 40'1 i08 Ross, J. P. 1970. Effect of temperature on mottling of soybean seed caused by soybean mosaic virus. Phytopathology 60 : 1 7981 8OO Rui, D. i960. Apporto alle conoscenze sulle virosi dei vegetal i. Informatore Agrac. Veroma 23:15-17. Schippers, B. 1 963. Transmission of bean common mosaic virus by seed of Phaseolus vulgaris L. cultivar Beka. Acta Bot. Neerl. 12:^33^97. Scott, H. A. 1961. Serological detection of barley stripe mosaic virus in seeds and in dehydrated leaf tissue. Phytopathology 51200-201. Sharma, S. R. and A. Varma. 1975. Cure of seed transmitted cowpea banding mosaic viruses. Phytopathol. Z. 83:l4it-151. Sheffield, F. M. L. 19^1. II. The cytoplasmic and nuclear inclusions associated with severe etch virus. J. Roy. Microscop. Soc. 6130-45. Shepard, J. F. 1969Serod iagnos i s of PVX in potato tuber sprouts. Plant Dis. Rep. 53:845-848. Shepard, J. F. 1970. A radial immunodiffusion test for the simultaneous diagnosis of potato viruses S and X. Phytopatholoqy 601669-1671.

    PAGE 163

    150 Shepard, J. F. 1972. Gel -d i f f us ion methods for the serological detection of potato viruses X. S, and M. Montana Agric. Exp. Stn. Bull. No. 662. 22 p. Shepard, J. F. J. W. Jutila, J. E. Catlin, F. S. Newsman, and W. H. Hawkins. 1971. Immunodiffusion assay for potato virus M infection Phytopathology 61 :873-87'4. Shepard, J. F. G. A. Secor, and D. E. Purcifull. 197^. Immunochemical cross-reactivity between the dissociated capsid proteins of PVY group plant viruses. Virology 58 : '46^-^*75 Shepherd, R. J. I963. Serological relationship between bean pod mottle virus and cowpaa mosaic viruses from Arkansas and Trinidad. Phytopathology 53:865-866. Shepherd, R. J. 196'4. Properties of a mosaic virus of cowpea and its relationship to the bean pod mottle virus. Phy topatholoriy S^: i66-^73. Shepherd, R. J. 1971. Southern bean mosaic virus. No. 57 in Descriptions of plant viruses. Commonw. Mycol Inst., Assoc~Appl. Biol., Kew, Surrey, England. ^4 p. Shepherd, R. j. 1972. Transmission of viruses through seed and pollen. Pages 267-292 iji C Kado, and H. 0. Agrawal, eds. Principles and Techniques in Plant Virology. Van Nostrand Reinhold Company, New York. 688 p. Shepherd, R. J., and R. W. Fulton. I962. Identity of a seed-borne virus of cowpea. Phytopathology 52:^89-493. Shepherd, R. J., and G. S. pound. i960. Purification of turnip mosaic virus. Phytopathology 50:797-803. Shepherd, R. J., and D. E. Purcifull. 1971. Tobacco etch virus. No. 55 J_n Descriptions of plant viruses. Commonw. Micol. Inst., Assoc. Appl. Biol., Kew, Surrey, England, k p. Sinclair, J. B. and M. C. Shurtleff. 1975. Compendium of soybean diseases. Am. Phytopathol Soc. St. Paul, Minnesota. 69 p. Slack, S. A., and R. J. Shepherd. 1975. Serological detection of seed-borne barley stripe mosaic virus by a simplified radialdiffusion technique. Phytopathology 65:9'*8-955. Smith, C. E. 1924. Transmission of cowpea mosaic by bean leaf-beetle Science 60:268. Smith, F. L., and W. B. Hewitt. 1938. Varietal susceptibility of common bean mosaic and transmission through seed. Calif Aqric Exp. Stn. Bull. 612:3-18. • y •

    PAGE 164

    151 Snyder, W. C \Sm. A seed-borne mosaic of asparagus bean, V i gna s esqui pedal is Phytopathology 32;5)8-523. ~ Takahashi, K, Y. Tanaka, and Y. Tsuda, 197^. Soybean mild mosaic virus. Ann. Phytopathol. Soc. Japan. kO:\Q3-]OS. Tamada, T. 19/7, The virus diseases of soybean in Japan. Food Fertilizer Technology Center. Tech, Bull. 33. II p, Thomas. W, D., Jr,, and R. W. Graham. 1951, Seed transmission of red node in Pinto bean. Phytopathology '1:959-962. Toler, R. W. S, S. Thompson, and J. M. Barber. 1963. Cowpea (southern pea) diseases in Georgia, 1961-62, plant Dis, Rep, hi Jhb-'Jkl Toms, G, C, and T, 0. Turner. 1965. The seed haemagg I u t in i ns of some PhasejDjus^ vo^ljaris L. cultivars, J. Pharm. Pharmacol. 17: I I8S-125S Tosic, M., R, E. Ford. H. E. Moline, and D. E. Mayhew, 197^. Comparison of techniques for pur i f i ca t ion of maize dwarf and sugarcane mosaic viruses. Phytopathology 6'*;439-'('j2 Tosic, M., and Z. Peiic, 1975, Investigation of alfalfa mosaic virus transmission through alfalfa seed. Phytopathol. Z. 83:320-327. Tremair.e, J. H., and N. S. Wright. I967. Cross-reactive antibodies in antisera to strains of southern bean mosaic virus. Viroloav 31 :'8|-/488. Tsuchizaki, T., K, Yora, and H. Asuyama. 1970, The viruses causing mosaic of r.owpeas and adzuki beans, and their t ransm i s s i b i I i t y through seeds. Ann. Phytopathol. Soc, Japan 36:112-120. Tu, J. C. 1973.^ Electron microscopy of soybean root and nodules infected with soybean mosaic virus. Phytopathology 63:1011-1017, Tu, J. C, 1975. Localizations of infectious soybean mosaic virus in mottled soybean seeds. Microbios 1^:151-156. Uyeda I M. Kojima. and D. Murayama. 1975, Purification and serology of bean yellow mosaic virus, Ann. Phytopathol. Soc. Japan i>l • 192-203. Uyemoto, J. K. R. Provvidenti, and D. E. Purcifull. 1973, Host range and serological properties of a seed-borne cowpea virus Phytopathology 63:208-209. (Abstr.), Uyemoto, J. K. R. Provvidenti, and W. T. Schroeder. 1972. Serological relationship and detection of bean common mosaic and bean yellow mosaic viruses in agar gel, Ann. AppI, Biol, 71:235-2^42.

    PAGE 165

    152 van Kammen, A. 1971. Cowpea mosaic virus. No. In Descriptions of plant viruses. Commonw. Mycol. Inst., Assoc. App'l Biol., Kew Surrey, England, ^ p. van Kammen, A. 1972. Plant virus with a divided genome. Annu. Rev Phytoparhol. 10:125-151. van Oosten, H. J. 1972. Purification of plum pox (sharka) virus with the use of Triton X-100. Neth. J, PI. Pathol. 78:33-i^^. van Regenmortel, M. H. V. 1966. Plant virus serology. Pages 207-271 in K. M. Smith, and M. A. Lauffer, eds. Advances in virus research Vol. 12. Academic Press, New York. 39^* p. van Regenmortel, M. H. V., and M. B. von Wechmar. 1970. A reexamination of the serological relationship between tobacco mosaic virus and cucumber virus k. Virology ^1:330-338. van Velsen, R. J. I962. Cowpea mosaic, a virus disease of Vigna ^'"g^sis in New Guinea. Papua New Guin. Agric. J. l4:T53'H6l, Verma, V. S. 1971. Effect of heat on seed transmission of mosaic disease of cowpt-a (V^igna s inensis Savi). Acta M i crob i o 1 oq i ca Polonicd 3: 163-166. Vidano, C. 1959. Indagini sopra un deperimento del la Vigna sinensis Endlicher in coltura italiana. Boll. Zool. Agr. Bachic~3 : 1 -97. Vidano, C, and M. Conti. I965. Transmiss ion i con afidi di un "cowpea mosaic virus" isolato da Vigna sinensis Endl. in Italia. Atti Accad. Sci. 99 : I O^i 1 1 050^ Voller A., A. Bartlett. D. E. Bidwell, M. F. Clark, and A. N. Adams. 1976. Ihe detection of viruses by enzyme1 inked immunosorbent assay (ELISA). J. Gen. Virol. 33:165-167. Vovlas, C., and A. Avgelis. 1972. Le virosi delle piante ortensi in Puglia. VIII. Un mosaico del Fagiol ino dal 1 'occhio. Phytopathol Medit. 11:117-118. ^ Walters, H. J., and 0. W. Barnert, Jr. 1964. Bean leaf beetle transmission of Arkansas cowpea mosaic virus. Phytopathology 5^:911 (Abstr.). ^' Weber K. and M. Osborn. I969. The reliability of molecular weight determinations by dodecyl su 1 fate-pol y-acrylamide gel electrophoresis. J. Biol. Chem. l^ik : kkOe-kh]2 Wells, D G., and R. Deba. I96I. Sources of resistance to the cowpea yellow mosaic virus. Plant Dis. Rep. 45:878-881.

    PAGE 166

    153 Wetter, C. I967. Immunodiffusion of tobacco mosaic virus and its interaction with agar. Virology 31:'<98-507. Williams, R. J. 1973. Diseases of cowpea ( Vigna unguiculata (L.) Walp.) in Nigeria. PANS 21:253-267. Williams, R. J. 1977a. Identification of multiple disease resistance in cowpea. Trop. Agric. 5^:53-59. Williams, R. J. 1977b. identification of resistance to cowpea (yellow) mosaic virus. Irop. Agric. 5'*:6l-67. Yu, T. F. 19^*6. A mosaic disease of cowpea ( Vi gna sinensis Endl .) Ann. Appl. Biol. 33 : '50-^)5'* ~ Zaumeyer, W. J., and L. L. Harter. 19^3Two new virus diseases of beans. J. Agrit. Res. 67-305-327. Zettler, F. W. I966. Aphid populations in central New York as related to epiphytology of bean common mosaic virus. Ph.D. Dissertation. Cornell University, Ithaca, New York. 120 p. Zettler, F. W. R. G. Christie, and J. R. Edwardson. I967. Aphid transmission of virus from leaf sectors correlated with intracellular inclusions. Virology 33:5^9-552. Zettler, F. W. I. R. Evans. 1972. Blackeye cowpea mosaic virus in Florida: Host range and incidence in certified cowpea seed. Proc. Fla. State Hortic. Soc. 85:99-101, Ziemiecki, A., and K. R. Wood. 1975. Serological demonstration of virus-specific proteins associated with cucumber mosaic virus infection of cucumber cotyledons. Physiol. Plant Pathol. 7:171Zink, F. W. R. G. Grogan, and J. E. Welch. 1956. The effect of percentage of seed transmission upon subsequent spread of lettuce mosaic virus. Phytopathology ^6:662-664.

    PAGE 167

    BIOGRAPHICAL SKETCH Jose Albersio rie Araujo Lima was bopn July 12, 19^*0, in Santana do Caiiri, Ceara, Brazil. He graduated from high schcjol in December, 1959, at the Colegio Estadual do Ceara in Fortaleza, Ceara, Brazil. He joined the Army in December, 1958, and was honorably dismissed as an Army Reserve Officer in December, I960. He attended the Universidade Federal do Ceara, receiving the Bachelor of Science degree in Agronomy, along with a diploma of Agronomic Engineering, in December, 1966. In March, 1967, he was hired as an Auxiliary Professor of Plant Pathology by the Universidade Federal do Ceara, and in 1973, he was promoted to Assistant Professor, a position that he has held up to the present time. He started a graduate program in Plant Pathology at the University of Arizona, Tucson, in September. 1970, and received his Master of Science degree in 1972. In September, 197'*, he entered the University of Florid^, rereiving the Doctor of Philosophy degree in March, 1978. He is the author or co-author of approximately ten publications of researches in Phuit Pathology. J, Albersio A. Lima is married to the former Diana MarTa de Gurgel Caracas, and he is the father of a son, Roberto. He is a member of the American Phytopatho I og i ca 1 Society, the Brazilian Phytopathological Society, the Association of Agronomic Engineering from Ceara, the Association of University Professors from Ceara, and the Latinoamerican Association of Plant Pathology. 15^

    PAGE 168

    I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Dan E. Pure i fu 1 1 Cha i rman Professor of Plant Pathology i certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. RaghaTi^an Charudattan Assistant Professor of Plant Pathology i certify that 1 have read this study and that in my opinion, it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Jfifhn R. Edward son 'rofessor of Agronomy I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Ernest Hiebert ~ Associate Professor of Plant Pathology

    PAGE 169

    I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Daniel A. Roberts Professor of Plant Pathology I certify that I have read this study and that in my opinion conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a 44'S5jej-tat ion for the degree of Doctor of Philosophy, It Francis W. Zettler Professor of Plant Pathology This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate Council, and was accepted as partia fulfillment of the requirements for the degree of Doctor of Philosophy March 1978 Dean, College of Agr^i culture Dean, Graduate School


    Figure 28 Photomicrographs showing different views (A, B, C, D) of
    cytoplasmic inclusions (arrows) induced by B1CMV in epi
    dermal strips of cowpea hypocotyl tissue stained with a
    combination of calcomine orange and luxol brilliant green.
    (Cl) cytoplasmic inclusions, (CW) plant cell wall, and
    (Nu) nucleus.


    33
    tissues. With this method, a clear sap was obtained after the first
    low speed centrifugation but the virus zone in the CsCl gradient was
    not very well separated from the host components.
    Plants of C, amarant ¡color and \/_. unguiculata mechanically
    inoculated with purified preparations of B1CMV showed the first symp
    toms of local lesions and systemic mosaic (Fig. I) ^ and 7 days after
    inoculation, respectively. The ultraviolet absorption curve (Fig, 5)
    obtained for the purified preparations of B1CMV had a maximum between
    260 and 262 nm, and a minimum at 2^ to 2^5 nm. The ratio between
    the absorption at wavelengths of 260 and 280 nm was approximately 1,2
    after correction for light scattering, as would be expected for a
    member of the PVY group. This value is consistent and agrees with those
    of other long flexuous rod-shaped viruses (Shepherd and Purcifull, 1971;
    Tosic et a 1., 197^; and Barnett and Alper, 1977), The virus solutions
    showed strong stream birefringence and electron microscopic examinations
    indicated that 73% of the 190 virus particles examined were between
    700 and 800 nm (Figs, 6, 7, 8). The rods observed in the purified
    preparations (Fig. 6) indicated a low percentage of virus fragmentation
    during the purification processes. As the result of end-to-end virus
    aggregation, a few particles with 1^00 to 1500 nm were also observed.
    Purified virus preparations usually were relatively free of normal plant
    constituents when examined with the electron microscope and in the
    spect rophotometer,
    Sedimentation coefficients determined for the virus at 20 C either
    in 0.02 M Tris buffer, pH 8.2, or in 0.05 M borate buffer, pH 8.2,
    indicated that B1CMV sedimented as a single species with the s^q values
    of 157 159 S. On the other hand, the Schlieren pattern (Fig. 9)


    2
    an aphid-transmitted, filamentous virus approximately 750 nm long
    (Harrison and Gudauskas, 1968a; 1968b; Gay and Winstead, 1970; Zettler
    and Evans, 1972; and Uyemoto et a 1., 1973). This virus was first
    isolated in Florida by Anderson (1955a), who designated it "blackeye
    cowpea mosaic virus" (BICMV) (Anderson, 1955b), a name that has been
    retained by Zettler and Evans (1972) and Edwardson et al. (1972).
    Because no antiserum specific for BICMV was available, and because
    only sparse information about the virus properties could be found in
    the literature, the first part of this research was undertaken to
    purify and characterize BICMV jn vitro and in vivo. Antisera prepared
    for BICMV and its cytoplasmic inclusions were used for serological
    characterization of the virus. Some methods for virus and inclusion
    purification, as well as certain physical, biological, immunochemical
    and cytological properties of BICMV were described in the present
    investigation. An abstract of part of this research was already
    published (Lima et al., 1976).
    Literature Review
    Several viruses infect cowpea, V. ungu ¡culata, causing different
    types of mosaic. The first report about mosaic of cowpea was published
    in 1921 by Elliot, who reported a high incidence of cowpea virus
    disease in Arkansas (Elliot, 1921). Smith (192A) demonstrated ex
    perimentally that the cowpea virus was transmitted either by rubbing
    the leaves of diseased and healthy plants together or by the bean leaf
    beetle, Ceratoma trifurcata Forst. Subsequently, Gardner (1927) working
    with a cowpea virus, observed that it was transmitted through seeds
    of certain cowpea varieties.


    0
    >
    §
    o o
    +75


    Figure II
    - Electron micrographs of purified preparations of B1CMV
    cytoplasmic inclusions stained with molybdate. All
    purified preparations consisted of tubes, most of which
    were fragmented during the purification process. Note
    striations (St) on high magnification (D).


    121
    SoyMV-infected soybean Jupiter1 seed lot were also estimated by the
    simplified double immunodiffusion test using hypocotyl discs (Table ll).
    Serology and Microscopy of Cytoplasmic Inclusions Induced by B1CMV
    and SoyMV in HypocotyIs of Germinated Seeds
    Cytoplasmic inclusions induced by B1CMV in cowpea and by SoyMV
    in soybean were detected by serology, light microscopy and electron
    microscopy in hypocotyls of 4-5-day-old seedlings. In double immuno
    diffusion tests, specific precipitin lines were observed with the in
    clusion antisera and extracts of vi rus-infected hypocotyls but not
    with extracts from healthy hypocotyls (Fig. 27).
    Light microscopic observations of epidermal strips of hypocotyls
    from B1 CMV-infected cowpea and SoyMV-infected soybean seedlings
    readily revealed the presence of cytoplasmic inclusions (Figs. 28, 29).
    Groups of inclusions induced by B1CMV and by SoyMV were abundant in
    cells of virus-infected hypocotyls (Figs. 28, 29), but were not ob
    served in cells of noninfected hypocotyls. Pinwheels with scrolls
    were observed in ultrathin sections of hypocotyl tissues infected
    either with BICMV or SoyMV (Figs. 30, 31), A large number of longi
    tudinal sections of pinwheel inclusions in hypocotyl tissues showed
    that they were abutted to the cell wall close to the plasmodesmata
    (Fig. 31-C, -D). S imilarly, tubes were observed in molybdate-treated
    extracts from hypocotyls infected with either virus.


    631


    20
    consisting of a center well with six peripheral wells spaced 4 5 mm
    from the center well as measured from the edges of the wells. Different
    gel patterns were also used in certain tests. Antigens used as reactants
    were prepared either in deionized water or in 1.5% SDS solution, ac
    cording to Purcifull and Batchelor (1977). In the first case, fresh
    tissue was ground with a mortar and pestle in deionized water (1/2,
    w/v) and expressed through cheesecloth. The second method which was
    more commonly used, consisted of grinding fresh tissue in 1.0 ml of
    water per gram of tissue and adding 1.0 ml of 3-0% SDS per gram of
    tissue prior to expressing the sap through cheesecloth. The antigens
    and undiluted antisera were pipetted directly into the appropriate
    wells, and the. plates were incubated in a moist chamber at 2b C for
    24 48 hr. The development of precipitation patterns was observed
    by looking at the plates, which were illuminated from the bottom with
    indirect lighting. The reactants were removed and 15% charcoal (Nor it
    A) in water (w/v) was added into the wells before photographs were
    taken.
    Single radial immunodiffusion tests were conducted in agar media
    containing 0.8% Noble agar, 1.0% NaN^, 0.3 or 0.5% SDS, and 10, 15, or
    20% B1CMV antiserum. Media were prepared either with antiserum obtained
    for untreated B1CMV and antiserum for pyrrolidine degraded B1CMV-
    protein. Each SDS concentration in the media was tested with antigens
    prepared in distilled water or in 1.5% SDS. During medium preparation,
    care was taken to avoid heating the antisera over 50 C and while ex
    posed to SDS, the antisera were maintained at 50 C for less than 2 min.
    Single radial diffusion plates were also prepared with a mixture
    of antisera to BICMV and CPMV. The CPMV-ntiserum was prepared by


    131


    Figure Page
    12 Double immunodiffusion tests in agar medium containing
    0.8* Noble agar, 1.0* NaN^, and 0.5% SDS 51
    13 Single radial diffusion tests in agar media containing
    different concentrations of SDS and antisera for BICMV
    and cowpea mosaic virus (CPMV) 54
    1 ^ Single radial diffusion tests with SDS and pyrrolidine
    degraded capsid protein of BICMV and CPMV 56
    15 Reciprocal double immunodiffusion tests with B1CMV
    and other potyviruses in medium containing 0.8* Noble
    agar, 1.0* NaN^, and 0.5% SDS prepared in 0.05 M Tris-
    HC1 buffer, pHj7.2 6l
    16 Immunodiffusion tests with B1CMV, Moroccan isolate of
    cowpea aphid-borne mosaic virus (CAMV) and siratro
    strain of bean common mosaic virus (BCMV-S) in agar
    medium containing 0.8* Noble agar, 1.0* NaN_, and
    0.5% SDS prepared in 0.05 M Tris-HCl buffer, pH 7.2. 63
    17 Photomicrographs of cytoplasmic inclusions in
    epidermal strips of cowpea leaves systemically in
    fected with B1CMV, stained with a combination of
    calcomine orange and luxol brilliant green 67
    18 Electron micrographs of ultrathin sections of cowpea
    leaf cells infected with BICMV showing cross-sections
    and longitudinal sections of pinwheel inclusions 69
    19 Double immunodiffusion tests with extracts from
    different portions of B1CMV-infected and healthy
    4-5-day-old cowpea seedlings 101
    20 Diagram showing methods for assaying legume seeds
    by single and double radial immunodiffusion 104
    21 Double immunodiffusion tests with hypocotyls from
    healthy and B1CMV-infected 4-5-day-old cowpea
    seedlings in medium containing 0.8* Noble agar,
    1.0* NaN^, and 0.5* SDS, prepared in water 106
    22 Single radial immunodiffusion tests with hypocotyl
    extracts from healthy and B1CMV-infected, 4-5-day-
    old cowpea seedlings 110
    23 Electron micrographs of BICMV in hypocotyl extracts
    from the same cowpea seedling using different
    preparat ions 113
    v i i i


    58
    observed when the antigens were prepared in 1,5% SDS and the medium
    used had 0.3% SDS and 10% antiserum (Fig. 13-A) or 0.5% SDS and 15%
    antiserum (Fig. 13~E). The same results were consistently observed
    with different batches of plates with the same medium compositions.
    Similar results were also observed with CPMV using antiserum obtained
    for SDS-treated virus. On the other hand, different results were ob
    served in SDS medium containing a mixture of BICMV and CPMV antisera.
    All media containing 0.3% SDS were cloudy with all combinations of
    BICMV and CPMV antisera used (Fig. 13-1, -J), indicating some type of
    interaction between SDS and antiserum proteins. Flowever, even with
    the cloudy appearance, some virus-specific reactions were still ob
    served (Fig. 13-1, -J). Clearer media were obtained with 0.5% SDS
    and 10 or 15% of each antiserum. The best reactions, however, were
    observed when both BICMV and CPMV antisera were used at concentrations
    of 10% in media containing 0.5% SDS (Fig. 13~H). Strong precipitin
    rings were observed around the wells containing BICMV or a combina
    tion of BICMV and CPMV whereas weaker reactions were observed around
    the wells containing only CPMV (Figs. )3*H, 1A-C). Unexpectedly, no
    reactions were observed around the wells containing only CPMV in a
    medium containing 15% of each antiserum and 0.5% of SDS (Fig. 13-G).
    Virus-specific reactions using BICMV and CPMV antisera were also
    obtained with the single radial diffusion method described by Shepard
    (1972). Precipi tin rings around the virus-wells were observed when
    the antigens were prepared in 1.5 or 2.5% pyrrolidine and the medium
    used contained 0.8% Noble agar, 0.2% NaN^, and 10 to 15% virus anti
    sera prepared in 0.05 M Tris-HCl buffer, pH 7.2, containing 0.85%
    NaCl. Sharp, white precipitin rings were formed close to edges of


    Figure 6 Electron microscopy of B1CMV in a purified preparation
    and in cowpea leaf extracts.
    A) Purified preparation of BlCMV negatively stained
    with 2% phosphotungstic acid, pH 6.5, containing
    0.]% BSA;
    B) Serologically specific electron microscopy (SSEM)
    of leaf extract from cowpea plants systemically
    infected with BlCMV. Antiserum for BlCMV diluted
    1/1000 in 0,05 M Tris buffer, pH 7.2, was used
    to sensitize the grid and the virus particles
    were positively stained with 1% uranyl acetate.
    Note the considerable increase in virus concentra
    tion compared with the normal leaf-dip preparation
    (0;
    C) Leaf-dip preparation of cowpea leaf tissue
    systemically infected with BlCMV, negatively
    stained with 2% phosphotungstate.


    59
    the wells containing 61CMV in agar medium prepared with BICMV antiserum,
    whereas whitish halos with greater diameters were formed around the
    wells containing CPMV in agar medium impregnated with CPMV antiserum
    (Fig. 1**-D, -E, -F). The same distinction between these two types of
    precipitin rings was observed when both antisera were added into the
    same medium, so that two concentric rings were formed around the
    wells containing both viruses (Fig. I^-F). The inner ring was the
    result of B1CMV-ant¡body specific reactions and the larger halos re
    sulted from CPMV-speoific reactions. This difference in types of
    precipitin rings could be related to the concentration of the antigens
    placed in the wells and to the reciprocal of antibody concentration
    (Shepard, 1972). St ronger and more compact rings were observed with
    CPMV when the antigens were diluted or the antiserum concentration
    was increased.
    Reciprocal double immunodiffusion tests with SDS-treated antigens
    showed that BICMV is serologically related to, but distinct from, the
    following potyviruses: BCMV-BV-1, BCMV-S, BYMV, DMV, LMV, PVY, SoyMV,
    TEV, and WMV-2 (Fig. 15). No reactions were detected, however, with
    certain potyviruses, including BiMV, PeMV, TuMV, WMV-1, CoMV, PVMV,
    and PWMV. Antiserum for BICMV also reacted specifically with CAMV
    forming a distinct spur which extended past the heterologous reaction
    (Fig. 16-A). In all positive serological relationships observed in
    the reciprocal serological tests, spurs were formed in both directions
    (Fig. 15).
    The serological distinctions observed between BICMV and BCMV-S,
    and CAMV by spur formation were demonstrated by the intragel cross
    absorption technique (Fig. 16-B, -D). The heterologous antigens,


    Figure 31
    - Electron micrographs of ultrathin sections of hypocotyl
    cells of ^-5ciay-o 1 d soybean seedlings grown from SoyMV-
    infected seeds. Note cross-sections (A, B) of the pin-
    wheels and longitudinal views of the inclusions abutted
    to the plant cell wall at the piasmodesmata (C, D).
    (CW) plant cell wall, (pi) plasmodesma, and (pw) pinwheel
    inc1 usions.


    71
    annuurn L. 1 Ear I y Ca I wonder 1 ; Cucumi s sat ¡ yus L. ; Cucurb i ta pepo L.
    'Small Sugar'; Lupnus angustifoljus L. 'Bitter Blue'; Lupinus luteus
    L. 'Sweet Yellow'; Phaseolus vulgaris 'Black Turtle-1', 'Green Northern
    1140', 'Improved Tendergreen', 'Lake Shasta', 'Michelite 62', 'Pink
    Rosa', 'Pink Viva1, 'Puregold Wax', 'Red Mexican U-3 ^' 'Red Mexican
    U-3 51, 'Top-crop* and 'VC 1822'; Pi sum sativum L. 'Alaska', 'Bonneville'
    and 'Ranger'; and Vicia faba L.
    The reactions of cowpea varieties to mechanical inoculations of
    B1CMV, BCMV-S, CAMV, and CPMV are indicated in Table I. All inoculated
    plants were assayed serologically for the presence of the viruses
    (Table l).
    Discuss ion
    Blackeye cowpea mosaic virus and its cytoplasmic inclusions were
    successfully purified from systemically infected cowpea or N^. benthamiana
    leaf tissues with the procedures outlined herein. The first method of
    virus purification (Fig. 2) gave good yields of highly purified B1CMV,
    and the combination of n butanol and chloroform-carbon tetrachloride
    (Fig. 4) was the better procedure for purification of B1CMV and its
    cytoplasmic inclusions from the same batch of tissue. The high degree
    of purity of the B1CMV preparations indicated by spectrophotometry,
    analytical centrifugation and PAGE analyses, as well as serological
    studies and electron microscopic observations, confirmed the efficiency
    of the pur ification procedures.
    Aggregation of virus particles and virus and host components
    during purification appears to be a limiting factor for obtaining high


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    132
    Discussion
    Seed-transmission of plant viruses is of agricultural importance
    because efficient virus transmission in space and time can be provided
    through seeds of infected host plants. The international exchange of
    several legume seeds is probably partly responsible for the worldwide
    distribution of important virus diseases of legumes. The occurrence
    of SoyMV in soybean and BCMV in bean in most countries where these
    crops have been tested for viruses (Bos, 1971, 1972) are good examples
    to illustrate this point. According to Phatak (197*0, several legumes
    are propagated in association with their seed-borne viruses on a wide
    scale in certain subtropical and tropical regions that lack organized
    seed certification and breeding programs. Brunt and Kenten (1973) ex
    plained the prevalence of cowpea mild mottle virus in cowpea in Ghana
    by the high rate of seed transmission in cowpeas and by the traditional
    practice of sowing seeds from crops previoysly grown in the same region.
    The control of virus diseases of legumes through the production
    of virus-free seeds or seed lots with very low percentage of infected
    seeds has been advocated frequently (Fajardo, 1930; Thomas and Graham,
    1951; Zettler and Evans, 1972; Baker, 1972 ; Phatak, 197**; Sinclair and
    Shurtleff, 1975; and Williams, 1975). The increase in percentage of
    infected seeds produced in crops with low initial infection and the
    efficient secondary spread of certain viruses into the growing crop
    from a small amount of primary inoculum randomly located throughout
    the planting, emphasize the importance of maintaining a low tolerance
    level in seed certification programs.


    9
    An aphid-transmitted virus was responsible for complete loss of cowpea
    in irrigated areas of northern Nigeria (Raheja and Leleji, 197*0. Based
    on the fact that the virus was neither mechanically transmitted nor
    seed-borne in cowpea, Raheja and Leleji (197**) concluded that it was
    either an atypical strain of CAMV or a new virus not previously described.
    A virus isolated from Crotalaria spectabi 1is Roth in a field at
    Gainesville, Florida, was studied by Anderson ( 1955b) and designated
    blackeye cowpea mosaic virus (B1CMV). Anderson (1955b, 1955c) reported
    that BICMV infected plants of cowpea, Crotalaria and Desmodiurn in the
    field, but considered Crota 1 aria and Desmodiurn as secondary hosts for
    the virus. In a subsequent study, Anderson (1959) observed that BICMV
    was transmitted by M. persi cae but not by the bean leaf beetle,
    £. trifurcata. Corbett (1956) found that BICMV was serologically
    related to BYMV and identified it as a strain of BYMV. Based on Corbett's
    conclusion, several subsequent reports have referred to BICMV as a cow
    pea strain of BYMV (Brierly and Smith, 1962; Kuhn, 196**a; Kuhn et a 1 ,
    1965; and Harrison and Gudauskas, 1968a).
    Light and electron microscopic studies of BICMV and BYMV showed
    marked cytological differences between these two flexuous rod-shaped
    viruses (Edwardson et al., 1972). According to Edwardson et al. (1972),
    the cytoplasmic inclusions induced by BICMV were consistently different
    from those induced by BYMV. In the light microscope, groups of plates
    were observed in cells of BYMV-infected tissues, whereas groups of
    tubes were seen in cells of epidermal strips obtained from B1CMV-infected
    tissue. Electron microscopy of ultrathin sections indicated that
    BICMV-induced cytoplasmic inclusions consisted of pinwheels with scrolls,
    whereas BYMV-induced cytoplasmic inclusions were made of pinwheels and


    141
    Cheo, P. C. 1955- Effect of seed maturation on inhibition of southern
    bean mosaic virus in bean. Phytopathology 45:17-21.
    Christie, R G. 1967. Rapid staining procedures for differentiating
    plant virus inclusions in epidermal strips. Virology 31:268-271.
    Christie, R G., and J. R. Edwardson. 1977. Light and electron micros
    copy of plant virus inclusions. Florida Agrie. Exp. Stn. Monogr.
    Ser. 9, 150 p
    Christie, S. R., and W E. Crawford. 1977. Plant virus range of
    Nicotiana benthamiana. Plant Dis. Rep. (in press).
    Clark, M. F., and A N. Adams. 1977. Characteristics of the microplate
    method of enzyme-1 inked immunosorbent assay for the detection of
    plant viruses. J. Gen. Virol. 34:475-483
    Clifford, H. T. 1977. Immunodiffusion techniques for the detection of
    the Cymbidium mosaic and Odontoglossum ringspot viruses of orchids.
    M. S. Thesis, University of Florida, Gainesville. 63 p.
    Cockbain, A. J., R. Bowen, and S. Vorra-Urai, 1976. Seed transmission
    of broad bean stain virus and Echtes ackerbohnenmosaik-vi rus in
    field bean (Vicia faba). Ann. Appl. Biol 84:321-332.
    Corbett, M. K. 1956. Serological and morphological relationships of
    plant viruses. Florida Univ. Agrie. Exp. Stn. Annu. Rep., p. 117-
    118.
    Crowley, N. C. 1957. Studies on the seed transmission of plant virus
    diseases. Aust. J. Biol. Sci. 10:449-464.
    Crowley, N. C. 1959. Studies on the time of embryo infection by seed-
    transmitted viruses. Virology 8:116-123.
    Dale, W. T. 1949- Observations on a virus disease of cowpea in Tri
    nidad. Ann. App. Biol. 36:327-333.
    Dale, W. T. 1953. The transmission of plant viruses by biting insects,
    with particular reference to cowpea mosaic. Ann. Appl. Biol. 40:
    385-392.
    Debrot, A. E., and C. E. Benitez de Rojas, 1967. Identificacin del
    virus del mosaico de la soya en Venezuela. Agron. Trop. 17:75-86.
    Demski, J. W., and H. B. Harris. 1974. Seed transmission of viruses
    in soybean. Crop Sci. 14:888-890.
    Derrick, K. S., and R. H. Brlansky. 1976. Assay for virus and myco-
    plasmas using serologically specific electron microscopy. Phyto
    pathology 66:815-820.


    ( 03 [c]
    MARKERS
    M. W. .
    EL 67000
    EL 53000
    2L 43000
    £ -SL 29000
    EL 17500
    6 8 10 12 14
    DISTANCE FROM TOP (cm)


    98
    extracting buffer ()/l, w/v) with mortars and pestles and small drops
    from these extracts were used to treat the antiserum-sensitized grids.
    Double Immunodiffusion Tests and SSEM for Detection of Other Viruses
    in Germinated Legume Seeds
    Double immunodiffusion tests using discs or extracts of hypocotyls,
    and SSEM using hypocotyl extracts were used to detect the presence
    of BCMV and SoyMV in germinated seeds. A seed lot of bean, P^. vulgaris
    Black Turtle' containing BCMV-infected seeds was obtained from Dr.
    Rosario Provvidenti, New York State Agricultural Experiment Station,
    Geneva. Infected seeds were detected by double immunodiffusion and
    SSEM using antiserum obtained for a severe strain of BCMV (BCMV-S)
    isolated from sirat.ro, Macroptilium atropurpureum, in Florida (Lima
    et al.,1977). Extracts of these germinated bean seeds prepared in
    0.05 M potassium phosphate buffer, pH 7-5, were also inoculated in a
    very sensitive local lesion bean line VC-1822 (Provvidenti and Cobb,
    1975). Antiserum for SoyMV was also used to detect SoyMV-infected
    seeds of soybean, Glycine max.
    Serology and Microscopy of Cytoplasmic Inclusions Induced by B1CMV
    and SoyMV in Hypocotyls of Germinated Seeds
    Germinated seeds of cowpea 'Knuckle Purple Hull' infected with
    B1CMV and germinated seeds of soybean 'Midwest' infected with SoyMV
    were serologically identified by double immunodiffusion tests using
    antiserum for B1CMV and SoyMV, respectively, and hypocotyl discs as
    assay antigens. Hypocotyl extracts from the vi rus-infected A-5-day-
    old seedlings were tested by SDS double immunodiffusion tests against
    B1CMV and SoyMV inclusion antisera.


    Lima, J. A. A., D. E. Purcifull, and E. Hiebert. 1976. Purification
    and serology of blackeye cowpea mosaic virus. Proc. Am. Phytopathol.
    Soc. 3:248 (Absrr.).
    Lima, J. A. A., D. E. Purcifull, and R. Sonoda. 1977. Some properties
    of a potyvirus isolated from siratro in Florida. Proc. Am. Phyto
    pathol. Soc. 4 (in press) (Abstr.).
    Lima-Neto, V. C., and A. S. Costa. 1976. Transmissao comparative do
    virus do mosaico comn da soja por sementes com manchas-caf e
    nao manchadas. Fitopatologa 11:20 (Abstr.).
    Lister, R. M. I960. Transmission of soil-borne viruses through seed.
    Virology 10:547-549-
    Lister, R. M. 1977- Detection of viruses in soybean seed by enzyme-
    linked immunosoibant assay. Proc. Am. Phytopathol. Soc. 4 (in
    press) (Abstr.)
    Lister, R. M., and A F. Murant. 1967- Seed-transmission of nematode-
    borne viruses. Ann. Appl. Biol. 59:49-62.
    Lister, R. H., and J M. Thresh. 1955. A mosaic disease of leguminous
    plants caused by a strain of tobacco mosaic virus. Nature 175:
    1047-1048.
    Lovisolo, 0., and M. Conti. 1966. Identification of an aphid-
    transmitted cowpea mosaic virus. Neth. J. PI. Pathol. 72:265-269.
    Lundsgaard, T. 1976 Routine seed health testing for barley stripe
    mosaic virus in barley seeds using the latex-test. J. Plant Dis.
    Protection 83:2/8-283.
    Markham, R. I960. A graphical method for rapid determination of
    sedimentation coefficients. Biochem. J. 77:516-519.
    Marshall, W. H and L. C. Norins. 1965- Antigenic properties of
    extract of Phaseolus vulgaris seeds (phytohaemagg1utinin) routinely
    used in leucocyte cultures. Aust. J. Exp. Biol. Med. Sci. 43:
    213-228.
    Martelli, G. P., and M. Russo. 1977- Plant virus inclusion bodies.
    Pages 175-266 jm M. A. Lauffer, F. B. Bang, K. Maramorosch, and
    K. M. Smith, eds. Advances in virus research, Vol. 21. Academic
    Press, New York. 409 p.
    Matthews, R. E. F. 1970. Plant Virology. Academic Press, New York.
    778 p.
    McDonald, J. G., and R. I. Hamilton. 1972. Distribution of southern
    bean mosaic virus in the seed of Phaseolus vulgaris. Phyto
    pathology 62:387-389.


    Figure k Flow diagram outlining the steps carried out during the
    purification of BICMV and its cytoplasmic inclusions by
    a combination of the first (Fig. 2) and second (Fig. 3)
    methods for purification of virus and inclusions.


    150
    Shepard, J. F. 1972. Gel-d¡ffus ion methods for the serological detec
    tion of potato viruses X, S, and M. Montana Agrie. Exp. Stn. Bull.
    No. 662. 22 p.
    Shepard, J. F., J. W. Jutila, J. E. Catlin, F. S. Newman, and W. H.
    Hawkins. 1971. Immunodiffusion assay for potato virus M infection
    Phytopathology 61:873-874.
    Shepard, J. F., G. A. Secor, and D. E. Purcifull. 1974. Immunochemical
    cross-reactivity between the dissociated capsid proteins of PVY
    group plant viruses. Virology 58 : 464-475.
    Shepherd, R. J. 1963. Serological relationship between bean pod mottle
    virus and cowpea mosaic viruses from Arkansas and Trinidad. Phyto
    pathology 53:865-866.
    Shepherd, R. J. 1964. Properties of a mosaic virus of cowpea and its
    relationship to the bean pod mottle virus. Phytopathology 54:
    466-473.
    Shepherd, R. J. 1971. Southern bean mosaic virus. No. 57 |n Descrip
    tions of plant viruses. Commonw. Mycol. Inst., Assoc. Appl. Biol.
    Kew, Surrey, England. 4 p.
    Shepherd, R. J. 19/2. Transmission of viruses through seed and pollen.
    Pages 267-292 m C. Kado, and H. 0. Agrawal,eds. Principles and
    Techniques in Plant Virology. Van Nostrand Reinhold Company, New
    York. 688 p.
    Shepherd, R. J., and R. W. Fulton. 1962. Identity of a seed-borne
    virus of cowpea. Phytopathology 52:489-493.
    Shepherd, R. J., and G. S. Pound. I960. Purification of turnip mosaic
    virus. Phytopathology 50:797-803.
    Shepherd, R. J., and D. E. Purcifull. 1971. Tobacco etch virus. No.
    55 jjt Descriptions of plant viruses. Commonw. Micol. Inst., Assoc.
    Appl. Biol., Kew, Surrey, England. 4 p.
    Sinclair, J. B., and M. C. Shurtleff. 1975. Compendium of soybean
    diseases. Am. Phytopathol. Soc., St. Paul, Minnesota. 69 p.
    Slack, S. A., and R. J. Shepherd. 1975. Serological detection of
    seed-borne barley stripe mosaic virus by a simplified radial-
    diffusion technique. Phytopathology 65:948-955.
    Smith, C. E. 1924. Transmission of cowpea mosaic by bean leaf-beetle
    Science 60:268.
    Smith, F. L., and W. B. Hewitt. 1938. Varietal susceptibility of
    common bean mosaic and transmission through seed. Calif. Agrie.
    Exp. Stn. Bull. 612:3-18.


    52
    respectively, with 30% of the particle lengths ranging from 700 to
    800 nm (Figs. 7 and 8). Some variation was observed with the particle
    size of purified virus stained with PTA and virus particles in leaf
    extracts prepared by SSEM and stained with uranyl acetate (Figs. 7
    and 8). On the other hand, grids with less plant debris and higher
    concentrations of virus particles were obtained with SSEM than with
    the conventional leaf-dip preparation (Fig. 6). Using normal leaf-dip
    preparations at least four grids were prepared and 10 electron micro
    graphs were taken to measure a maximum of 25 virus particles. On the
    other hand, 132 virus particles were measured by examining two micro
    graphs obtained by SSEM.
    In cowpea leaf extracts, BICMV had a TIP of 65 C, LIV of 48 hr,
    -4
    and DEP of 10 Blackeye cowpea mosaic virus was still infectious
    after 10 min at 60 C but not at 65 C and lost its infectivity after
    48 hr at room temperature, but not at 24 hr. Sap of cowpea leaves
    systemically infected with B1CMV lost infectivity when diluted more
    than 10 ^ with distilled water.
    Serology
    Antisera specific for B1CMV were obtained against untreated
    virions and pyrrolidine degraded viral protein. Both antisera reacted
    with SDS- or pyrrolidine-treated BlCMV in purified preparations or
    in plant sap in double and single radial diffusion tests (Figs. 12,
    13, 14). Most bleedings were specific for viral antigens; however,
    some bleedings also reacted with extracts from healthy plants, sug
    gesting the presence of antibodies specific for normal plant components.
    To remove these antibodies the antiserum was absorbed with plant


    92
    Seed-Borne Viruses in Phased us vulgaris
    A large body of evidence (Bos, 1971) has confirmed the first
    demonstration of seed transmission of BCMV in P. vulgaris by Reddick
    and Stewart (1919). Burkholder and Muller (1926) reported that seeds
    from bean plants showing mosaic gave rise to 50% diseased plants.
    The virus remained viable within bean seeds for a period of at least
    30 years (Pierce and Hungerford, 1929). Fajardo (1930) observed
    that both infected and noninfected bean seeds germinated with equal
    readiness and vigor, and concluded that the viability of the seeds
    was not directly affected by the presence of BCMV. Fajardo (1930) and
    others (Crowley, 1957, 1959; and Schippers, 1963) found that plants
    infected at early vegetative growth produced more vi rus-infected seeds
    than those infected at later stages.
    It has been demonstrated that BCMV is transmitted irregularly
    through seeds harvested from vi rus-infected plants. Fajardo (1930)
    observed that some of the seeds in a single pod from bean plants in
    fected with BCMV were v i rus-infected and some were noninfected. By
    crossing vi rus-infected and healthy bean varieties, Nelson and Down
    (1933) provided evidence that BCMV was equally transmitted by ovule
    or pollen from infected parent plants. Similarly, Medina and Grogan
    (i960 obtained high percentage of seed transmission of BCMV through
    either the pollens or ovules of infected plants, but also observed
    that the pollens usually transmitted the virus to a largernumber of
    progeny plants than the ovules. Cross-pollination experiments per
    formed by Schippers (1963) revealed that the embryo infection
    with BCMV and consequently the seed-transmission might originate from


    149
    Reddick, D., and V. B. Stewart. 1919. Transmission of virus of bean
    mosaic in seed and observations on thermal death-point of seed and
    virus. Phytopathology 9:445-450.
    Richter, J., J. Polak, and E. Proll. 1975. Ausarbeitung von Verfahren
    zum Serologischen sche1lnachweis des Gurkenmosaik-vi rus in naturlich
    infizierten krautigen Pflanzen. Arch. Phytopathol. v. Pfanzenschutz
    11:307-318.
    Robertson, D. G. 1965- The local lesion reaction for recognizing cowpea
    varieties immune from and resistant to cowpea yellow mosaic virus.
    Phytopathology 55:923-925.
    Ross, J. P. 1963. Interaction of the soybean mosaic and bean pod mottle
    viruses infecting soybeans. Phytopathology 53:887 (Abstr.).
    Ross, J. P. 1968. Effect of single and double infections of soybeans
    with soybean mosaic and bean pod mottle viruses on yields and seed
    characters. Plant Dis. Rep. 52:344-348.
    Ross, J. P. 1969. Effect on time and sequence of inoculation of soy
    beans with soybean mosaic and bean pod mottle viruses on yields
    and seed characters. Phytopathology 59:1404-1408.
    Ross, J. P. 1970. Effect of temperature on mottling of soybean seed
    caused by soybean mosaic virus. Phytopathology 60:1798-1800.
    Rui, D. I960. Apporto alie conoscenze sulle virosi dei vegetal i. In-
    formatore Agrac., Veroma 23:15-17.
    Schippers, B. 1963. Transmission of bean common mosaic virus by seed
    of Phaseolus vulgaris L. cultivar Beka. Acta Bot. Neerl. 12:433-
    497.
    Scott, H. A. 1961. Serological detection of barley stripe mosaic
    virus in seeds and in dehydrated leaf tissue. Phytopathology 51:
    200-201 .
    Sharma, S. R., and A. Varma. 1975- Cure of seed transmitted cowpea
    banding mosaic viruses. Phytopathol. Z. 83:144-151.
    Sheffield, F. M. L. 1941. II. The cytoplasmic and nuclear inclusions
    associated with severe etch virus. J. Roy. Hicroscop. Soc. 61:
    30-45.
    Shepard, J. F. 1969. Serodiagnosis of PVX in potato tuber sprouts.
    Plant Dis. Rep. 53:845-848.
    Shepard, J. F. 1970. A radial immunodiffusion test for the simulta
    neous diagnosis of potato viruses S and X. Phytopathology 60:
    1669-1671.


    Figure 24 Electron micrographs of serologically specific electron
    microscopy with BICMV, BCMV-S, and CPMV.
    A) BICMV antiserum grid and extracts from a B1CMV-
    infected cowpea hypocotyl ;
    B) BICMV antiserum grid and extracts from one gram of
    BICMV-infected hypocotyl and 49 g of healthy cowpea
    hypocotyls. Arrows point to virus particles;
    C) BCMV-S antiserum grid and leaf extract from BCMV-S
    infected bean;
    D) CPMV antiserum grid and leaf extract from CPMV-
    infected cowpea.


    63


    137
    first part of this research, an agar medium impregnated with a mixture
    of antisera was used for serodiagnosis of two morphologically distinct
    viruses in cowpea (Figs, 13, 1*0-
    The presence of cytoplasmic inclusions in tissues of germinated
    seeds is also useful for detection of vi rus infected seeds by light
    microscopy and serology. Cytoplasmic inclusions induced by B1CMV in
    cowpea and by SoyMV in soybean were detected by serology, light micros
    copy and electron microscopy in hypocotyls of germinated seeds. Al
    though the cytoplasmic inclusions induced by potyviruses were found to
    be widespread in systemically infected plants (Sheffield, 19^1; Hampton
    et al., 1973; and Tu, 1973), no previous report has indicated their
    presence in either infected seed per se or in germinated vi rus-infected
    seeds (Sheffield, 19^1; Camargo et al,, 1968; Edwardson, 197^; Andrews
    and Shalla, 197t; Tu, 1975; Martel 1 i and Russo, 1977; and Christie and
    Edwardson, 1977).
    In summary, the following techniques have been demonstrated to be
    useful for detecting potyviruses and/or their cytoplasmic inclusions
    in hypocotyl tissues of A-5-day-old seedlings grown from vi rus-infected
    legume seeds: a) conventional double immunodiffusion, b) hypocotyl-disc-
    double immunodiffusion, c) conventional single radial immunodiffusion,
    d) hypocotyl-disc-single radial immunodiffusion, e) SSEM of virus par
    ticles; and g) light microscopy of cytoplasmic inclusions. The hypo
    cotyl -disc-doub1e immunodiffusion technique is the simplest method to
    use when it is necessary to determine the percentage of vi rus-infected
    seeds.


    24
    The cowpea varieties were also inoculated with CPMV, CAMV, and
    BCMV-S. Crude sap from all inoculated cowpea plants were also tested
    in double immunodiffusion against antisera specific for CPMV, B1CMV,
    and BCMV-S, respectively. Since CAMV was shown to be serologically
    related to B1CMV, the serological tests to detect its presence in the
    inoculated plants were done with BlCMV antiserum.
    Result s
    Purification and Properties of Blackeye Cowpea Mosaic V i rus
    Purified preparations of BlCMV were obtained from systemically in
    fected leaves of either V. unguiculata 'Knuckle Purple Hull' (Fig. 1-A)
    or N^. benthamiana using the purification procedures diagrammed in
    Figures 2, 3, and 4. The best yield with the highest degree of purity
    was obtained using the first method of virus purification (Fig. 2) and
    infected cowpea leaves (Fig. 1-A) as a source of virus. The first
    trifol¡oate cowpea leaves collected 15 to 18 days after inoculations
    gave the highest yield of virus (8 10 mg) per 100 g (fresh weight)
    of infected tissue and n-butanol proved to be the best clarifying agent
    for cowpea tissue. An opalescent,sharp virus-band was usually obtained
    after equilibrium density gradient centrifugation in 30% CsCl. The
    virus zone was located at 12 to 15 nm from the bottom of the tube
    while most of the green host components stayed at the top portion of
    the gradient. The clear pellet obtained after a high speed centrifuga
    tion of virus removed from CsCl gradients confirmed the absence of
    colored host components. The combination of chloroform and carbon
    tetrachlor¡de,a 1 though necessary for inclusion purification, was an
    inferior method of clarification for obtaining virus from cowpea


    142
    Desjardins, F. R,, R. L. Latterell, and J. E. Mitchell. 1954. Seed
    transmission of tobacco-ringspot virus in Lincoln variety of
    soybean Phytopathology 44 ; 86.
    Doi, Y., M. U. Chang, and K. Yora. 1977- Orchid fleck virus. No. 183
    in Descriptions of plant viruses. Commonw. Mycol. Inst., Assoc.
    Appl. Biol., Kew, Surrey, England. 3 p>
    Drijfhout, E and L Bos. 1977. The identification of two new strains
    of bean common mosaic virus, Neth. J PI Pathol, 83:13_25-
    Edwardson, J. R. 1974. Some properties of the potato virus-Y group.
    Fla. Agrie. Exp Stn, Monogr. Ser, 4. 398 p.
    Edwardson, J, R., D E, Purcifull, and R. G. Christie. 1968. Structure
    of cytoplasmic inclusions in plants infected with rod-shaped viruses
    Virology 34:250-263.
    Edwardson, J. R., F, W. Zettler, R. G. Christie, and I. R. Evans. 1972.
    A cytological comparison of inclusions as a basis for distinguishing
    two filamentous legume viruses. J, Gen. Virol. I 5:113 ~118.
    Ekpo, E. I. A., and A W Saettler. 1974 Distribution pattern of bean
    common mosaic virus in developing bean seed. Phytopathology 64:269
    270.
    Elliot, J. A. 1921 A mosaic of sweet and red clovers. Phytopathology
    11:146-148
    Fajardo, T. G 1930. Studies on the mosaic disease of the bean.
    Phytopathology 2 0:4 69-494
    Fenner, F. 1976. Classification and nomenclature of viruses. Inter-
    virology 7:4-115.
    Fischer, H 0 and B E. Lockhart. 1976a. A strain of cowpea aphid-
    borne mosaic virus isolated from cowpeas in Morocco. Phytopathol.
    Z 85:43-48.
    Fischer, H. U., and B. E. Lockhart. 1976b. A strain of cucumber mosaic
    virus isolated from cowpeas in Morocco. Phytopathol. Z. 85:132-138
    Fountain, D. W,, and W Yang. 1977. Isolectins from soybean (Glycine
    max) Biochfm. Biophys. Acta 492:176-185
    Fulton, R. W 1964. Transmission of plant viruses by grafting, dodder,
    seed, and mechanical inoculation. Pages 39-67 hi M, K. Corbett,
    and H. D. Sfsler, eds. Plant Virology. Univ. Florida Press, Gaines
    ville, 527 p,


    N. benthamiana used for virus purification. Electron microscopy of
    purified BlCMV-l negatively stained with molybdate revealed the
    presence of tubular inclusions with only trace amounts of host com
    ponents (Fig. 11). At high magnification, striations of protein sub
    units were observed on individual tubes (Fig. 11-D). These regularly
    spaced striations were estimated to have a periodicity of approximately
    5 nm. Striations with similar periodicity have been observed in
    cytoplasmic inclusions induced by several other potyviruses (Edwardson
    et al., 1968; Hiebert and McDonald, 1973; and Morales and Zettler,
    1977). Few virus particles were observed in the purified preparations
    of BlCMV-l which were not reactive to B1CMV-As (Fig. 12). Purified
    preparations of BlCMV-l with the highest degree of purity were obtained
    from N. benthamiana, with yields of 5 to 20 A^g^ units were usually
    obtained from 100 g of fresh weight of N_. benthamiana or \l_. unguiculata
    tissues. The ultraviolet absorption spectrum obtained for SDS disas
    sociated BlCMV-l was typical of proteins, with a maximum at 277 nm
    and a minimum at 246 248 nm (Fig. 5). Polyacrylamide gel electro
    phoresis of SDS-disrupted inclusion proteins revealed a single subunit
    component estimated to have a MW of 70,000 dal tons (Fig. 10).
    Virus Particle Size and Stabi1ity in Sap
    Electron microscopic examinations of purified preparations of
    BICMV negatively stained with PTA indicated that 73% of 190 virus
    particles measured were between 700 and 800 nm with a modal length
    of 753 nm. Particle measurements of several leaf-dip preparations
    negatively stained with PTA and of grids prepared for SSEM with
    infected cowpea leaf tissue gave modal lengths of 758 and 780 nm,


    Figure 22 Single radial immunodiffusion tests with hypocotyl extracts
    from healthy (H) and B1CMV-infected (l), 4-5-day-old cow-
    pea seedlings in media containing 0.8% Noble agar, 1.0%
    NaN^, 0.5% SOS, and 15% B1CMV antiserum (A, C) and 15%
    normal serum (B). The top row and the two bottom rows
    of wells in C were charged with extracts from healthy
    hypocotyls and the others were randomly filled with ex
    tracts from groups of 5 hypocotyls containing 1 or 2
    B1CMV-infected hypocotyl per group.


    Figure 9 Schlieren patterns from sedimentation velocity experiment
    with stored (A), and fresh (B) purified preparations
    of B1CMV. Photograph was taken 8 minutes after the rotor
    reached a speed of 27,690 rpm. Sedimentation is from
    left to right.


    Polyacrylamide gel electrophoresis of SDS dissociated cytoplasmic
    inclusions and viral coat proteins clearly indicated that ths viral
    coat protein subunit was smaller than the inclusion subunit (Fig. 10).
    The PAGE results revealed that the inclusions were made of a single
    kind of protein with an estimated molecular weight of 70,000 daltons.
    Polyacrylamide gel electrophoresis of cytoplasmic inclusion prepara
    tions conducted by Hiebert and McDonald (1973) showed one protein
    component with molecular weight of 67,000 daltons for PVY; 67,000 for
    PeMV; 69,300 for BiMV; 69,600 for TEV; and 70,300 for TuMV. The PAGE
    studies also indicated that freshly purified B1CMV consisted of a main
    protein component with a molecular weight around 34,000 daltons. Two
    smaller protein components were also revealed by PAGE analysis of SDS
    denatured viral coat protein (Fig. 10). Since only traces of the
    faster moving proteins were observed with fresh purified B1CMV, and
    greater amounts of these proteinaceous components were revealed by
    PAGE analyses of stored purified B1CMV preparations (Fig. 10), it is
    assumed that the smaller components are due to the degradation of the
    slow moving protein during purification and storage. Hiebert and
    McDonald (1976) observed that some possible enzymatic degradation of
    TuMV capsid protein occurred during storage of purified virus prepara
    tions. The lower sedimentation coefficient estimated for stored
    purified B1CMV preparations (Fig. 9) is further evidence of proteolytic
    degradation of viral coat protein during storage at 4 C. According
    to Hiebert and McDonald (1976), it is likely that '^q values reported
    for potyviruses that are near 140 S represent virus with partially
    degraded capsid protein, whereas those near 160 S represent virus with
    intact capsid protein."
    This proteolytic degradation also changes


    97
    light box or with a binocular microscope (6 to 15 x) in which the
    plates were illuminated from the bottom. Extracts of groups of 5
    hypocotyls were placed into wells (3 mm in diameter) punched in the
    agar medium in a row arrangement. The wells were spaced 3 to 4 mm
    from each other as measured from the edges of the wells and as many
    as 230 germinated seeds could be tested in a 90 x 15 mm plastic petri
    dish. Precipitin rings around the wells charged with extracts from
    infected tissue could be detected anywhere between one to 24 hr.
    Serologically Specific Electron Microscopy
    Hypocotyl extracts prepared in 0.05 M Tris buffer, pH 7-2, con
    taining 0.15 M NaCl and 0.4 M sucrose were examined for the presence
    of B1CMV by SSEM as described previous1 y (oerrick and Brlansky, 1976).
    Copper grids with Parlodion film coated with carbon were treated with
    B1CMV antiserum diluted to 1/1000 in 0.05 M Tris buffer, pH 7.2. The
    grids were washed with 0.05 M Tris buffer and floated on drops of
    hypocotyl extracts lor 3 to 4 hr at room temperature. After washing
    with approximately 2 ml of the extracting buffer and then with ap
    proximately I ml of deionized water, the grids were positively stained
    with 1.0% uranyl acetate in 50% ethanol. The grids were washed again
    with 50% ethanol, dried and examined in the Philips Model 200 electron
    microscope. The sensitivity of SSEM to detect the presence of B1CMV
    particles in extracts from a mixture of hypocotyls was determined by
    diluting infected 'Knuckle Purple Hull' hypocotyl tissue with non-
    infected tissues up to a dilution of 1/50 (w/w). The different mix
    tures of infected and noninfected hypocotyl tissues were ground in


    73
    by magnesium ions were reported by Govier and Woods (1971). They in
    dicated that in the presence of Mg ions the particles were straight,
    contrasting with the flexuous particles observed in the absence of
    Mg ions. On antiserum-coated grids several antibodies combine with
    a single virus particle and, possibly, increase; its length. Because
    of the specific antigen-antibody reaction the B1CMV particles were so
    strongly attached to the surface of the antiserum-coated grids that
    they could not be removed by repeated washing. On the other hand,
    positive staining of B1CMV particles with ethanolic uranyl acetate
    may have induced some changes in their lengths. Measurements of 25
    B1CMV particles on grids prepared by conventional leaf-dip preparation
    with PTA gave a modal length similar to that estimated for purified
    B1CMV negatively stained with PTA. Milne and Luisoni (1977) emphasized
    that negative staining gives better preservation and better resolution
    of viral capsids than positive staining. Using SSEM with uranyl
    acetate as a negative stain, Milne and Luisoni (1977), observed no
    change in the normal lengths of TMV and a potexvirus. However, leaf-
    dip preparations often contain too few particles to photograph con
    veniently for virus particle measurements, whereas relatively large
    numbers of particles can be photographed by using the serologically
    specific electron microscopic technique. As indicated by Derrick
    and Brlansky (1976), the addition of sucrose in the extracting and
    washing buffers greatly reduced the amount of plant debris on the
    SSEM grids. High amounts of plant debris in electron microscopic
    preparations are frequent problems in establishing the dimensions of
    a virus.


    148
    Phatak, H. C. 1974. Seed-borne plant virus identification and
    diagnosis in seed health testing. Seed Sci. Technol. 2:3~155.
    Phatak, H. C., J. R. Diaz-Ruiz, and R. Hull. 1976. Cowpea ringspot
    virus: a seed transmitted cucumovirus. Phytopathol. Z. 87:132-
    142.
    Pierce, W. H., and C. W. Hungerford. 1929. A note on the longevity
    of the bean mosaic virus. Phytopathology 19:605-606.
    Porto, M. D. M., and D. 1. Hagedorn. 1975. Seed transmission of a
    Brazilian isolate of soybean mosaic virus. Phytopathology 65:
    713-716.
    Provvidenti, R., and E. D. Cobb. 1975- Seed transmission of bean
    common mosaic virus in tepary bean. Plant Dis. Rep. 59:966-969.
    Purcifull, D. E. 1966. Some properties of tobacco etch virus and its
    alkaline degradation products. Virology 29:8-14.
    Purcifull, D. E., and D. L. Batchelor. 1977. Immunodiffusion tests
    with sodium dodecyl sulfate (SDS)-treated plant viruses and plant
    viral inclusions. Univ. Florida Agrie. Exp. Stn. Bull. No. 788
    (Tech.). 39 p
    Purcifull, D. E., S. R. Christie, and D, L. Batchelor. 1975. Preser
    vation of plant virus antigens by freeze-drying. Phytopathology
    65:1202-1205.
    Purcifull, D. E., J. R. Edwardson, and S. R. Christie. 1970. A morpho
    logical comparison of inclusions induced by tobacco etch and
    potato Y viruses. Phytopathology 60:779~803.
    Purcifull, D. E., and G. V. Gooding. 1970. Immunodiffusion tests
    for potato Y and tobacco etch viruses. Phytopathology 60:1036-
    1039.
    Purcifull, D. E., E. Hiebert, and J. G. McDonald, 1973- Immunochemical
    specificity of cytoplasmic inclusions induced by viruses in the
    potato Y group. Virology 55 = 275~279-
    Purcifull, D. E., and R. J. Shepherd. 1964. Preparation of the protein
    fragments of several rod-shaped plant viruses and their use in
    agar-gel diffusion tests. Phytopathology 54:1102-1108.
    Quacquarel 1 i A., and A. Avgelis. 1975. Ni cot ana bent hamiana Domin,
    as host for plant viruses. Phytopathol. Medit. 14:36-39-
    Raheja, A. K., and 0. I. Leleji. 1974. An aphid-borne virus disease
    of irrigated cowpea in Northern Nigeria. Plant Dis. Rep. 58:1080-
    1084.


    400 600 800 1000 1200 l400 1600
    PARTICLE LENGTH (nm)
    NUMBER
    OF PARTICLES


    96
    tests in agar medium containing 0.8% Noble agar, 0.5% SDS, and 1.0%
    pH 7.2. Eight to twelve discs of individual hypocotyls were embedded
    in the agar medium, k to 5 mm away from each antiserum well. Undiluted
    antiserum for BlCliV was routinely used in these tests, but dilutions
    of 1/2, 1/4, and 1/8 of the antiserum with either normal serum or
    0,05 M Tris buffer, pH 7.2 were also tested against hypocotyl discs
    and extracts of hypocotyl tissue. As controls each hypocotyl was also
    tested against normal serum, and hypocotyls from noninfected seeds
    were also included in each test. Extracts from groups of hypocotyls
    were tested by double immunodiffusion against B1CMV antiserum. Hypo
    cotyl extracts were pipetted into the antigen wells distributed in an
    hexagonal arrangement around the antiserum well. All plates were in
    cubated in a moist chamber at 2k C for 2k to k8 hr. The sensitivity
    of double immunodiffusion for detection of B1CMV in extracts of bulked
    hypocotyls of germinated 'Knuckle Purple Hull' seeds was tested by
    mixing 1.0 g of infected hypocotyl tissue with different amounts of
    noninfected hypocotyls.
    Single Radial Immunodiffusion Tests
    Discs and extracts of hypocotyls were tested by single radial
    immunodiffusion in medium containing 0.8% Noble agar, 0.5% SDS,
    1.0% NaN^ and 15% antiserum for B1CMV buffered with 0.05 M Tris-HCI,
    pH 7.2. Hypocotyl discs were embedded directly into the solidified
    agar medium with the aid of forceps and the plates were incubated
    in a moist chamber at 2k C for 2k to 72 hr. Precipitin reactions
    were detected by direct observation of the plates on a darkfield


    Figure
    Page
    24
    Electron micrographs of serologically specific
    electron microscopy with BlCMV, BCMV-S, and CPMV
    115
    25
    Double immunodiffusion tests with hypocotyls of 4-5-
    day-old bean and soybean seedlings using antiserum
    for BCMV-S and for SoyMV,
    118
    26
    Electron micrographs of serologically specific
    electron microscopy with extracts from BCMV- and
    SoyMV-infected hypocotyls
    120
    27
    Double immunodiffusion tests with hypocotyl extracts
    from 4-5-day-old seedlings of cowpea and soybean
    using antisera for B1CMV, SoyMV, and their cyto
    plasmic inclusions
    123
    28
    Photomicrographs showing different views of cytoplas
    mic inclusions induced by BlCMV in epidermal strips
    of cowpea hypocotyl tissue stained with a combination
    of calcomine orange and luxol brilliant green
    125
    29
    Photomicrographs showing different views of epidermal
    cells of hypocotyls from 4-5-day-old soybean seedlings
    containing cytoplasmic inclusions induced by SoyMV...
    127
    30
    Electron micrographs of ultrathin sections of cells
    from hypocotyls of 4-5-day-old seedlings infected
    with BlCMV
    129
    31
    Electron micrographs of ultrathin sections of
    hypocotyl cells of 4-5~day-o|d soybean seedlings
    grown from SoyMV-infected seeds...
    131
    i X


    Figure 27 Double immunodiffusion tests with hypocotyl extracts from
    ^-5-day-old seedlings of cowpea (A) and soybean (B) using
    antisera for BICMV, SoyMV, and their cytoplasmic inclusions.
    A) Serological test for detecting BICMV cytoplasmic
    inclusions (BICMV-l) in hypocotyls of ^-S-day-old
    cowpea seedlings grown from B1CMV-infected seeds.
    The center wells were charged with: (is) B1CMV- I
    antiserum, (Vs) BICMV antiserum, and (Ns) normal
    serum. The peripheral wells were charged with
    SDS-treated extracts from: (l) B1CMV-infected
    hypocotyls, and (H) healthy hypocotyls.
    B) Serological test for detection of SoyMV cyto
    plasmic inclusions (SoyMV-l) in hypocotyls of
    ^-S-day-old soybean seedlings grown from SoyMV-
    infected seeds. The center wells were charged
    with: (is) SoyMV-l antiserum, (Vs) SoyMV anti
    serum, and (Ns) normal serum. The peripheral
    wells were charged with SDS-treated extracts from:
    (l) SoyMV-infected hypocotyls, and (H) healthy
    soybean hypocotyls.


    ]k
    Clarification with n-but.anol and chloroform-carbon tetrachloride.
    Because n-butanol resulted in virus preparations of higher purity, but
    chloroform-carbon tetrachloride was superior for preservation of in
    clusion proteins (Hiebert, unpublished), these solvent systems were
    combined for purification of virus and inclusions from the same batch
    of tissue. Infected tissue was homogenized with two parts (w/v) of
    0.5 H KPO^, pH 7.5, containing 0 5 1.0% Na^SO^. The homogenate was
    filtered through cheesecloth and subjected to centrifugation at 11,700 g
    for 10 min. The supernatant was used for virus purification as de
    scribed previously using rvbutanol for clarification. The pellet was
    resuspended in approximately 2 volumes of 0.5 M KP0(, pH 8.2, 0.5%
    Na2S0^, homogenized with one volume of chloroform-carbon tetrachloride
    (1:1, v/v) and centrifuged at 5,000 rpm for 5 min in a Sorvall Centri
    fuge. The aqueous phase was subjected to a centrifugation at 11,700 g
    for 15 min. The supernatant was collected for additional virus puri
    fication using PEG, equilibrium density-gradient centrifugation and
    differential centrifugation. The pellet was resuspended in 0.05 M
    KP0,, pH 8.2, containing 0.1% 2-ME and treated with 5% Triton X-100.
    The inclusions were then purified by sucrose step gradient centrifuga
    tion as described above.
    Virus Particle Size Determination
    Crude leaf extracts from systemically infected cowpea plants and
    purified virus preparations were negatively stained in 2% potassium
    phosphotungstate (PTA), pH 6.5, containing 0.1% bovine serum albumin
    (BSA) prior to photography in an electron microscope. The procedure
    used was similar to those previously described (Edwardson et al., 1968
    and Purcifull et al., 1970). Small pieces of B1CMV-infected leaf were


    LIST OF TABLES
    Table Page
    I Symptoms and results of serological assays on
    varieties of cowpea, Vigna unguiculata mechanically
    inoculated with B1CMV, BCMV-S, CMV, and CPMV 70
    II Comparison of immunodiffusion tests with hypocotyl
    discs and growirig-on tests for detection of virus-
    infected seeds 1 08
    v i


    57
    components purified from V. ungu¡culata by high speed centrifugation
    according to the method used by Purcifull et al. (1973)- The high
    specificity of most of the antisera obtained against purified B1CMV
    preparations confirmed the efficiency of the methods used for its
    purification. The titers of antiserum varied depending on the bleeding
    date and on the rabbits, but 32 was the highest antiserum titer es
    timated by SDS-gel double immunodiffusion tests with a series of dilu
    tions (1/2, 1/4, 1/d, 1/16, and 1/32) of B1CMV-infected cowpea tissue
    prepared in 1.5% SDS.
    Antiserum specific for cytoplasmic inclusions induced by B1CMV
    was obtained from a rabbit injected with preparations of BICMV-I
    purified from infected tissue of hL benthamiana. The B1CMV-I antiserum
    reacted specifically with purified preparations of B1CMV-1 and crude
    sap of B1CMV-infected cowpea, but not with either purified preparations
    of B1CMV or crude sap of noninocu1ated plants (Fig. 12-B). The posi
    tive reactions with BICMV-I were more evident after 48 hr of incuba
    tion. The results obtained with B1CMV antiserum also indicated that
    B1CMV was not serologically related to its cytoplasmic inclusions
    (Fig. 12-A). Attempts to obtain specific antiserum by injecting
    rabbits with BICMV-I purified from infected cowpea tissue were unsuc
    cessful. All three rabbits injected with BICMV-I purified from in
    fected cowpea developed high titers of antibodies specific for normal
    plant tissue antigens.
    Single radial immunodiffusion studies in SDS-agar medium im
    pregnated with the virus antiserum indicated that appropriate SDS and
    antiserum concentrations need to be previously selected for highest
    sensitivity and to avoid spurious reactions. The best results were


    76
    (Tremaine and Wright, 1967; and Koenig and Givord, 197*0- The results
    of the present study indicated that the immunization program and the
    conditions of the antigenic solution used for rabbit immunization may
    also affect the antiserum specificity. A highly specific antiserum
    for BCMV-S was obtained from a rabbit immunized with freshly purified
    BCMV-S, whereas antiserum with a broader cross-reactive spectrum was
    obtained from the same rabbit after a booster injection with a purified
    preparation of BCMV-S stored at 4 C for more than one month. The use
    of such antisera would make it difficult to distinguish between certain
    plant viruses in SDS immunodiffusion tests. The serological distinc
    tion between BICMV and BCMV-S was impossible to detect in SDS double
    immunodiffusion when the BCMV-S antiserum with a wider cross-react ivity
    was used. On the other hand, an antiserum with a wide spectrum of
    activity should be useful for identification of vi rus-infected tissue
    used for plant propagation and possibly for identification of virus
    at the group level. As any vi rus-infected plant organ is undesirable
    for plant propagation the specific virus identification may not be
    necessary in such cases. For example, the BCMV-S antiserum was suc
    cessfully used to identify cowpea seeds infected with B1CMV.
    Unilateral serological relationships observed between B1CMV and
    SoyMV (Fig. 15G) and with B1CMV and BYMV (Fig. 15-H) showed the ne-
    nessity of reciprocal tests for demonstrating the absence of serologi
    cal relationship between two viruses. According to Matthews (1970)
    "to demonstrate that two viruses are serologically unrelated, reactive
    antisera must be prepared against each of the viruses under test."
    Reciprocal tests are also important, to show distinction between two
    closely related viruses. It was more difficult to observe a spur


    86
    The main purpose of the present investigation was to develop ef
    ficient and rapid serodiagnostic techniques to assay legume seed lots
    for the presence of vi rus-infected seeds. Seeds of cowpea, \l_. unguiculata
    'Knuckle Purple Hull* infected with B1CMV were used as a model host-
    virus combination. Immunochemical techniques for detection of B1CMV,
    BCMV, and SoyMV in hypocotyls of germinated vi rus-infected seeds of
    \l_. unguiculata, P_. vulgaris, and G^. Max, respectively, are described
    in this chapter. Abstracts of portions of this research have already
    been published (Lima and Purcifull, 1977a, 1977b),
    Literature Review
    The phenomenon of seed transmission of plant viruses was first
    demonstrated by Reddick and Stewart (1919), who presented strong
    evidence of seed transmission of BCMV in Phaseolus vulgaris. Since
    then, a large body of information has been accumulated about the
    transmission of numerous plant viruses through the seeds of infected
    host plants (Fulton, 1961*; Bennett, 1966; Shepherd, 1972; Baker, 1972;
    and Phatak, 1971*). Among the 183 plant viruses described in the
    Commonwealth Mycological Institute up to September, 1977, (Doi et al.,
    1977), 51 viruses have been experimentally demonstrated to be seed-
    borne to some extent. Several plant viruses are known to be seed-borne
    in many leguminous host plants, but this review will cover only those
    viruses transmitted through seeds of Vigna spp., Glycine max, and
    Phaseolus vu1 garis.


    78
    unabsorbed TMV antiserum. For a full precipitation of the crossreacting
    antibodies close to the center well, a fairly high concentration of
    the heterologous antigen should be used to fill the antiserum well. This
    is illustrated by the intragel cross-absorption tests with B1CHV anti
    serum shown in Figure 16. A precipitin ring was formed very close to
    the center well when a highly concentrated purified preparation of
    BCMV-S (0.5 1.0 mg/ml) was used to absorb B1CMV antiserum (Fig. 16-D)
    whereas the ring formed approximately 2 mm away from edge of the well
    when B1CMV antiserum was absorbed with a less concentrated preparation
    of CAMV (0.01 0.05 mg/ml) (Fig, 16-B). In both cases, though, the
    intragel cross-absorption test showed serological distinction between
    the viruses. The intensity of the reaction between the homologous
    antigen and the cross absorbed antiserum may give some information
    about the degree of relationship between the viruses. Weaker homologous
    reaction indicates closer serological relationship. Based on this,
    the results of the present study clearly indicate that B1CHV is more
    closely related serologically to BCMV-S than to CAMV (Fig. 16-B, -D).
    The different degrees of serological relationships are also indicated
    by the intensity of the precipitin lines spurring over the heterologous
    virus reactions in straight diffusion tests (Fig. 16-A, -C). Serological
    relationship between different potyviruses has been commonly observed
    (Bercks, I960; Purcifull and Shepherd, 196*4; Purcifull and Gooding,
    1970; (Jyemoto et a I 1972; and Shepard et al., 197*0, and the cross -
    absorption of an antiserum with heterologous viruses has also been
    used to study serological relationship between plant viruses in tube
    precipitin tests (Wetter, 1967; and Alba and Oliveira, 1977), and in
    combination with gel diffusion tests (van Regenmortel, 1966; Wetter,


    Figure 8 Histograms of BICMV particle lengths from two different
    electron microscopic preparations to show particle length
    distribution from 600 to 900 nm. Class interval = 20 nm.
    A) Particle length distribution of BICMV from purified
    preparation negatively stained with phosphotungstate;
    B) Particle length distribution of BICMV from cowpea
    leaf extract prepared on grids sensitized with BICMV
    antiserum and positively stained with uranyl acetate.


    26
    \


    CHAPTER I
    PURIFICATION, PARTIAL CHARACTERIZATION, AND
    SEROLOGY OF BLACKEYE COWPEA MOSAIC VIRUS
    Introduction
    Cowpea, Vigna unguiculata (L.) Walp. (=Vigna sinensis (L.) Endl.),
    is grown as a crop in high-temperature areas of tropical and subtropi
    cal countries. Cowpea seeds constitute a source of good quality pro
    tein and dried seeds are an important part of the diet of many people
    in the tropical and subtropical world, particularly in Africa and the
    rural zone of northeastern Brazil. The fresh seeds and immature pods
    are also eaten and they can be frozen or canned as is sometimes done
    in the United States. Cowpeas are also grown as fodder plants for hay,
    silage or pasture and used as a green manure and cover crop. When
    grown under optimum conditions, cowpea can produce seed yields as high
    as 2,600 Kg/ha. However, several factors limit cowpea yields in most
    fields. Virus diseases are considered as a major limiting factor to
    the production of cowpeas in several countries (Dale, 1949; Wells and
    Deba, 1961; Toler et al., 1963; Brantley et al., 1965; Kuhn et al.,
    1966; Harrison and Gudauskas, 1968a; Harrison and Gudauskas, 1968b;
    Gay and Winstead, 1970; Zettler and Evans, 1972; Bock, 1973; Phatak,
    1974; Haque and Persad, 1975; Kaiser and Mossahebi, 1975; and Lima and
    Nelson, 1977). Several viruses infect cowpea, and many of them can be
    transmitted through seeds from infected cowpea plants. The most im
    portant cowpea seed-borne virus in the southeastern United States is
    1


    82
    Serological studies with such decomposed leaf tissue and B1CMV
    antiserum gave results similar to those obtained with the CAMV isolate
    (Fig. 16-A) obtained originally from Morocco. As the inactivation
    of the virus in the decomposed tissue may have destroyed some of its
    antigenic determinants, no conclusive results about its serological
    relationship with BICMV can be derived from these tests.
    Light and electron microscopy of cowpea and other host cells in
    fected with any one of these three legume potyviruses revealed that
    their cytoplasmic inclusions are morphologically similar. In ultrathin
    sections, their inclusions consisted of pinwheels similar to those
    induced by the potyviruses from Edwardson's subdivision-I (Edwardson,
    197*0. The cytoplasmic inclusions induced either by BCMV-S or CAMV,
    however, failed to react with antiserum for B1CMV induced inclusions.
    The low titer of the inclusion antiserum, however, may be one of the
    reasons for the absence of reactions. Despite the great similarity
    in the ultrastructures of pinwheel inclusions induced by B1CMV and CAMV,
    they showed some difference at the light microscope level. Whereas
    B1CMV induced big masses of cytoplasmic inclusions in 'Knuckle Purple
    Hull1 (Fig. 17), only scattered small bundles of inclusions were ob
    served in the cells of this host infected with CAMV. This is also an
    indication of no direct correlation between the severity of the symp
    toms and abundance of cytoplasmic inclusions induced by these poty-
    viruses, since 'Knuckle Purple Hull' is more susceptible to CAMV than
    to B1CMV (Table I). A similar phenomenon was observed with these two
    viruses in spectabilis. In addition to this, the nuclear inclu
    sions readily observed in cells of £. spectabilis systemically




    I certify that I have read this study and that in my opinion it
    conforms to acceptable standards of scholarly presentation and is
    fully adequate, in scope and quality, as a dissertation for the
    degree of Doctor of Philosophy.
    /7__
    Daniel A. Roberts
    Professor of Plant Pathology
    I certify that I have read this study and that in my opinion it
    conforms to acceptable standards of scholarly presentation and is
    This dissertation was submitted to the Graduate Faculty of the College
    of Agriculture and to the Graduate Council, and was accepted as partial
    fulfillment of the requirements for the degree of Doctor of Philosophy.
    March 1978
    Dean, College of Agriculture
    Dean, Graduate School


    Figure 21 Double immunodiffusion tests with hypocotyl from healthy
    and Bl CHV-infected, 4-5-day-old cowpea seedlings in medium
    containing 0.8% Noble agar, 1.0% NaN^ and 0.5% SDS, pre
    pared in water.
    A B) Serological tests with hypocotyl extracts prepared
    in water. The center wells were charged with:
    (As) B1CHV antiserum, and (Ns) normal serum. The
    peripheral wells were charged with extracts from:
    (1) B1CHV-infected hypocotyls, (H) healthy hypo-
    cotyls, (5) one gram of B1CHV-infected hypocotyl
    mixed with 4 g of healthy hypocotyls, (10) one
    gram of B1CMV-infected hypocotyl and 9 g of
    healthy hypocotyls, (15) one gram of infected
    hypocotyl and 14 g of healthy hypocotyls, (20)
    one gram of infected hypocotyl and 19 g of healthy
    hypocotyls, (25) one gram of infected hypocotyl
    and 24 g of healthy hypocotyls, and (30) one gram
    of infected hypocotyl and 29 g of healthy hypo
    cotyl s.
    C D) Serological tests with saml1 discs of different
    hypocotyls embedded into the agar medium 4 5 mm
    away from the antiserum well. Ten (c) or twelve
    (d) hypocotyl discs are embedded around the cen
    ter wells and the hypocotyl discs are numbered
    from the top in a clockwise direction (arrows).
    Wells were charged with: (As) B1CMV antiserum, and
    (Ns) normal serum. Note virus-antiserum specific
    reactions with hypocotyls: 1, 2, 8, 11, 14, 16,
    18, 19 (0, and 6, 8, 9, 14, 16, 20, and 23 (D).
    E F) Serological tests with hypocotyl extracts (E) and
    hypocotyl discs (F) from healthy and B1CHV-
    infected cowpea seedlings. The center wells were
    charged with: (A) undiluted BICMV antiserum, (A:2)
    B1CMV antiserum diluted 1/2 with normal serum,
    (A:4) B1CMV antiserum diluted 1/4 with normal
    serum, and (A:8) B1CMV antiserum diluted 1/8 with
    normal serum. The peripheral wells were charged
    with extracts from: (5) one gram of B1CHV-infected
    hypocoty1s,and 4 g of healthy hypocotyls, (10) one
    gram of B1CHV-infected hypocotyls and 9 g of healthy
    hypocotyls, and (H) healthy hypocotyls. Hypocotyl
    discs from healthy (h) and B1CHV-infected (i)
    seedlings were alternated around the antiserum wells
    in F.


    139
    Athow, K. L,. and J, B. Bancroft. 1959- Development and transmission
    of tobacco ringspot virus in soybean, Phytopathology 49:697-701.
    Baker, K. F. 1972. Seed pathology. Pages 317-416 j'r^ T. T. Kozlowski,
    ed. Seed Biology Vol, II. Academic Press, New York, 447.
    Bancroft, J, B 1970. Brome mosaic virus. No, 3 j_n_ Descr¡ptions of
    plant viruses. Commonw. Mycol, Inst., Assoc. Appl. Biol, Kew
    Surrey, England. 4 p,
    Bancroft, J. B. 1971. Cowpea chlorotic mottle virus. No. 49 j_n^ De
    scriptions of plant viruses. Commonw. Mycol, Inst,, Assoc, Appl.
    Biol,, Kew, Surrey, England. 4 p.
    Barnett, 0. W., and M. Alper. 1977, Characterization of Iris fulva
    virus. Phytopathology 67:448-454,
    Batchelor, D. L. 1974. Immunogenicity of sodium dodecyl sulfate
    denatured plant viruses and paint viral inclusions, Ph.D. Dis
    sertation, University of Florida, Gainesville, 8] p,
    Beier, H., D J, Siler, M. L. Russell, and G, Bruening. 1977. Survey
    of susceptibility to cowpea mosaic virus among protoplasts and
    intact plants from V i gna sinensis lines. Phytopathology 67:917-921,
    Bennett, C. W. 1966 Seed transmission of plant virus. Pages 221-
    261 jm K, M. Smith and M, A Lauffer, eds. Advances in virus
    research, Vol. 14. Academic Press, New York. 350 p.
    Bercks, R. I960. Serological relationships between beet mosaic virus,
    potato virus Y, and bean yellow mosaic virus. Virology 12:311-313.
    Bock, K. R. 1973. East African strains of cowpea aphid-borne mosaic
    virus. Ann. Appl. Biol, 74:75-83.
    Bock, K, R,, and M. Conti, 1974. Cowpea aphid-borne mosaic virus,
    No, 134 j_n_ Descr i pt ions of plant viruses. Commonw, Mycol, Inst.,
    Assoc, Appl. Biol,, Kew, Surrey, England, 4 p.
    Bos, L 1971. Bean common mosaic virus. No, 73 in Descriptions of
    plant viruses. Commonw. Mycol, Inst., Assoc. Appl. Biol,, Kew,
    Surrey, England, 4 p.
    Bos, L. 1972, Soybean mosaic virus. No. 93 _ij^ Descriptions of plant
    viruses. Commonw. Mycol. Inst., Assoc, Appl. Biol,, Kew, Surrey
    England, 4 p.
    Bos, L., and D. Z. Maat, 1974. A strain of cucumber mosaic virus,
    seed-transmitied in beans, Neth, J, PI. Pathol, 80:113-123.
    Brandes, J. 1964. I dentif i zierung von gestreckten pflanzenpathogenen
    viren auf morphologischer Grundlage. Mitt. biol. BundAnst. Ld u.
    Forstw. 110:1-130.


    revealed a difference in S values between B1CMV in fresh preparations
    and B1CHV in purified preparations stored at A C for more than 30
    days. Both virus preparations showed a single sedimenting peak, but
    the s2q values for BICMV in fresh preparations and at a concentration
    varying from 0.5 to 1.0 mg/ml ranged from 157 to 159 S while the S2Q
    values for the virus in the stored preparations and at the same con
    centrations ranged from 1 AO to IA2 S (Fig. 9). The lower sedimenta
    tion coefficients obtained for the stored virus suggested that a
    change in virus mass (MW) had occurred. Hiebert and McDonald (1976)
    observed some possible enzymatic degradation of capsid protein of
    purified turnip mosaic virus. Proteolytic degradation of capsid
    protein of stored purified preparations of B1CMV was also observed
    by polyacrylamide gel electrophoresis (PAGE) studies. Polyacrylamide
    gel electrophoresis analysis of SDS-degraded virus of a freshly
    purified preparation of B1CMV revealed a main protein component with
    an estimated molecular weight (MW) of 3^,000 daltons and two smaller
    ones with MWs of 29,000 and 27,000 daltons (Fig. 10). These smaller
    components may have arisen by degradation of the slow moving component
    during storage (Hiebert and McDonald, 1976). Stored BICMV preparations
    contained only the faster moving protein components with MWs of 29,000
    and 27,000 daltons (Fig. 10), presumably derived from 3^,000 daltons
    component.
    Purified Inclusion Preparations
    Using either of the methods outlined in Figs. 3 and A, purified
    cylindrical inclusions induced by BICMV were obtained from the same
    batches of systemically infected leaf tissue of V. unguiculata or


    szi


    Abstract of Dissertation Presented to the Graduate Council
    of the University of Florida in Partial Fulfillment of the Requirements
    for the Degree of Doctor of Philosophy
    BLACKEYE COWPEA MOSAIC VIRUS: PURIFICATION, PARTIAL CHARACTERIZATION,
    SEROLOGY, AND IMMUNOCHEMICAL AND CYTOLOGICAL TECHNIQUES
    FOR DETECTION OF VI RUS-INFECTED LEGUME SEEDS
    By
    J. ALBERS 10 A. LIMA
    March, 1978
    Chairman; Dan E. Pureifull
    Major Department: Plant Pathology
    Blackeye cowpea mosaic virus (B1CMV) was increased in cowpea, Vigna
    ungui cu lata (L.) Walp., 'Knuckle Purple Hull1, and infected leaves were
    used for virus and cytoplasmic inclusion purification. Either n-butano]
    or a combination of chloroform and carbon tetrachloride was used in the
    clarification process. Polyacrylamide gel electrophoresis of sodium
    dodecyl sulfate (SDS) dissociated inclusions and virus revealed that
    the inclusions were made of a single protein estimated to have a molecu
    lar weight (MW) around 70,000 daltons whereas freshly purified B1CMV
    consisted of a main protein component with a MW of 3^,000 daltons and
    two smaller proteins with MWs of 29,000 and 27,000 daltons. Purified
    B1CMV had a 260/280 nm absorption ratio of 1.2 and a modal length of
    753 nm. Freshly purified BICMV preparations showed a single sedimenting
    peak with s2157-159 S. The purified BICMV cytoplasmic inclusions
    had absorption spectra characteristic for proteins. Electron micro
    scopy of purified inclusions revealed the presence of tubes showing
    striations with periodicities of approximately 5 nm.


    Figure 12 Double immunodiffusion tests in agar medium containing 0.8% Noble agar, 1.0% NaN and
    0.5% SDS. 3
    A B) Serological tests to demonstrate specificity of B1CMV antiserum (Vs), and
    antiserum obtained against B1CMV induced cytoplasmic inclusions (Is). The
    peripheral wells were filled with the following antigenic solutions prepared
    in 1.5% SDS: (V) purified B1CMV, (l) purified preparations of B1CMV inclusions,
    (Co) extracts of B1CMV-infected cowpea, and (H) crude extract of healthy cow-
    pea. All antigens were also tested against normal serum (Ns).
    C) Serological relationship between B1CMV and a New York isolate of B1CMV.
    The center wells were filled with: B1CMV antiserum (Bis), and normal serum
    (Ns). The peripheral wells were charged with SDS-treated extracts from:
    (Bl) B1CMV-infected cowpea, (NY) isolate of B1CMV from New York in cowpea,
    and (H) healthy cowpea.
    D) Serological relationship between a strain of bean common mosaic virus isolated
    from siratro (BCMV-S) and the B1CMV isolate from New York. The center wells
    were charged with: (Ss) BCMV-S antiserum, and (Ns) normal serum. The peri
    pheral wells were charged with SDS-treated extracts from: (S) bean leaves
    infected with BCMV-S, (NY and N) cowpea leaves infected with the New York
    isolate of B1CMV, (H-lower) healthy bean,and (H-upper) healthy cowpea.


    93
    an infected egg-cell or an infected pollen grain. Studies to determine
    the distribution of BCMV in developing reproductive tissues suggested
    that successful seed transmission was insured by the presence of in
    fective virus within the embryo itself (Ekpo and Saettler, 197*0.
    A correlation between symptom severity and percentage of seed
    transmission of BCMV has been observed in different bean varieties
    (Smith and Hewitt, 1938). Medina and Grogan (1961) suggested that the
    percentage of seed transmission of BCMV was greatly affected by the
    differences in varietal susceptibility of the beans. However, Zettler
    (1966) found similar percentages of seed transmission of BCMV in 5
    different bean varieties.
    Using the bean cultivar VC 1822 as an indicator host for BCMV,
    Provvidenti and Cobb (1975) demonstrated a rate of seed transmission
    from 7 to 22% for BCMV in tepary bean (Phaseolus acutifolius Gray var.
    1 atifo 1ius Freem.), and observed that the virus was carried in the
    embryo but not in the testa. Two BCMV isolates obtained from bean
    grown in the Netherlands were observed to be transmitted through ap
    proximately 20 to 8t)% of seeds harvested from infected bean plants
    (Drijfhout and Bos, 1977).
    A strain of CMV isolated from bean plants grown in Spain was also
    demonstrated to be seed-borne in one of twelve bean varieties studied
    by Bos and Maat (197*0. As they tested only a few seeds from virus-
    infected plants of each bean variety, they suggested that a low per
    centage of seed transmission could possibly occur in other varieties.
    The transmissib i 1ity of SBMV through seeds of bean was first
    demonstrated by Zaumeyer and Harter (19*+3) Cheo (1955) found a very


    115
    t .
    *
    j
    l
    * s*
    Ji
    t,
    r
    f

    1000 rfhv ,
    t :
    I :
    ;vv
    -O^rv^
    y \ .
    r'^ .
    v


    152
    van Kammen, A. 1971. Cowpea mosaic virus. No. 47 in Descriptions of
    plant viruses. Commonw. Mycol. Inst., Assoc. Appl. Biol., Kew,
    Surrey, England, A p.
    van Kammen, A. 1972. Plant virus with a divided genome. Annu. Rev.
    Phytopathol. 10:125-151.
    van Oosten, H. J. 1972. Purification of plum pox (sharka) virus with
    the use of Triton X-100. Neth. J, PI. Pathol. 78:33-44.
    van Regenmortel, M. H. V. 1966. Plant virus serology. Pages 207-271
    j_r^ K. M. Smith, and M. A. Lauffer, eds. Advances in virus research
    Vol. 12. Academic Press, New York. 394 p.
    van Regenmortel, M. H. V., and M. B. von Wechmar. 1970. A reexamina
    tion of the serological relationship between tobacco mosaic virus
    and cucumber virus 4. Virology 41:330-338.
    van Velsen, R. J. 1962. Cowpea mosaic, a virus disease of Vigna
    s i nensis in New Guinea. Papua New Gn. Agrie. J. 14:153-161.
    Verma, V. S., 1971. Effect of heat on seed transmission of mosaic
    disease of cowpea (V igna s i nensis Savi). Acta Microbiologica
    Polonica 3:163-166.
    Vidano, C. 1959. Indagini sopra un deperimento della V igna sinens i s
    Endlicher in cultura italiana. Boll. Zool. Agr. Bachic. 3:1-97.
    Vidano, C., and M. Conti, 1965. Transmission i con afidi di un "cowpea
    mosaic virus" isolato da Vigna sinensis Endl. in Italia. Atti
    Accad. Sci. 99:1041-1050.
    Voller, A., A. Bartlett, D. E. Bidwell, M. F. Clark, and A. N, Adams.
    1976. The detection of viruses by enzyme-1 inked immunosorbent
    assay (ELISA). J. Gen. Virol. 33:165-167.
    Vovlas, C,, and A. Avgelis. 1972. Le virosi delle piante ortensi in
    Puglia, VIII. Un mosaico del Fagiolino dall'occhio. Phytopathol.
    Medit. 11:117-118.
    Walters, H. J., and 0. W. Barnett, Jr. 1964. Bean leaf beetle
    transmission of Arkansas cowpea mosaic virus. Phytopathology
    54:911 (Abstr.).
    Weber, K., and M, Osborn. 1969. The reliability of molecular weight
    determinations by dodecyl sulfate-poly-acrylamide gel electro
    phoresis. J. Biol. Chem. 244:4406-4412.
    Wells, D. G., and R. Deba. 1961. Sources of resistance to the cowpea
    yellow mosaic virus. Plant Dis. Rep. 45:878-881.


    Table II Comparison of immunodiffusion tests with hypocotyl discs and growing-on tests for
    detection of vi rus-infected seeds.
    Seed Source
    V i rus
    Seed
    Assay
    Methods
    Tested
    Sero1ogy
    Growi ng'
    -on Test
    Proportion of
    Infected Seeds
    Percentage of
    Infected Seeds
    Proportion of
    Infected Seeds
    Percentage of
    Infected Seeds
    Cowpea (V. unguiculata)
    Knuck1e Purple Hull
    Lot A
    B1CMV
    72/323*
    22.3
    33/160
    20.6
    Lot B
    B1CMV
    12/160
    7.5
    13/173
    7.5
    Lot C
    B1CMV
    5/115
    4.3
    8/145
    5.5
    Lot D
    B1CMV
    17/160
    10.6
    13/141
    9.2
    Early Ramshorn
    B1CMV
    5/100
    5.0
    34/545**
    6.3**
    Soybean (G. max)
    Jupiter
    SoyMV
    4/176
    2.8
    2/89
    2.2
    Bean (P. vulgaris)
    Black Turtle
    BCMV
    3/79
    3-8
    Not tested
    Not tested
    (*) Number of vi rus-infected seeds over number of seeds tested;
    (**)- Pio-Ribeiro, G. (unpublished).


    o

    D O
    Q
    ,
    0.
    o 0 g>

    .N
    L
    .w


    85
    Barley stripe mosaic virus has no known vector, such as insects or
    mites, but it has a high rate of seed transmission, which severely
    reduces crop production. On the other hand, LMV is transmitted by
    a low percentage of seeds, but is regularly spread further in the
    field by aphids, resulting in substantial losses (Zink et al., 1956).
    Several methods (Phatak, 197*0 have been developed to detect the
    presence of infected seeds to control plant, diseases caused by seed-
    borne viruses: Growing-on tests. Seeds are planted in greenhouses
    or under other insect-proof conditions and the first leaves of the
    seedlings are observed for the characteristic symptoms which vary
    according to the host-virus combination This method can fail under
    environmental conditions that adversely affect the symptom development
    and with latent strains of a virus that do not produce visible symp
    toms. Indicat or-inoculation tests. Seeds are ground up with buffer
    solution and mechanically inoculated in the indicator hosts. Although
    this method has been used extensively for LMV (Phatak, 197**), it is
    very time consuming and requires considerable greenhouse space.
    Serological tests Immunochemical tests have also been developed
    for detecting virus in extracts from single seeds (Scott, 1961, and
    Lister, 1977), and individual seed embryos (Hamilton, 1965). However,
    no successful results have been obtained in double immunodiffusion
    systems with long flexuous rod viruses such as those from the potyvirus
    group (Phatak, 197A). Electron microscopy. A serologically specific
    electron microscopic technique developed by Derrick and Brlansky (1976)
    has been successfully used to detect the presence of virus particles
    in extracts of groups of seeds (Brlansky and Derrick, 1976).


    101


    Figure 23 Electron micrographs of B1CMV in hypocotyl extracts from
    the same cowpea seedlings using different preparations,
    (A) serologically specific electron microscopy (SSEM)
    with B1CMV antiserum diluted 1/1000 in 0.05 M Tris-HCl
    buffer, pH 7.2, and positively stained with uranyl
    acetate, (B) dip preparation with 2% phosphotungstic acid
    pH 6.5, containing 0.1% BSA, (C) preparation similar
    to SSEM using normal serum instead of BICMV-ant¡serum,
    and (O) preparation similar to SSEM using Tris buffer
    instead of antiserum. The micrograph A represents a
    typical view of the entire grid whereas micrographs B,
    C, and D are selected areas of the grids showing virus
    part icles (arrows).


    72
    yields of viruses in the PVY group (Shepherd and Pound, I960; van
    Oosten, 1972; Hiebert and McDonald, 1973; Uyeda et al., 1975; and
    Barnett and Alper, 1977). Hiebert and McDonald (1973) reported
    aggregation of virus particles after PEG precipitation. The losses
    of BICMV by low speed centrifugation due to aggregation of virus
    particles were reduced by maintaining the virus in KPO^, buffer, pH
    8.2, after precipitation with PEG.
    Another critical aspect on purification of potyviruses for ob
    taining maximum virus yield is the host used for virus increase. In
    order to obtain a good yield of BICMV from the 'Knuckle Purple Hull1
    variety of cowpea the virus was inoculated into the source plants
    at the age of 3 to 4 days after emergence and the systemically in
    fected leaf tissues were harvested 15 to 18 days after inoculation.
    Attempts to purify the virus from plants inoculated later than that
    or from tissue harvested more than 30 days after inoculation resulted
    in very poor yields of virus and cytoplasmic inclusions.
    Electron microscopic examinations of purified preparations of
    BICMV indicated a low percentage of virus fragmentation during the
    purification processes (Fig. 6-A). Particle measurements of purified
    BICMV and of BICMV particles on grids prepared for SSEM with infected
    cowpea leaf tissue gave two modal lengths (Figs. 7, 8) which differed
    by approximately 30 nm. Variations in lengths of virus particles have
    been extensively observed (Edwardson, 1974). As reviewed by Edwardson
    (1974), virus length variations may be attributed to several factors,
    including sample preparation, host influence, virus strain differences,
    and normal fluctuations in the electron microscope magnification. In
    crease of 50 to 100 nm in certain potyvirus particle lengths induced


    Wave Length (nm)
    Absorbance of Purified B1CMV
    0.40


    153
    Wetter, C. 1967- Immunodiffusion of tobacco mosaic virus and its
    interaction with agar. Virology 31:^98-507.
    Williams, R. J. 1975. Diseases of cowpea (Vigna unguiculata (L.)
    Walp.) in Nigeria. PANS 21:253-267.
    Williams, R. J. 1977a. Identification of multiple disease resistance
    in cowpea. Trop. Agrie. 5^-53-59-
    Williams, R. J. 19771>. Identification of resistance to cowpea (yellow)
    mosaic virus, lrop. Agrie. 5^:61-67.
    Yu, T. F. 19^+6. A mosaic disease of cowpea (Vigna sinensis End 1.) .
    Ann. Appl. Biol. 33:450-^5**-
    Zaumeyer, W. J., and L. L. Harter. 19^3- Two new virus diseases of
    beans. J. Agrie. Res. 67 305~327-
    Zettler, F. W. 1966. Aphid populations in central New York as related
    to epiphytology of bean common mosaic virus. Ph.D. Dissertation.
    Cornell University, Ithaca, New York. 120 p.
    Zettler, F. W. R. G. Christie, and J. R. Edwardson. 1967. Aphid
    transmission of virus from leaf sectors correlated with intra
    cellular inclusions. Virology 33:5^9-552.
    Zettler, F. W., I. R. Evans. 1972. Blackeye cowpea mosaic virus in
    Florida: Host range and incidence in certified cowpea seed.
    Proc. Fla. State Hortic. Soc. 85:99-101.
    Ziemiecki, A., and K. R. Wood. 1975. Serological demonstration of
    virus-specific proteins associated with cucumber mosaic virus
    infection of cucumber cotyledons. Physiol. Plant Pathol. 7:171-
    177.
    Zink, F. W., R. G. Grogan, and J. E. Welch. 1956. The effect of
    percentage of seed transmission upon subsequent spread of lettuce
    mosaic virus. Phytopathology ^+6 :662-66^4.


    600 700 800 900
    PARTICLE LENGTH (nm)
    NUMBER OF PARTICLES
    o
    PO -Pi
    o o
    O'
    o
    oo
    o
    o
    no -P cn
    o o o
    00
    o
    -F-


    83
    infected with BlCMV (Christie and Edwardson, 1977) were not seen in
    leaf tissue of this host infected with CAMV.
    In summary, BlCMV is a potyvirus that belongs to Edwardson's
    subdivision-I (Edwaidson, 197*0 and has a modal length of approximately
    750 nm. The BlCMV particles have a single sedimenting peak with s^q =
    157 159 S and have a main protein component with a MW of 3*t,000
    daltons. Its cytoplasmic inclusions are made of tubes which show
    striations with periodicities of approximately 5 nm and consist of a
    single type of protein estimated to have a MW of 70,000 daltons. The
    virus also induces nuclear inclusions in certain hosts including C.
    spectabi 1is. Blackeye cowpea mosaic virus is serologically unrelated
    to seven potyviruses and serologically related to, but distinct from
    eight other potyviruses in SDS-immunodiffus ion. The virus has a narrow
    host range outside leguminosae, is seed-borne in at least two cowpea
    varieties and is transmitted by aphids in a nonpersistent manner.
    Based on its physical, biological, cytological and immunochemical
    properties, BlCMV can be differentiated from any other virus that
    infects cowpea. The antisera prepared for BlCMV and its cytoplasmic
    inclusions were essential tools for the development of the serological
    techniques for detection of vi rus-infected seeds described in Chapter II.


    10
    laminated aggregates. Light and electron microscopic investigations re
    vealed that B1CMV induces nuclear inclusions in C_. spectabi 1 is (Zettler
    et al., 196?; Edwardson et al., 1972; and Christie and Edwardson, 1977),
    while no such inclusions were observed in cells of spectab i 1 i s
    infected with BYMV. Based on those cytological distinctions, Edwardson
    et al. (1972) concluded that BICMV and BYMV are distinct members of
    the potyvirus group Subsequently, Zettler and Evans (1972) demonstrated
    that BICMV and BYMV had dissimilar host ranges, providing additional
    evidence that they are distinct viruses.
    In host range studies, BICMV was shown to be very similar to BCMV,
    but different from watermelon mosaic virus-2 (WMV-2), (Uyemoto et al.,
    1973). Leaf-dip preparations of B1CMV-infected tissue revealed the
    presence of flexuous rods, 750 nni long, and double immunodiffusion
    tests with BCMV arid WMV-2 antisera indicated that BICMV was serologi
    cally identical to BCMV and related to, but distinct from WMV-2
    (Uyemoto et al., 1973).
    Materials and Methods
    Source of Virus Isolate
    The blackeye cowpea mosaic virus used in this study was isolated
    from infected seeds of cowpea V_. ungu ¡culata 'Knuckle Purple Hull'
    harvested from a field in Gainesville, Florida. The virus was trans
    mitted by aphids from infected cowpea plants grown from infected seeds
    to non-infected 'Knuckle Purple Hull1 plants. Two aphids (M. persicae)
    were used per test plant and each aphid was allowed to have an acqui
    sition period of 30 to 60 sec. A single test plant showing typical
    mosaic was assayed by leaf-dip electron microscopy for the presence of


    18
    Schlieren optics. The data were corrected for standard water viscosity
    conditions at 20 C, but not for the effect of virus concentration. The
    virus concentrations used varied from 0.5 to 1.0 mg/ml.
    Serology
    Antiserum product ion for virus and cytoplasmic inclusions. Antisera
    were obtained by injecting a New Zealand white rabbit with untreated
    virions and a second rabbit with pyrrolidine-degraded virus protein.
    All rabbits selected for immunization were first bled to produce normal
    sera. The concentrations of untreated B1CMV in 0.02 M Tris buffer,
    pH 8.2, used in the immunization process varied from 1.0 to 2.0 mg of
    nuc1eoprotein per ml of purified solution. B1CMV used for pyrrolidine
    degradation was suspended in 0.005 M borate buffer, pH 8.2. The virus
    protein was degraded according to the method used by Shepard (1972).
    A virus solution was mixed with an equal volume of 5% pyrrolidine in
    distilled water (v/v). The mixture was then immediately dialyzed
    against two liters of 0.05 M borate buffer, pH 8.2, containing 0.37%
    actual formaldehyde for approximately A8 hr at A C to remove the
    pyrrolidine and fix the protein subunits.
    A series of A to 5 intramuscular injections was given to each
    rabbit with an interval of 10 to 15 days between the injections. Each
    injection consisted of 1.0 to 2.0 ml preparations of virus or degraded
    viral protein vigorously emulsified with equal volume of Freund's com
    plete or incomplete adjuvants (Difco). Booster injections were given
    at intervals of about 2 months.
    The immunized animals were bled every week, starting 10 to 15
    days after the last injection of the initial series of A 5 injections.


    67


    4
    that 6 varieties and 16 pure lines were resistant. Robertson (1965)
    screened 79 cowpea varieties for resistance to CYMV in a screened
    greenhouse. Those varieties that showed no local or systemic reactions
    when inoculated with the virus were classified as immune; those that
    developed necrotic lesions but did not become systemically infected
    were classified as resistant; and those that showed systemic infection
    were classified as susceptible. Chant (1962) found that the cowpea
    virus from Trinidad caused local lesions on Chenopodiurn ama rant¡color
    Coste and Reyn., Mucuna atterrina Holland, Petunia hybrida Vilm., and
    P. vulgaris, and that the virus was polyhedral with a mean diameter
    of approximately 25 nm.
    Doub1e-immunodiffusion tests showed that a cowpea virus from
    Arkansas and the Trinidad cowpea mosaic virus were closely related,
    but not identical serologically and that both were anti gen i cal Iy related
    to bean pod mottle virus (BPMV) (Shepherd,1963). Studying other proper
    ties of the virus, Shepherd (1964) confirmed a close similarity of the
    Arkansas virus with the cowpea mosaic virus from Trinidad (Dale, 1949).
    Walters and Barnett (1964), working with a cowpea mosaic virus serologi
    cally identical to the Arkansas isolate, demonstrated also that it was
    efficiently transmitted by the bean leaf beetle, C. trifurcata. A
    detailed study of three cowpea mosaic virus isolates from Surinam
    (South America), along with the previously reported cowpea viruses
    from Trinidad (Dale, 1949) and Nigeria (Chant, 1959, I960, 1962) re
    vealed that they are strains of cowpea mosaic virus (Agrawal, 1964).
    Detailed descriptions of host range, biophysical, biochemical, and
    immunochemical properties of cowpea mosaic virus were reported, and
    the abbreviation CPMV was proposed to eliminate any possible confusion


    133
    Several techniques have been developed for diagnosing seed-borne
    viruses in routine seed health testing laboratories. Phatak (197*0
    grouped those techniques into dry examination (visual inspection of
    dry seeds), biological tests (growing-on and infectivity tests), bio
    chemical tests (colorimetric, histochemica1, and serological tests),
    and biophysical tests (electron microscopy). According to Phatak
    (197**) the highly specific serological tests are the best among the
    tests available to assay seed lots for the presence of vi rus-infected
    seeds. Several serological tests have been used for detecting virus-
    infected seeds (Scott, 1961; Hamilton, 1965; Phatak, 197**; Slack and
    Shepherd, 1975; Lundsgaard, 1976; and Lister, 1977). However, the
    present investigation contains the first successful results with
    double immunodiffusion tests for detecting potyviruses in germinated
    seeds.
    The use of hypoc.otyl tissue of germinated legume seeds eliminated
    the problem of nonspecific reaction commonly observed with extracts
    from seeds per se or germinated seeds, including roots, cotyledons,
    and primary leaves in immunodiffusion tests (Fig. 19). Cockbain et
    al. (1976) reported that when embryos of Vicia faba L. minor seeds
    were tested by double immunodiffusion, the agar became clouded,
    obscuring the virus-specific precipitation lines. The nonspecific
    precipitates that interfered with the vi rus-spec ific reactions (Fig. 19)
    may be related to the high concentration of haemagg1utin ins (lectins)
    reported to be present in legume seeds (Toms and Turner, 1965; Moreira
    and Perrone, 1977; and Fountain and Yang, 1977). It has been demon
    strated that lectins bind specifically to mono and polysaccharides as


    Page
    CHAPTER II IMMUNOCHEMICAL ANO CYTOLOGICAL TECHNIQUES FOR
    DETECTION OF LEGUME VIRUSES IN INFECTED SEEDS.... 84
    I nt roduct ion., 84
    Literature Review.....,....,,.,, 86
    Seed-Borne Viruses in V i gna spp 87
    Seed-Borne Viruses in Glycine max 90
    Seed-Borne Viruses in Phaseolus vulgaris... 92
    Materials and Methods 94
    Source of Seed and Seed Germination 94
    Preparation of Antigens for Serology 95
    Double Immunodiffusion Tests 95
    Single Radial Immunodiffusion Tests 96
    Serologically Specific Electron Microscopy. 97
    Double Immunodiffusion Tests and SSEM for
    Detection of Other Viruses in Germinated
    Legume Seeds 98
    Serology and Microscopy of Cytoplasmic
    Inclusions Induced by B1CMV and SoyMV
    in Hypocotyls of Germinated Seeds......... 98
    Results,.., 99
    Preparation of Antigens for Serological
    Tests 99
    Double Immunodiffusion Tests 102
    Single Radial Immunodiffusion Tests 107
    Serologically Specific Electron Micro
    scopy. Ill
    Double Immunodiffusion Tests and SSEM
    for Detection of Other Viruses in
    Germinated Legume Seeds...,.., 116
    Serology and Mycroscopy of Cytoplasmic
    Inclusions Induced by BlCMV and SoyMV in
    Hypocotyls of Germinated Seeds 121
    Discuss ion.. 132
    LITERATURE CITED. 138
    BIOGRAPHICAL SKETCH 154
    v


    Figure 29 Photomicrographs showing different views (A, B, C, D) of
    epidermal cells of hypocotyls from ^5_day-old soybean
    seedlings containing cytoplasmic inclusions (arrows)
    induced by SoyMV. The hypocotyl epidermal strips were
    stained with a combination of calcomine orange and luxol
    brilliant green. (Cl) cytoplasmic inclusions, (CW)
    plant cell wall, and (Nu) nucleus.


    13
    The homogenized mixture was centrifuged in a Sorvall Centrifuge at
    5,000 rpm for 5 niin and the pellet containing the organic solvents was
    discarded. The aqueous phase was centrifuged at 13,200 g for 15 min
    to precipitate the virus induced inclusions. The supernatant was
    treated as previously described for virus purification and the pellet
    containing the inclusions was resuspended in 0.05 M KPO^, pH 8.2, and
    0.1% 2-ME. The inclusion suspension was homogenized in a Sorvall
    Omni-mixer homogenizer for 2 min and enough Triton X-100 was added to
    make a final concentration of St (v/v). After stirring for one hour
    at 4 C this mixture was subjected to a low speed centrifugation of
    27,000 g for 15 min to precipitate the inclusions. The pellet was
    resuspended in 10 to 20 ml of 0.02 M KPO^, pH 8.2, containing 0.1%
    2-ME, and homogenized for 30 sec. The inclusions were sedimented
    again by centrifugation of 27,000 g for 15 min. The pellet was homogen
    ized for 30 sec and the homogenate was layered on a sucrose step
    gradient made up of 10 ml of 80%, 7 ml of 60%, and 7 ml of 50% (w/v)
    sucrose in 0.02 M KPO^, pH 8.2. The gradient was centrifuged for one
    hour at 27,000 rpm in a Beckman SW 25.1 rotor. The inclusions layered
    on top of the 80% sucrose zone and were collected by droplet from the
    bottom of the tube. To remove the sucrose, the inclusions were diluted
    in 0.02 M KPO^, pH 8 2, and precipitated by a centrifugation at 27,000
    for 15 min. The pellet was resuspended in 0,02 M Tris, pH 8,2, and
    inclusion yield was estimated spectrophotometrica11y after being dis
    rupted in 2% sodium dodecyl sulfate (SDS). The inclusion preparations
    were either immediately used for immunization of rabbits or stored at
    -20 C by either freezing directly or by freeze-drying.


    BIOGRAPHICAL SKETCH
    Jos Albersio ele Araujo Lima was born July 12, 19*40, in Santana
    do Cariri, Cear, Brazil, He graduated from high school in December,
    1959, at the Colegio Estadual do Cear in Fortaleza, Cear, Brazil.
    He joined the Army in December, 1958, and was honorably dismissed as
    an Army Reserve Officer in December, I960. He attended the Universidade
    Federal do Cear, receiving the Bachelor of Science degree in Agronomy,
    along with a diploma of Agronomic Engineering, in December, 1966. In
    March, 1967, he was hired as an Auxiliary Professor of Plant Pathology
    by the Universidade Federal do Cear, and in 1973, he was promoted to
    Assistant Professor, a position that he has held up to the present
    time. He started a graduate program in Plant Pathology at the Univer
    sity of Arizona, Tucson, in September, 1970, and received his Master
    of Science degree in 1972, In September, 197*4, he entered the Univer
    sity of Florida, receiving the Doctor of Philosophy degree in March,
    1978. He is the author or co-author of approximately ten publications
    of researches in Plant Pathology.
    J, Albersio A. Lima is married to the former Diana Mara de
    Gurgel Caracas, and he is the father of a son, Roberto. He is a mem
    ber of the American PhytopathoIogica1 Society, the Brazilian Phyto-
    pathological Society, the Association of Agronomic Engineering from
    Cear, the Association of University Professors from Cear, and the
    Latinoamerican Association of Plant Pathology.
    15*4


    99
    Cytoplasmic inclusions were examined in epidermal strips of the
    hypocotyls of germinated cowpea seeds infected with B1CMV, and of
    SoyMV-infected hypocotyls of germinated soybean seeds, after staining
    with a combination of calcomine orange and "luxol" brilliant green
    (Christie, 1967).
    Small pieces of B1CMV-infected and noninfected cowpea hypocotyls
    were prepared for ultrathin sectioning as described previously in
    Chapter I. Similarly, healthy and SoyMV-infected soybean hypocotyls
    were prepared for ultrathin sectioning. Sections were cut with a
    diamond knife and stained with potassium permanganate, uranyl acetate,
    and lead citrate. All specimens were examined with a Philips Model
    200 electron microscope.
    Resu 1ts
    Preparation of Antigens for Serological Tests
    Antigens prepared from hypocotyls of germinated seeds proved to
    be very satisfactory for indexing seeds in double and single immuno
    diffusion tests. Neither small discs nor extracts from hypocotyl
    tissues showed any kind of nonspecific reaction that could interfere
    with the virus-specific reaction in double or in single immunodiffusion
    tests. On the other hand, extracts obtained from whole seedlings,
    including roots, cotyledons and primary leaves showed several types
    of precipitat ion patterns when tested against any serum in double
    diffusion tests (Fig. 19). Such nonspecific precipitations were also
    observed with extracts obtained from cotyledons, primary leaves or
    root tissues. These nonspecific reactions were reduced when the agar


    6
    had properties somewhat similar to the cowpea strain of SBMV, the two
    viruses were not serologically related (Shepherd, 1963)-
    A carlavirus isolated from cowpea in Ghana was described and
    designated as cowpea mild mottle virus (CMMV) by Brunt and Kenten
    (1973) and Brunt (197*0- Cowpea mild mottle virus is seed-borne in
    cowpeas, is 650 nm in length and is apparently not transmitted by
    aphids.
    A virus with small isometric particles, isolated from Iranian
    cowpea seeds was considered as new and named cowpea ringspot virus
    (CpRV) on the basis of symptomatology and particle morphology, which
    were similar to other ringspot viruses (Phatak, 197*+; and Phatak et
    al., 1976). According to Phatak (197*+) the virus was not transmitted
    by aphids, induced intracellular inclusions in cowpea, had a wide
    experimental host range and was serologically unrelated to AO other
    isometric viruses most of which commonly infect various legumes. Cow
    pea ringspot virus was also transmitted in 15-20% of the seeds of three
    cowpea cultivars (Phatak et al., 1976).
    McLean (1 9*+1) studied some physical and biological properties of
    a cowpea virus and found that it was transmitted by the following
    species of aphids: Macrosiphum solani Ashm., Acynthosiphon pisum
    (Harris), Aphis gossypii Glover, Myzus persi cae (Sulz.) but not by
    the bean leaf hopper (Empoasca fabae Le. B.), the tarnished plant bug
    (Lygus pratensis L.), the Mex¡can bean beetle (Epilachra corrupta Mis.),
    and the striped cucumber beetle (Diabrotica vittata Faba). Snyder
    (19*+2) described a mosaic disease of asparagus bean, Vigna sesquipedal is
    Wight, and also studied some biological and physical properties of
    the causal agent. His positive results obtained with aphid transmission


    123


    Figure 5 Absorption spectra of purified preparations of B1CMV
    in 0.02 H Tris buffer, pH 8.2, and B1CMV cytoplasmic
    inclusions in the same buffer.


    32
    INFECTED TISSUE


    135
    detect TRSV and SoyMV in individual soybean seeds (Lister, 1977)- The
    high sensitivity of SSEM for detection of particles of B1CMV, BCMV,
    and SoyHV in infected hypocotyI extracts as well as of other seed-borne
    viruses in seed extracts (Brlansky and Derrick, 1976) make it suitable
    for identification of vi rus-infected seeds when an electron microscope
    is available. The ELISA and SSEM techniques, however, are more complex
    than the double immunodiffusion tests and/or require the use of sophis
    ticated equipment such as an electron microscope.
    A close correlation was observed between the percentages of virus-
    infected seeds estimated by the simplified double immunodiffusion tech
    nique with discs of individual hypocotyls and the growing-on test in
    different legume seed lots (Table ll). The advantages of the hypocotyl
    disc-double immunodiffusion technique over the growing-on test are
    that: a) it is faster and more specific, b) it does not require green
    house space, and c) it does not depend on environmental conditions for
    symptom development. The growing-on test is largely used, sometimes
    in combination with serology, for indexing seed lots for the presence
    of v i rus-infected seeds. The growing-on test, however, requires
    suitable temperature and light under insect-proof conditions for a
    period varying from 2 to 6 weeks. Kaiser and Mossahebi (197*0 reported
    that at times, virus symptoms did not appear until the second or third
    trifoliolate leaf showed up on bean plants grown from seeds infected
    with BCMV. According to Sharma and Varma (1975), the symptoms of cow-
    pea banding mosaic virus appeared only on the first trifoliolate or
    the subsequent leaves of cowpea under low temperature conditions. Tosic
    and Pesie (1975) observed that the greatest number of alfalfa seedlings


    Table I. Symptoms and results of serological assays on varieties of cowpea, Vigna unguiculata
    mechanically inoculated with B1CMV, BCMV-S, CAMV, and CPMV.
    Cowpea Varieties
    Symptoms
    (a)
    Serology
    (b)
    BlCMV
    BCMV-S
    CAMV
    CPMV
    B! CMV
    BCMV-S
    CAMV
    CPMV
    Black Local
    Mt
    M
    +
    _
    +
    _
    Bola de Ouro
    -
    -
    Mt
    Mt
    -
    -
    +
    +
    CE-73
    -
    Mt
    Mt
    Mt
    4-
    4-
    4-
    CE-74
    Mt
    -
    Mt
    -
    +
    -
    +
    -
    CE-175
    M
    -
    Mt
    Mt
    +
    -
    +
    +
    CE-89
    M
    -
    Mt
    -
    +
    -
    +
    -
    CE-3-53
    Mt
    -
    M
    -
    +
    -
    +
    -
    Cream 40
    Mt
    -
    M, Ld
    M Ld.De
    +
    -
    +
    +
    Early Ramshorn
    Mt
    M
    M
    Ne, De
    +
    4-
    +
    +
    Crowder Pea
    -
    -
    Mt
    M, Ld
    -
    -
    +
    +
    1 peane V11
    Mt
    -
    Mt
    -
    +
    -
    +
    -
    Jaguaribe
    M
    -
    Mt
    -
    +
    -
    +
    -
    Knuckle Purple Hul1
    M
    -
    M, Ld
    M,Ld,De
    +
    -
    +
    4-
    Pitiuba
    Mt
    -
    M
    -
    +
    -
    +
    -
    Potomac
    M
    -
    Mt
    -
    +
    -
    +
    -
    Ser ido
    -
    -
    Mt
    Mt
    -
    -
    +
    +
    Snapper Long Pod
    -
    -
    M, Ld
    A.
    -
    -
    +
    -U
    Sete Semanas
    -
    -
    Mt
    -
    -
    -
    +
    -
    V-4 Alagoas
    M
    -
    Mt
    Chi
    +
    -
    +
    +
    V-5 Parayba
    M
    M
    Mt
    +
    -
    +
    +
    (a) Chl= systemic chlorosis, De= plant death; Ld= leaf deformation, M= mosaic, Mt= mottle, Ne=
    systemic necrosis, and = no symptoms.
    (b)
    positive serological reaction in double diffusion tests,
    no serological reaction in double diffusion tests.
    -'j
    o
    not tested


    19
    The rabbits were fasted for 4 12 hr prior to each bleeding and 30 -
    50 ml of blood were collected into glass tubes according to the proce
    dure described by Purcifull and Batchelor (1977). Blood samples were
    allowed to clot for approximately 45 min at 37 C in a waterbath. The
    clotted blood was subjected to a centrifugation of 2,000 rpm in a
    Sorvall table centrifuge for 10 min. The antisera were transferred
    with a Pasteur pipette to conica1-bottomed tubes and clarified by a
    second centrifugation at 5,000 rpm for 10 min. Antiserum specificity
    and titer were determined by Ouchterlony (1962) double-diffusion tests
    in SDS-agar plates. The antisera were stored at -20 C by either
    freezing directly or after freeze-drying.
    The B1CMV-induced cytoplasmic inclusions (BICMV-l) used for anti
    serum production were purified from N_, bent ham i ana. Freshly purified
    cylindrical inclusions, which were unreactive with antiviral sera, were
    used for immunization and the foot pad route of immunization (Ziemiecki
    and Wood, 1975) was used. The rabbit received three injections into
    the foot pad, each containing 0.1 ml of purified inclusions (0.1 0.2
    O.D. units/ml at 280 nm) in 0.02 M Tris, pH 8.2, emulsified with an
    equal volume of either Freund's complete or incomplete adjuvants.
    Serological tests. Both double and single immunodiffusion tests
    in agar gel were used in the present study. Most double immunodiffusion
    tests were performed in agar medium containing 0.8% Noble agar (Difco);
    0.5% SDS (Sigma) and 1.0% NaN^ (Sigma) in deionized water (Purcifull
    and Batchelor, 1977), or 0.05 M Tris-HCl buffer pH 7-2. Reactant wells
    were punched in the solidified agar medium with an adjustable gel
    cutting device made by Grafar Corp., Detroit, Mich. Routinely the
    wells (7 mm in diameter) were punched in an hexagonal arrangement


    LIST OF FIGURES
    Figure Page
    1 Systemic and localized symptoms induced by blackeye
    cowpea mosaic virus (BICMV) in cowpea, V. ungui culata
    'Knuckle Purple Hull' and C. amarant ¡color 26
    2 Flow diagram outlining the procedure of purification
    of BICMV using n-butanol as clarifying agent 28
    3 Flow diagram outlining the procedure for purification
    of BICMV and its cytoplasmic inclusions, using
    chloroform and carbon tetrachloride as clarifying
    agents . 30
    4 Flow diagram outlining the steps carried out during
    the purification of BICMV and its cytoplasmic inclu
    sions by a combination of the first and second methods
    for purification of virus and inclusions 32
    5 Absorption spectra of purified preparations of BICMV
    in 0.02 M Tris buffer, pH 8,2, and BICMV cytoplasmic
    inclusions in the same buffer ^
    6 Electron microscopy of BICMV in a purified preparation
    and in cowpea leaf extracts ^7
    7 Histograms of lengths of BICMV particles from purified
    preparation negatively stained with phosphotungstate,
    and cowpea leaf extract using the serologically spe
    cific electron microscopy and uranyl acetate as a 39
    positive stain
    8 Histograms of BICMV particle lengths from two different
    electron microscopic preparations to show particle
    length distribution from 600 to 900 nm 41
    9 Schlieren patterns from sedimentation velocity experi
    ment with stored and fresh purified preparations of
    BICMV 44
    10 Electrophoretic analyses of BICMV induced cytoplasmic
    inclusions and BICMV capsid protein in 6% polyacryl
    amide gel 46
    11 Electron micrographs of purified preparations of
    BICMV cytoplasmic inclusions stained with molybdate.. 49
    v 1 1


    65
    leaves (Fig.17), and at high magnification end views of them could
    be seen as small dots by changing the microscope focus. In ultrathin
    sections of B1CMV-infected tissue, these inclusions consisted of tubes
    attached to a central core, forming pinwheels (Fig. 18), similar to
    those induced by the potyviruses from Edwardson's subdivision-I
    (Edwardson, 197*0- As reported by Edwardson (197*0, the pinwheels
    contained arms with pronounced curvatures and tight scroll-like
    tubular inclusions. Only tubes were observed in purified preparations
    of cytoplasmic inclusions induced by B1CMV (Fig. 11).
    Host Range and Resistant Cowpea Varieties
    Blackeye cowpea mosaic virus was readily transmitted mechanically
    from cowpea 'Knuckle Purple Hull' to the following plants in which it
    was detected serologically and caused the following symptoms:
    Crotalaria spectabilis (mosaic); Glycine max (L.) Mer. (mild mottle
    and chlorotic spots); Macroptilium atropurpureum (DC.) Urb. (mosaic);
    Macroptilium bracteatum (L.) Urb. (mosaic); Nicotiana benthamiana
    (mottle); 0cimum basi 1icum (local lesions); Phaseolus vulgaris 'Black
    Turtle-2' (epinasty, necrosis, yellowing) and 'Bountiful' (chlorotic
    spots on inoculated leaves); Vigna unguiculata 'Black Local' (mosaic),
    'Early Ramshorn' (mottle), 'Knuckle Purple Hull' (mosaic), and 12
    Brazilian cowpea cultivars in which the reactions varied from symptom
    less to mosaic (Table I). Small chlorotic lesions were found on the
    leaves of Chenopodium amarant¡color inoculated with purified prepara
    tions of B1CMV or cowpea sap containing BICMV (Fig. 1-B).
    Based on failure to induce symptoms and on negative serological
    results, BICMV did not infect Arachis hypogaea L. 'Florunner', Capsicum


    Figure 17
    - Photomicrographs (A, B, C, D) of cytoplasmic inclusions
    in epidermal strips of cowpea leaves systemically infected
    with B1CMV, stained with a combination of calcomine orange
    and luxol brilliant green. A) cells with masses of cyto
    plasmic inclusions, B) details of mass of inclusions seen
    in "A", c) general view of the inclusion distribution in
    epidermal cells, and D) phase contrast micrograph of the
    area photographed in "A". (Ci) cytoplasmic inclusions,
    (CW) plant cell wall, (G) guard cells, and (Nu) nucleus.
    f


    5
    with CMV (cucumber mosaic virus). Cowpea mosaic virus (CPMV) has been
    extensively studied in different laboratories and was fully described
    by van Kammen (1971, 1972). It was selected as the type member of the
    comovirus group (Fenner, 1976) and reported from several other parts
    of the world, including Brazil (earner et al., 1969; and Lima and
    Nelson, 1977), Nigeria (Williams, 1975), Venezuela (Debrot and Rojas,
    1967), and Puerto Rico (Perez and Cortes-Mon1 lor, 1970; and Alconero
    and Santiago, 1973).
    Kuhn (1964b) purified and characterized a new virus isolated
    from cowpea in Georgia and named it cowpea chlorotic mottle virus
    (CCMV), which was subsequently described by Bancroft (1971). This
    virus belongs to the bromovirus group (Fenner, 1976) and is physically
    similar to brome mosaic virus (Bancroft, 1970) and broad bean mottle
    vi rus (Gibbs, 1972) neither of which produces symptoms in cowpea
    (Bancroft, 1971).
    Strains of cucumber mosaic virus (CMV) are also known to infect
    cowpea. Cucumber mosaic virus strains have been isolated from naturally
    infected cowpeas showing mosaic symptoms in southeastern United States
    (Anderson, 1955a; Kuhn, 1964a; and Harrison and Gudauskas, 1968a),
    Italy (Vovlas and Avgelis, 1972), Morocco (Fischer and Lockhart, 1976b),
    and South Africa (Klesser, i960). An aphid-transmitted, spherical
    virus, approximately 25 run in diameter, was also reported from India
    by Chenulu et al. (1968). According to their descriptions, the virus
    closely resembles a strain of CMV.
    Shepherd and Fulton (1962) identified a seed-borne virus of cowpea
    as a strain of southern bean mosaic virus (SBMV) (Shepherd, 1971).
    Although a virus isolated from naturally infected cowpea in Arkansas


    145
    Karnovsky, M. J. 1965. A formaldehyde-glutaraldehyde fixative of high
    osmolality for the use in electron microscopy. J. Cell Biol.
    27:137A.
    Kendrick, J. B., and M. W. Gardner. 1924, Soybean mosaic: seed
    transmission and effect on yield. J. Agr. Res. 27:91-98.
    Kennedy, B. W., and R. L. Cooper. 1967. Association of virus infection
    with mottling of soybean seed coats. Phytopathology 57:35-37.
    Khatri, H. L., and L. Singh. 1974. Studies on a mosaic disease of
    cowpea. J. Res. 11:289-294,
    Klesser, P. J. I960. Viruses diseases of cowpea. Bothalia 7:233-251.
    Koenig, R., and L. Givord. 1974. Serological interrelationships in
    the turnip yellow mosaic virus group. Virology 58:119-125.
    Koshimizu, S., and T. lizuka. 1957. Relationship between the brown
    speck of soybean seed and soybean mosaic. Ann. Phytopathol. Soc.
    Japan 22:18 (Abstr.).
    Koshimizu, Y., and N. lizuka. 1963- Studies on soybean virus diseases
    in Japan. Tohoku Natl. Agrie. Expt. Stn. Bull. 27:1-103.
    Kuhn, C. W. 1964a. Separation of cowpea virus mixtures. Phytopa
    thology 54:739-740.
    Kuhn, C. W. 1964b. Purification, serology, and properties of a new
    cowpea virus. Phytopathology 54:853-857.
    Kuhn, C. W., B. B. Brantley, and G. Sowell. 1965. Immunity to bean
    yellow mosaic virus in cowpea. Plant Dis. Rep. 49:879-881.
    Kuhn, C. W., B. B. Brantley, and G. Sowell. 1966. Southern pea
    viruses: identification, symptomatology, and sources of resis
    tance. Univ. Georgia Agrie. Exp. Stn. Bull. 157. 22 p.
    Lima, J. A. A., and M. R. Nelson. 1975- Squash mosaic virus variabil
    ity: nonreciproca1 cross-protection between strains. Phytopa
    thology 65:837-8^0.
    Lima, J. A. A., and M. R. Nelson. 1977. Etiology and epidemiology of
    mosaic of cowpea in Cear, Brazil. Plant Dis. Rep. 61:864-867.
    Lima, J. A. A., and D. E. Purcifull. 1977a. Immunochemical tests
    for detection of blackeye cowpea mosaic virus in infected seeds.
    Proc. Am. Phytopathol. Soc. 4 (in press) (Abstr.).
    Lima, J. A. A., and D. E. Purcifull. 1977b. Serology and microscopy
    of cylindrical inclusions induced by blackeye cowpea mosaic and
    soybean mosaic viruses in hypocotyls of germinated seeds. Proc.
    Am. Phytopathol. Soc. ** (in press) (Abstr.).




    Figure 15
    - Reciprocal double immunodiffusion tests with B1CMV and
    other potyviruses in medium containing 0.8% Noble agar
    1.0% NaNj, and 0.5% SOS prepared in 0.05 M Tris-HCl
    buffer, pH 7-2. All the antigenic solutions were pre
    pared in 1.5% SOS. The center wells were charged with:
    (l) B1CMV antiserum, (2) PVY antiserum, (3) TEV antiserum,
    (A) WMV-2 antiserum, (5) DMV antiserum, (6) BbMV-BV-l
    antiserum, (7) BCMV-S antiserum, (8) SoyMV antiserum,
    (9) BYMV antiserum, (10) BiMV antiserum, and (ll) l.MV
    antiserum. The top rows of wells in all cases were
    charged with SDS-treated extracts from: (a) BICMV-
    infected cowpea, and (c.) healthy cowpea. The bottom rows
    of wells were charged with SOS-treated extracts from:
    A) PVY-infer ted tobacco (b), and healthy tobacco (d);
    B) TEV-infected tobacco (b), and healthy tobacco (d);
    C) WMV-2-infected pumpkin (b), and healthy pumpkin (d);
    D) DMV-infected dasheen (b), and healthy dasheen (d);
    E) BCMV-BV-1 infected bean (|>),and healthy bean (d) ;
    F) BCMV-S-infected bean (b), and healthy bean (d);
    G) SoyMV-in(ected N. benthamiana (b), and healthy
    N. bent hamiana (d); H] BYMV-infected pea (b) and
    healthy pea Td); l) BiMV-infected Nicotiana hybrid (b) ,
    and healthy Nicot¡ana hybrid (d); and J) LMV-infected
    pea (b), and healthy pea (d).


    107
    normal serum (Fig. 2 I -F). The percentages of B1CMV-infected seeds in
    four different lots of cowpea 'Knuckle Purple Hull' and one 'Early
    Ramshorn' seed lot were estimated by this simplified double immuno
    diffusion test and by the growing-on test (Table ll). Over a hundred
    seedlings tested by this serological technique were randomly checked
    by SSEM for the presence of BICMV particles and in all cases the results
    corresponded.
    Single Radial Immunodiffusion Tests
    The presence of BICMV in cowpea hypocotyl extracts was detected
    by single radial immunodiffusion tests in agar medium impregnated
    with B1CMV antiserum. Precipitin rings were observed around the
    wells charged with extracts from groups of 5 individual hypocotyls
    in which at least one was infected with BICMV, but not around those
    wells charged with extracts of noninfected hypocotyls (Fig. 22). The
    virus-specific reactions started to appear at approximately one hour
    after the hypocotyl extracts had been added into the wells and became
    very clear and evident 3 to 23 hr later. The reactions were still
    very distinct 24 hr after the hypocotyl extracts had been added into
    the wells.
    When small discs of individual hypocotyls were directly embedded
    into the agar medium containing BICMV antiserum, the virus-specific
    reactions took longer to appear. These specific reactions were re
    cognized as opalescent precipitates around infected hypocotyl discs
    that could be better detected under a binocular microscope. The
    opalescent precipitates which usually were located at the ends of the
    hypocotyl discs, started to appear in 24 hr, but were more evident


    87
    Seed-Borne Viruses in Vigna spp.
    Gardner (1927) apparently was the first to report the transmission
    of a cowpea virus through seeds of cowpea. Since then, many viruses
    which naturally infect cowpea have been demonstrated to be seed-borne
    in this host.
    A virus isolated from cowpea in Trinidad was demonstrated to be
    transmitted through 8% of seeds of asparagus-bean (Vigna sesquipedal is)
    obtained from vi rus-infected plants (Dale, 1949). The virus is be
    lieved to be a representative strain of cowpea mosaic virus (Agrawal,
    1964; and van Kammen, 1971, 1972). It seems that the seed transmis-
    sibility of CPMV is erratic and depends on the type of virus isolate
    and the cowpea variety involved. A cowpea mosaic virus isolated from
    cowpea grown in Arkansas was seed-borne in this host (Shepherd, 1964).
    Approximately 620 Blackeye1 cowpea plants grown from seeds harvested
    from plants artificially inoculated with CPMV failed to develop mosaic
    symptoms (Perez and Cortes-Mon11 or, 1970). On the other hand, Haque
    and Persad (1975) observed that the rate of seed transmission of CPMV
    varied from zero to 5-8% depending on the cowpea varieties and selec-
    t ions.
    Anderson (1957) reported the seed transmission of three cowpea
    viruses, including a strain of CMV, which was transmitted through
    4 28% of cowpea seeds from artificially infected plants, A virus
    closely resembling a strain of CMV was seed-borne in cowpea with a
    transmission rate of 5 to 16% (Chenulu et a 1. 1968). On the basis
    of symptoms observed on cowpea plants grown from commercial seeds,
    Gay and Winstead (1970) reported seed transmission of CMV, a cowpea


    91
    which is not seed-borne in soybean, also increased the percentage of
    mottled seeds (Ross, 1963).
    Ghanekar and Schwenk (197**) showed a rate of seed-transmission
    for tobacco streak virus varying from 2.6 to 30.6% according to the
    soybean cultivar.
    Among the nine viruses known to infect and cause diseases in soy
    bean in Japan, SoyMV, soybean stunt virus, soybean mild mosaic virus,
    peanut stunt virus, southern bean mosaic virus, and a strain of alfalfa
    mosaic virus were reported to be transmitted to some extent through
    seeds of soybean varieties (Koshimizu and lizuka, 1963; I izuka and
    Yunoki, 197**; Takahashi et a I. 197**; lizuka, 197**; and Tamada, 1977).
    The transmission of nepoviruses through seeds of their host is
    well documented. Tobacco ringspot virus (TRSV), a nepovirus (Fenner,
    1976) responsible for a disease commonly called bud blight of soybean,
    was first reported to be transmitted through seeds of artificially
    inoculated soybean plants in 195** (Desjardins et al., 195**). These
    results were confirmed by subsequent studies (Kahn, 1956; Owusu et al.,
    1968; Athow and Bancroft, 1959; and Demski and Harris, 197**). Kahn
    (1956) also demonstrated the seed-transmissib i 1 i ty of tomato ringspot
    virus in soybean and observed that soybean seeds from plants infected
    with either tobacco or tomato ringspot virus had a lower percentage
    of emergence than seeds from virus-free plants. Three other nepo
    viruses: arabis mosaic, raspberry ringspot and tomato black ring
    viruses were experimentally demonstrated to be transmitted through
    seeds of soybean plants artificially inoculated with these viruses
    (Lister, 1960; and Lister and Murant, 1967).


    113


    69


    116
    other grid preparations (Fig. 23). As the antiserum sensitized grids
    were washed several times during their preparation, a great reduction
    in the amount of plant debris on the grids was observed. On the other
    hand, when grids not previously treated with the antiserum were washed,
    the virus particles were also removed (Fig. 23-C, -D).
    The SSEM seemed to be also adequate to assay seeds for polyhedral
    viruses since it was successfully used to observe CPMV particles in
    cowpea leaf extracts using grids treated with antiserum specific for
    CPMV (Fig. 24-D).
    Double Immunodiffusion Tests and SSEM for Detection of Other Viruses
    in Germinated Legume Seeds
    The double immunodiffusion tests and SSEM used to detect B1CMV
    in germinated cowpea seeds were also useful for detecting BCMV in in
    fected germinated bean seeds and SoyMV in hypocotyls of germinated
    soybean seeds infected with SoyMV. Both legume viruses were detected
    by double immunodiffusion tests using hypocotyl extracts from groups
    of 5 seedlings and small discs of individual hypocotyls as assay
    antigens (Fig. 25). The results obtained in the simplified double
    immunodiffusion tests with hypocotyl discs were confirmed by SSEM,
    which also proved to be a very good technique to assay bean and soy
    bean seed lots for the presence of BCMV (Fig. 26-A) and SoyMV (Fig.
    26-B), respectively.
    The results obtained from the inoculation of the hypersensitive
    bean line VC-1822 with extracts from germinated bean seeds were also
    in agreement with those obtained by the immunochemical tests for BCMV.
    The percentages of BCMV-infected bean 'Black Turtle' seed lot and of


    Figure 20 Diagram showing methods for assaying legume seeds by
    single and double radial immunodiffusion: I) seeds
    are placed on moistened paper towels, 2) the towels
    are rolled up and placed upright in beakers, 3) ger
    minated seeds after four to five days, b~S) individual
    seedlings are divided into three parts: roots (r) ,
    hypocotyl (hy) and epicotyl (ep) consisting of
    cotyledons and primary leaves, 6) hypocotyl tissue
    is ground with mortar and pestle, 7) hypocotyl ex
    tracts are rested in double radial immunodiffusion
    (DRD), 8) hypocotyl extracts are also tested in single
    radial immunodiffusion (SRD), 3) small discs are cut
    from each hypocotyl, 10) discs from different hypo-
    cotyls are tested by DRD, and 11) discs from different
    hypocotyls are tested by SRD.


    17
    for 1.5 to 4.0 hr at 160 V with a pulsed constant power supply at
    300 pulses per second and about 90 mA current.
    Prior to being used for electrophoresis, the protein was disasso
    ciated by mixing 0.2 ml of protein solution with 0.1 ml of 10% SDS and
    10 20 pi 2-ME and heating this mixture in boiling water for 1 to 2
    min. Samples of 20 50 pi of disassociated proteins were added to
    0.1 ml of one-fifth of the electrophoresis buffer concentration, con
    taining 30% sucrose and 0.15% SDS.
    Serum albumin (MW 67,000), glutamate dehydrogenase (MW 53,000),
    ovalbumin (MW 43,000), carbonic anhydrase (MW 29,000), and TMV coat
    protein (MW 17,500) were used as protein markers to estimate the
    molecular weight values for inclusions and virus coat protein subunits.
    After electrophoresis, the gel slabs were stained and fixed
    overnight in a staining solution containing 50% methanol, 10% glacial
    acetic acid, and 0.1% Coomassie brilliant blue R250. Before photog
    raphy, the gels were destained by soaking them for 8 hr in a solution
    made up of 10% methanol and 7-5% acetic acid followed by several
    changes in the solution over a period of several days. The distances
    migrated by the protein subunits into the running gels were measured
    from the photographs of the stained gels.
    Sedimentation Coefficient Determination
    The sedimentation rates of fresh and stored purified B1CMV in
    either 0.02 M Tris buffer, pH 8.2 or 0.05 M borate buffer, pH 8.2
    were measured with a Beckman Model E analytical ultracentrifuge ac
    cording to the method of Markham (i960). After the rotor reached a
    speed of 27,690 rpm photographs were taken at 4 min intervals using


    21
    immunizing a rabbit with CPMV degraded by SDS according to a procedure
    described by Purcifull and Batchelor (1977). A lyophilized, purified
    preparation containing approximately 3 mg of CPMV was resuspended in
    1 ml of 1.0% SDS solution containing 2.0% 2-ME, and boiled for ap
    proximately 5 min before emulsification with Freund's adjuvant and
    intramuscular injection into a rabbit. Three similar injections were
    given into the same rabbit with 7~day intervals between injections.
    Serological relationship between B1CMV and other potyviruses. Re
    ciprocal double imiTuinod i f fus ion tests with B1CMV and the following
    potyviruses were conducted in SDS-contain ing media: bean yellow mosaic
    virus (BYMV) bean common mosaic virus (BCMV-BV-1), bean common mosaic
    virus-siratro isolate (BCMV-S), bidens mottle virus (BiMV), dasheen
    mosaic virus (DMV) lettuce mosaic virus (LMV) pepper mottle virus
    (PeMV), potato virus Y (PVY) soybean mosaic virus (SoyMV) tobacco
    etch virus (TEV), turnip mosaic virus (TuMV) watermelon mosaic virus-1
    (WMV-l), and watermelon mosaic virus-2 (WMV-2). The source of each
    antiserum was as follows: BYMV (Jones and Diachun, 1977); BCMV-BV-1
    (j. K. Uyemoto, New York State Agricultural Experiment Station); BCMV-S
    (Lima et al., 1977); DMV (Abo El-Nil et al., 1977); BiMV, LMV, PeMV,
    PVY, SoyMV, TEV, TuMV, WMV-l, and WMV-2 (D. E. Purcifull, University
    of Florida, Gainesville).
    Using BICMV-As, the serological relationship of B1CMV with
    commelina mosaic virus (CoMV) (Morales and Zettler, 1977), a Moroccan
    isolate of CAMV (Fischer and Lockhart, 1976a), pepper veinal mottle
    virus (PVMV) and pokeweed mosaic virus (PWMV) were also studied in
    double diffusion tests with SDS-treated antigens. In all serological
    te3ts, the reactants were arranged so that B1CMV was always placed in


    ACKNOWLEDGEMENTS
    I wish to express my sincere gratitude and appreciation to Dr. Dan
    E. Purcifull, chairman of my supervisory committee, for his invaluable
    counsels, friendship, advice, and constant guidance during the course
    of this investigation.
    Appreciation is extended to other members of my supervisory com
    mittee, Drs. Ernest Hiebert, John R. Edwardson, Raghavan Charudattan,
    Francis W. Zettler, and Daniel A. Roberts for their helpful suggestions
    during the research and their efforts in criticizing the manuscript.
    I also wish to extend my appreciation to Mr. Richard G. Christie for
    his valuable help with the light microscope and for his constant en
    thusiasm for teaching useful cytological techniques for diagnosing
    plant virus diseases. The understanding and cooperation of Mr. S.
    Christie, Mr. W. Crawford, Mrs. J. Hill, and Mrs. D. Miller during
    the laboratory experiments are also greatly appreciated.
    1 further wish to extend my gratitude to Mrs. Maria I. Cruz for
    her understanding and cooperation as the Secretary of the International
    Programs of the University of Florida, Gainesville, and for her time
    spent in typing this dissertation.
    I was supported by funds from the United States Agency for Inter
    national Development (USAID), Universidade Federal do Cear, and Ford
    Foundation, to whom I wish to express my sincere thanks.
    Special recognition is expressed to my wife, Diana, whose patience
    friendship, and love made this work possible.
    i i i


    Oil


    Brantley, B. B., C. W. Kuhn, and G. Sowell. 1965. Effect of cucumber
    mosaic virus on southern pea (Vigna sinensis). Proc. Am. Soc.
    Hortic. Sci. 87:355-358.
    Brierley, P., and F. F. Smith. 1962. Three cowpea mosaic viruses from
    gladiolus. Plant Dis. Rep. 46:335-337.
    Brlansky, R. H., and K. S. Derrick. 1976. Detection of seed-borne
    plant viruses using serologically specific electron microscopy.
    Proc. Am. Phytopathol. Soc. 3:334-335 (Abstr.).
    Brunt, A. A. 1974_ Tropical leguminous crops. Glasshouse Crop Re
    search Institute. Annual Report. 1974 (Abstr.).
    Brunt, A. A., and R. H. Kenten. 1973. Cowpea mild mottle, a newly
    recognized virus infecting cowpeas (Vigna ungu i cu lata) in Ghana
    Ann. Appl. Biol. 74:67-74.
    Brunt, A. A., and R. H. Kenten. 1974. Cowpea mild mottle virus. No.
    140 Descript ions of plant viruses. Commonw. Mycol Inst.,
    Assoc. Appl. Biol., Kew, Surrey, England. 4 p.
    Burkholder, W. H., and A. S. Muller. 1926. Hereditary abnormalities
    resembling certain infectious diseases of beans. Phytopathology
    16:731-737.
    Camargo, I. J. B., E. W. Kitajima, and A. S. Costa, 1968. Estudo ao
    microscopio electrnico de tecidos de plantas ¡nfetadas pelo
    virus do mosaico comum e mosaico amarelo do feijoeiro. Bragantia
    27:409-415 -
    Carner, J., K. S i 1berschmidt, and E. Flores. 1969. Ocorroncia do
    virus do mosaico da Vigna no Estado de Sio Paulo. 0 Bioloqico
    35:13-16.
    Casper, R. 1974. Serodiagnosis of plum pox virus. Acta Horticulturae
    44:171-172.
    Chant, S. R. 1959- Virus of cowpea, Vigna ungu¡culata L. (Walp.)
    in Nigeria. Arm. Appl. Biol. 47 :565-572.
    Chant, S. R. I960. The effect of infection with tobacco mosaic and
    yellow mosaic viruses on the growth rate and yield of cowpea in
    Nigeria. Empire J. Exp. Agrie. 28:114-120.
    Chant, S. R. 1962. Further studies on the host range and properties
    of Trinidad cowpea mosaic virus. Ann, Appl. Biol. 50:159-162.
    Chenulu, V. V.s J. Sachchidananda, and S. C. Mehta. 1968. Studies on
    a mosaic disease of cowpea from India. Phytopathol. Z. 63:381-
    387.


    64
    which were placed in the center well prior to the antiserum, cross-
    reacted with and fully precipitated the cross-reacting antibodies at
    the region of optimal proportions close to the center well.
    Serological distinction was also observed between a freshly
    purified preparation of BICMV and purified B1CMV stored at 4 C for
    more than 30 days (Fig. 16-F). This suggested some enzymatic degrada
    tion of certain BICMV antigenic determinants during the storage period
    Serological relationship studies between CAMV and BCMV-S using
    BCMV-S antisera obtained by different bleedings of the same rabbit
    indicated that the antiserum specificity varied according to the
    immunization program and the conditions of the antigenic solution used
    A highly specific antiserum for BCMV-S was obtained from a rabbit in
    jected with approximately 8 mg of freshly purified preparations of
    BCMV-S. Using this specific antiserum it was possible to show a com
    plete serological distinction between BCMV-S and CAMV (Fig. 16-E).
    About three months after the initial immunization, a booster injection
    with a purified preparation of BCMV-S stored at 4 C for more than
    one month was given to the same rabbit. All antisera obtained 15 days
    or more after the booster injection reacted with CAMV, forming a spur
    between CAMV and BCMV-S when they were placed into adjacent antigen
    wells around the antiserum well (Fig. 1 6-E).
    Light and Electron Microscopy
    Light microscopic observations of epidermal leaf strip prepara
    tions from plants systemically infected with B1CMV revealed the pres
    ence of tubular cytoplasmic inclusions similar to those described by
    Edwardson et al. (1972) and Edwardson (1974) for B1CMV. Side views
    of groups of tubular inclusions were easily observed in B1CMV-infected


    Figure 18 Electron micrographs of ultrathin sections of cowpea leaf
    cells infected with BlCMV showing cross-sections (A, B,
    C) and longitudinal sections (D) of pinwheel inclusions.


    VIRUS ABBREVIATIONS
    Virus Names Abbreviation
    Bean common mosaic virus BCMV
    Bean common mosaic vi rus-siratro isolate BCMV-S
    Bean pod mottle virus BPMV
    Bean yellow mosaic virus BYMV
    Bidens mottle virus B i MV
    Blackeye cowpea mosaic virus B1CMV
    Clover yellow vein virus CYVV
    Commel ina mosaic virus CoMV
    Cowpea aphid-borne mosaic virus CAMV
    Cowpea chlorotic mottle virus CCMV
    Cowpea mild mottle virus CMMV
    Cowpea mosaic virus CPMV
    Cowpea ringspot virus CpRV
    Cowpea yellow mosaic virus CYHV
    Cucumber mosaic virus CMV
    Dasheen mosaic virus DMV
    Iris mosa ic virus I MV
    Lettuce mosaic virus LMV
    Pea seed-borne mosaic virus PSMV
    Pepper mottle virus PeMV
    Pepper vein mottle virus PVMV
    Pokeweed mosaic virus..... PWMV
    Potato vi rus X PVX
    Potato vi rus Y PVY
    Southern bean mosaic virus SBMV
    Soybean mosaic virus SoyMV
    Sugarcane mosaic virus SMV
    Tobacco etch virus TEV
    Tobacco mosaic virus TMV
    Tobacco ringspot virus TRSV
    Turnip yellow mosaic virus TuMV
    Watermelon mosaic virus-1 WMV-1
    Watermelon mosaic virus-2 WMV-2
    x


    Figure 3 Flow diagram outlining the procedure for purification
    of B1CMV and its cytoplasmic inclusions, using chloroform
    and carbon tetrachloride as clarifying agents. The pro
    cedure is described in the text.


    at 48 to 72 hr after the test had been set up. Usually after 72 hr,
    a nonspecific precipitate also started to appear throughout the agar
    medium.
    Serological 1y Specific Electron Microscopy
    The SSEM technique developed by Derrick and Brlansky (1976) was
    adapted with great success to identify seed lots of cowpea infected
    with BlCMV (Fig. 23). Virus particles were still observed in extracts
    from hypocotyl tissue which was mixed 1/90 (w/w) with noninfected
    hypocotyls (Fig. 24-B). This indicates that to test several seed
    lots by SSEM for the presence of BlCMV, germinated seeds can be divided
    into groups of up to 50 equal pieces of individual hypocotyls. Each
    group of hypocotyl-pieces is ground in the extracting buffer and a
    drop of the extract is then examined by SSEM using antiserum sensi
    tized grids. Virus particles will be observed in extracts obtained
    from those hypocotyl groups in which at least one hypocotyl is in
    fected with BICMV.
    To illustrate the sensitivity of SSEM for detection of virus
    particles in hypocotyl extracts, the same B1CMV-infected hypocotyl
    was used for four different electron microscopic preparations: a) SSEM;
    b) normal dip preparation on a carbon coated Formvar film supported
    in copper grids and negatively stained with 2% PTA; c) preparation
    similar to SSEM using normal serum instead of B1CMV-antiserum; and
    d) preparation similar to SSEM using grids with Parlodion film coated
    with carbon but treated with 0.05 M Tris buffer instead of antiserum
    (Fig. 23). The results showed a great increase of virus concentration
    on the grids sensitized with BlCMV antiserum when compared with the


    106


    7
    indicated that these viruses were not identical to the one described
    by Smith (1924). A cowpea virus similar to those described by McLean
    (1941) and Snyder (1942) was reported from China by Yu (1946). The
    virus which was transmitted by aphids was also seed-borne in cowpea.
    In addition to cowpea, the virus also infected lima bean and adzuki
    bean, Phaseolus angularis (Wi 1 Id.) Wight (=V igna angularis (WiI Id.)
    Ohwi. and Ohshi) (Yu, 1946), Cowpea viruses apparently similar to
    those were also reported from Ceylon (Abrygunawardena and Perera, 1964),
    Germany (Brandes, 1964), India (Nariani and Kandaswany, 1961), and New
    Guinea (van Velsen, 1962).
    An aphid-borne virus isolated from cowpea in northern Italy was
    studied by Lovisolo and Conti (1966), and designated as cowpea aphid-
    borne mosaic virus (CAMV). The virus was a rod, approximately 750 nm
    long, and was seed-borne in cowpea, but appeared to be clearly different
    from BICMV isolated in Florida (Anderson, 1955b), As reported by Lovisolo
    and Conti (1966), the virus was first recorded and described in Italy by
    Vidano (1959) and Rui (i960). The virus was transmitted in a non-persistent
    manner by M. Pers i cae, Aphis fabae Scop., A, medicaginis Koch, A. gossypii,
    and Macros iphum euphorb ? ae (Thomas) (Vidano and Conti, 1965). A similar
    virus was later isolated in East Africa and three strains of this virus
    were differentiated by host range and serology (Bock, 1973). It was
    also observed that CAMV is distantly serologically related to bean common
    mosaic virus (BCMV) (Lovisolo and Conti, 1966; and Bock, 1973), but
    no direct serological relationship was detected with the African
    type strain of CAMV and potato virus Y (PVY) bean yellow mosaic virus
    (BYMV), pea seed-borne mosaic virus (PSMV), clover yellow vein virus
    (CYVV) soybean mosaic virus (SoyMV) sugarcane mosaic virus (SMV) ,


    89
    The mosr important cowpea virus in the southeastern United States
    was first isolated in Florida by Anderson (1955a) who later designated
    it blackeye cowpea mosaic virus (BlCMV) (Anderson, 1955b) and de
    monstrated i ts seed transmissibi Iity in cowpea (Anderson, 1957). The
    seed transmissibi 1ity of BlCMV was confirmed by subsequent studies at
    the University of Florida, Gainesville, Zettler and Evans (1972) found
    as much as 18% of BICMV-infected seeds in lots of certified cowpea
    seeds, and Uyemoto et al. (1973) reported 28% of seed transmission for
    this virus in cowpea 'Knuckle Purple Hull*.
    Cowpea mild mottle virus, a carlavirus isolated from cowpea in
    Ghana, was demonstrated to be transmitted through seeds of cowpea, bean,
    and soybean in variable but sometimes large proportions (Brunt and
    Kenten, 1973, 1974). Cowpea banding mosaic virus, which was identified
    as a member of the cucumovirus group in India, was transmitted through
    high percentages of seeds from infected cowpea varieties (Sharma and
    Varma, 1975). A new isometric cowpea virus isolated from cowpea seeds
    from Iran and designated cowpea ringspot virus had a seed transmission
    rate of 15 to 20% in three cowpea cultivars (Phatak, 1974; and Phatak
    et al., 1976).
    Thus, among the four filamentous and the six isometric viruses
    known to naturally infect cowpea, only TMV (fi Iamentous)and cow
    pea chlorotic mottle virus (isometric) have not been demonstrated to
    be seed-borne in cowpea. Kuhn (1964b) found no evidence of seed
    transmission of cowpea chlorotic mottle virus (CCMV) in more than
    2,100 seeds harvested from infected cowpea, and Gay (1969) observed
    no virus symptoms in 3,000 cowpea plants grown from seeds harvested


    151
    Snyder, W. C. 19^2. A seed-borne mosaic of asparagus bean, Viftna
    sesqui peda lis. Phytopathology 32;5l8-523.
    Takahashi, K,, Y. Tanaka, and Y. Tsuda, 197^, Soybean mild mosaic
    virus. Ann. Phytopathol. Soc. Japan. AO:103-105-
    Tamada, T. 1977, The virus diseases of soybean in Japan, Food
    Fertilizer Technology Center. Tech, Bull. 33. II p,
    Thomas. W, 0., Jr,, and R. W. Graham. 1951, Seed transmission of
    red node in Pinto bean. Phytopathology 4 I : 959-962.
    Toler, R. W, S, S. Thompson, and J. M, Barber. 1963, Cowpea (southern
    pea) diseases in Georgia, 1961-62, Plant D i s Rep, **7 : 7^6 7^+ 7,
    Toms, G, C,, and T, D. Turner, 1965, The seed haemagg1utinins of
    some Phaseolus vulgaris L. cultivars. J. Pharm. Pharmacol. 17:
    1 18 S -12 5 S .
    Tosic, M., R E. Ford, H. E. Moline, and D, E, Mayhew, 197^, Comparison
    of techniques for purification of maize dwarf and sugarcane mosaic
    viruses. Phytopathology 6A;439"iti*2
    Tosic, M., and Z. Pesie, 1975, Investigation of alfalfa mosaic virus
    transmission through alfalfa seed. Phytopathol, Z. 83:320-327,
    Tremaine, J, H., and N. S. Wright. 1967- Cross-reactive antibodies
    in antisera to strains of southern bean mosaic virus. Virology
    31 : A81 -A88.
    Tsuchizaki, T,, K, Yora, and H. Asuyama, 1970, The viruses causing
    mosaic of cowpeas and adzuki beans, and their transmissibi 1ity
    through seeds. Ann. Phytopathol. Soc, Japan 36:112-120.
    Tu, J. C, 1973, Electron microscopy of soybean root and nodules
    infected with soybean mosaic virus. Phytopathology 63:1011-1017,
    Tu, J. C, 1975, Localizations of infectious soybean mosaic virus in
    mottled soybean seeds. Microbios 1b:151-156,
    Uyeda, I., M. Kojima, and D, Murayama. 1975, Purification and serology
    of bean yellow mosaic virus, Ann. Phytopathol. Soc. Japan 4l ;
    192-203.
    Uyemoto, J. K., R. Provvidenti, and D. E. Purcifull. 1973. Host range
    and serological properties of a seed-borne cowpea virus. Phyto
    pathology 63:208-209, (Abs t r-),
    yemoto, J. K,, R. Provvidenti, and W. T. Schroeder, 1972. Serological
    relationship and detection of bean common mosaic and bean yellow
    mosaic viruses in agar gel, Ann. Appl, Biol, 7I:235-2A2.


    To my wife, Diana, and my son, Roberto, who
    with understanding, friendship, and love
    helped to transform a goal into a reality.


    Figure 30 Electron micrographs of ultrathin sections of cells from
    hypocotyls of t-S-day-old cowpea seedlings infected with
    BlCMV. Note cross-sections (A, B, C) and longitudinal
    sections (D) of pinwheel inclusions induced by BlCMV.
    (CW) plant cell wall, (IS) intercellular space, (m) mito
    chondrion, and (pw) pinwheel inclusions.


    strain of southern bean mosaic virus and a virus referred to as a cow-
    pea strain of bean yellow mosaic virus. A strain of CMV isolated from
    cowpeas in Morocco was transmitted through approximately 25% of the
    seeds harvested from artificially inoculated cowpcas (Fischer and
    Lockhart, 1976b). the seed transraissibility of the cowpca strain
    of SBMV was firsc demonstrated by Shepherd and Fulton (1962).
    McLean (1961) observed that a cowpea virus was transmitted through
    approximately 5^ of seeds of highly susceptible cowpea-varietics, but
    slightly susceptible or somewhat resistant varieties produced lower
    percentages of virus-infected seeds. Snyder (1962) observed only 3
    to 61 transmission of a cowpca virus through commercial seeds and 37
    to 6IL transmission of the same virus through seeds obtained from
    virus-infected plants. Yu (1966), however, found no difference In
    the percentages of virus-infected cowpea plants grown from seeds ob
    tained from artificially or naturally infected plants. These three
    viruses were also transmitted by aphids (McLean, 1961; Snyder, 1962;
    and Yu, 1966). Similar aphid-transmitted viruses reported from
    India were also demonstrated to be seed-borne (liarlani and Kandaswamy,
    1961; and Yerma, 1971). A rod-shaped virus Isolated from cowpea in
    northern Italy was designated cowpea aphid-borne mosaic virus (CAMV)
    and studied by Lovisolo and Conti (1966) who demonstrated its seed
    transmissibility in cowpea. The transmissibility of CAMV through
    seeds of cowpea has been confirmed by several studies involving dif
    ferent strains of the virus and varieties of the host (Kaiser et al.,
    1966; Tsuchizaki et al., 1970; Bock, 1973; Sock and Conti, 1976;
    Phatak, 1976; Khatri and Singh, 1976; and Fischer and Lockhart, 1976a).


    Figure 25 Double immunodiffusion tests with hypocotyls of A-5-day-
    old bean and soybean seedlings using antiserum for
    BCMV-S and for SoyMV.
    A B) Serological tests with hypocotyl discs (A) and
    hypocotyl extracts (B) from healthy and BCMV-
    BV-I infected bean seedlings. The center wells
    were charged with: (As) BCMV-S antiserum, and
    (Ns) normal serum. The peripheral wells were
    filled with extracts from: (l) BCMV-BV-1-
    infected hypocotyls, and (H) healthy bean
    hypocotyls. The hypocotyl discs were embedded
    directly into the agar medium and numbered
    from the top in a clockwise direction (arrows).
    Note a virus-antiserum specific reaction with
    hypocotyl "l" in A.
    C D) Serological tests with hypocotyl discs (c) and
    hypocotyl extracts (D) from healthy and SoyMV-
    infected soybean seedlings. The center wells
    were charged with: (As) SoyMV antiserum, and
    (Ns) normal serum. The peripheral wells were
    filled with extracts from: (l) SoyMV-infected
    hypocotyls, (2) SoyMV-infected Nicotiana
    benrhamiana leaves, (3) healthy N^. benthamiana
    leaves, and (A) healthy soybean hypocotyls.
    Note a positive reaction with hypocotyl "IA"
    in C.


    13*
    well as to globulins of normal serum and to virus coat protein. Marshall
    and Nor ins (1965) showed that extracts of P. vulgaris seeds containing
    lectins precipitated a and 3 globulins of normal rabbit serum, Gumpf
    and Shannon (1977) demonstrated that a barley lectin interacted with
    purified BSMV vut_rx> and formed insoluble aggregates that greatly
    reduced the virus infectivity. Phatak (197*) assumed that the high
    content of lectin in soybean seeds was responsible for the unsatisfactory
    results of the passive haemagglutination test with SoyMV in seed ex
    tracts. Thus, the absence of nonspecific reactions with extracts of
    hypocotyl tissue in contrast to the nonspecific precipitates observed
    with extracts of root or cotyledons and primary leaves (Fig. 19) of
    *-5-day-old legume seedlings may be an indication that a very low
    content of lectin is present in the hypocotyl.
    In support of the use of germinated seed for serology, instead of
    seed per se, is the fact that if any seed certification program or
    seed producers will assay for the presence of vi rus-infected seeds,
    they will also test some other seed properties such as percentage of
    seed germination. Consequently, the germinated seeds could be used
    in a concomitant serological indexing program.
    The sensitivity of the double immunodiffusion test indicates that
    germinated seeds can be divided into groups of 5 to 30 seedlings and
    precipitin reactions will be observed with those groups containing at
    least one infected seedling (Fig. 2|). Immunochemical tests with
    higher sensitivity have been also used for indexing vi rus-infected
    seeds. The extremely sensitive enzyme-1inked immunosorbent assay
    (ELISA) (Voller et a I., 1976; and Clark and Adams, 1977) was used to


    120


    Figure 13
    - Single radial diffusion tests in agar media containing
    different concentrations of SDS and antisera for black-
    eye cowpea mosaic virus (BICMV-As) and cowpea mosaic
    vi rus (CPMV-As).
    The media in (A, B, C) contain 0,8% Noble agar,
    1.0% NaN 0.3% SDS, and 10% BICMV-As (A), 15% BICMV-As
    (B), and^20% BICMV-As (c). The media in (D, E, F)
    contain 0.8% Noble agar, 1.0% NaN^, 0.5% SDS, and 10%
    BICMV-As (D), 15% BICMV-As (E), and 20% BlCMV-As (F).
    The wells in (A, B, C, D, E, F) were charged with:
    (l) extracts from 81CMV-infected cowpea prepared in
    1.5% SDS 1/2 (w/v), (2) solution used in "1" diluted
    1/2 with 1.5% SDS, (4) solution used in "1" diluted
    1/A with 1.5% SDS, (8) solution used in "1" diluted
    1/8 with 1.5% SDS, and (H) extract from healthy cowpea
    prepared in 1.5% SDS.
    The media in (G, H) contain 0.8% Noble agar,
    1.0% NaN 0.5% SDS, and 15% BICMV-As + 15% CPMV-As
    (G), and 10% BICMV-As + 10% CPMV-As (H). The media
    in (l, J) contain 0.8% Noble agar, 1.0% NaN3, 0.3%
    SDS, and 10% BlCMV-As + 10% CPMV-As (l), and 8%
    BICMV-As + 8% CPMV-As (j). The wells in (G, H, I, J)
    were charged with SDS-treated extracts from: B1CMV-
    infected cowpea (row no. 1), CPMV-infected cowpea
    (row no. 2), cowpea leaf tissue containing both
    B1CMV and CPMV (row no. 3), and healthy cowpea (row
    no. A).


    79
    1967; Nelson and Knuhtsen, 1973; Shepard et a)., 197*1; and Jones and
    Diachun, 1977)- The intragel cross-absorption technique was also ob
    served to be useful in demonstrating cross-protection between sero
    logically distinct strains of plant viruses (Lima and Nelson, 1975).
    The fact that most of the antisera obtained against purified
    B1CMV preparations did not react with extracts of non infected cowpea
    tissue can be added to confirm the efficiency of the virus purifica
    tion procedures described herein. On the other hand, the high popula
    tion of antibodies for normal plant antigens developed by the rabbits
    injected with B1CMV-I purified from infected cowpea was an indication
    that vi rus-infected cowpea tissue may have a high concentration of
    host antigens, which were difficult to separate from the B1CMV cyto
    plasmic inclusions. However, using N, benthamiana as a source plant
    for B1CHV-I purification, antiserum specific for B1CMV-I was obtained.
    This is an additional indication of the useful application of N^.
    benthamiana in plant virus research. Nicotiana benthamiana has been
    artificially infected with more than 50 plant viruses (Quacquare 11i
    and Avgelis, 1975; and Christie and Crawford, in press), showing its
    great potential for cytological, serological, and physiological studies
    of different viruses in the same biological system.
    The foot-pad route of rabbit immunization (Ziemiecki and Wood,
    1975) used to obtain the antiserum specific for B1CMV-I was an efficient
    procedure. The high yield of antibodies obtained for BCMV-S using the
    same route of immunization (Lima et al., unpublished) is additional
    evidence that a high titer antiserum can be obtained at the expense
    of very little antigen.


    1A3
    Gardner, M. W. 1927 Seed transmission of cowpea mosaic. Proc. Ind.
    Acad. Sc i. A3.
    Gardner, M. W., and J. B. Kendrick. 1921. Soybean mosaic. J. Agrie.
    Res. 22:111-1 IA
    Gay, J. 0. 1969. Effect of seed maturation on the infectivity of
    cowpea chlorotic mottle virus. Phytopathology 59:802-8oA.
    Gay, J. D., and E. E. Winstead. 1970. Seed-borne viruses and fungi
    from southern pea seed grown in eight states. Plant Dis. Rep.
    5A:2A3-2A5.
    Ghanekar, A. M., and F. W. Schwenk. 197A. Seed transmission and dis
    tribution of tobacco streak virus in six cultivars of soybeans.
    Phytopathology 6A:112-11 A
    Gibbs, A. J. 1972. Broad bean mottle virus. No. 101 in Description
    of plant viruses. Commonw. Mycol. Inst., Assoc. Appl. Biol.,
    Kew, Surrey, England. A p.
    Govier, D. A., and R. D. Woods, 1971 Changes induced by magnesium
    ions in the morphology of some plant viruses with filamentous
    particles. J. Gen. Viol. 13:127-132.
    Granett, A. L., and T. A. Shalla. 1970. The relation of tobacco mosaic
    virus X-protein to amorphous cellular inclusions (X-bodies).
    Phytopathology 60:A19-A2A.
    Grogan, R. G., J. E. Welch, and R. Bardin. 1952. Common lettuce mosaic
    and its control by the use of mosaic-free seed. Phytopathology
    A2:573-578.
    Gumpf, D. J., and L M. Shannon. 1977. Interaction of barley stripe
    mosaic virus and barley lectin. Proc. Am. Phytopathol. Soc. A
    (i n press) (Abst r ).
    Hamilton, R. I. 1965. An embryo test for detecting seed-borne barley
    stripe mosaic virus in barley. Phytopathology 55:798-799.
    Hampton, R. 0., S. Phillips, J E. Knesek, and G. I. Mink. 1973.
    011rastructuraI cytology of pea leaves and roots infected by pea
    seed-borne mosaic virus. Arch. Ges. Virusforsch. A2:2A2-253.
    Haque, S. Q., and G. C. Persad. 1975. Some observations on the seed-
    transmission of beetle-transmitted cowpea mosaic virus. Pages
    119-121 jji J. Bird, and K. Maramorosh eds. Tropical Diseases of
    Legumes. Academic Press, New York, 171 p.
    Harrison, A. N., and R. T. Gudauskas. 1968a. Identification of
    viruses isolated from cowpeas in Alabama. Plant Dis, Rep. 52:
    3A-36.


    136
    infected with alfalfa mosaic virus could be detected only 1^ days after
    germination and symptom expression was not reliable enough for the es
    timation of percentage of vi rus-infected seeds. Phatak et al. (1976)
    used the indicator-inoculation test in association with the growing-on
    test to estimate the percentage of cowpea seeds infected with cowpea
    ringspot virus because infected seedlings were either symptomless or
    the symptoms were very mild. In assessing the proportion of V. faba
    minor seeds infected with broad bean stain virus, Cockbain et al.
    (1976) found that under high temperature the symptoms were often mild
    and evanescent, and vi rus-infected seedlings remained without obvious
    symptoms for more than 6 weeks. Thus, the quantitative estimation of
    vi rus-infected seeds of leguminous crops by the growing-on test may
    often be difficult due to symptomless infections. On the other hand,
    the reliability and simplicity of the hypocotyl-disc-double immuno
    diffusion test make it highly suitable for commercial certification
    programs. Observations during the course of this research indicated
    that a trained person can set up approximately 50-60 hypocotyl discs
    per hour in an agar plate to be tested by this technique. This sim
    plified double immunodiffusion technique does not require tissue
    grinding, antigen wells, and chemical treatment of the antigen prior
    to incorporation into the agar matrix.
    The single radial immunodiffusion test also proved to be satis
    factory for detection of B1CMV in cowpea hypocotyls. These results
    provide another option for a routine seed health testing program. Sin
    gle radial immunodiffusion in multiple-antisera media could be used
    for indexing legume seeds for more than one virus. As shown in the


    80
    Reciprocal immunodiffus ion tests with antisera specific for
    B1CMV and B1CMV-I (Fig. 12-A, -B) confirmed the findings of Hiebert
    et al. (1971), Purcifull et al. (1973), Batchelor (197*0, and McDonald
    and Hiebert (1975) that the inclusion body proteins are immuno
    chemical ly distinct from viral coat protein and host proteins.
    The results of single radial immunodiffusion tests indicated
    that agar-media impregnated with mono-specific antiserum or with a
    mixture of antisera can be used for serodiagnosis of two morphologically
    distinct legume viruses. Single radial immunodiffusion tests were
    first used in plant virology by Shepard (I9&9) for serodiagnosis of
    potato virus X in potato tuber sprouts. Subsequently the same method
    was successfully used to identify plant tissue infected with carla-
    viruses (Shepard, 1970; and Shepard et al. 1971) potyviruses (Uyemoto
    et al., 1972; and Casper, 197*), a cucumovirus (Richter et al., 1975),
    a hordeivirus (Slack and Shepherd, 1975), and tobamovirus (Granett and
    Shalla, 1970; and Clifford, 1977). Radia1 -immunodiffus ion plates con
    taining a mixture of antisera to two or three filamentous viruses have
    been used for detection of potato viruses X, S, and M (Shepard, 1972).
    This, however, appears to be the first report of a multiple-antisera
    medium for detection of both an isometric and a rod-shaped plant viruses.
    Shepard (1969) observed that single radial immunodiffusion was
    more sensitive than double immunodiffusion for detection of PVX in in
    fected plant tissue, but Richter et al. (1975) obtained better results
    with double diffusion tests than with single diffusion for serological
    detection of CMV in naturally infected herbaceous plants. No attempts
    to compare these two serological techniques were made in the present
    study. Some observations, however, indicated that single radial


    TABLE OF CONTENTS
    Page
    ACKNOWLEDGEMENTS ¡
    LIST OF TABLES vi
    LIST OF FIGURES vi i
    VIRUS ABBREVIATIONS x
    ABSTRACT xi
    CHAPTER I PURIFICATION, PARTIAL CHARACTERIZATION, AND
    SEROLOGY OF BLACKEYE COWPEA MOSAIC VIRUS I
    Introduction 1
    Literature Review 2
    Materials and Methods 10
    Sources of Virus Isolate 10
    Virus and Inclusion Purification II
    Virus Particle Size Determination 1 4
    Stability of Virus in Sap 15
    Polyacrylamide Gel Electrophoresis of Viral
    and Inclusion Proteins 16
    Sedimentation Coefficient Determination.... 17
    Sero 1 ogy 18
    Antiserum production for virus and
    cytoplasmic inclusions 18
    Serological tests 19
    Serological relationships between B1CMV
    and other potyvi ruses 21
    Light and Electron Microscopy of Virus
    Induced Pinwheel Inclusions.. 22
    Host Range and Screening Cowpea Varieties
    for Resistance 23
    Results 2 4
    Purification and Properties of Blackeye
    Cowpea Mosaic Virus 2b
    Purified Inclusion Preparations b2
    Virus Particle Size and Stability in Sap... 47
    Serology. 52
    Light and Electron Microscopy 64
    Host Range and Resistant Cowpea Varieties.. 65
    Discuss ion 71
    i v


    8
    tobacco severe etch virus (TEV), and iris mosaic virus (I MV) (Bock,
    1973; and Bock and Conti, 197*0- A seed-transmitted virus tentatively
    identified as CAMV was considered to be responsible for the most im
    portant and widespread disease of cowpeas in Iran (Kaiser et al., 1968).
    Additional studies about various properties of the Iranian isolate of
    CAMV indicated its similarity to the Italian and African isolates (Kaiser
    and Mossahebi, 1979). A CAMV isolate was also reported from Japan in
    fecting adzuki bean, P. angularis, under natural conditions (Tsuchizaki
    et al., 1970). Fisher and Lockhart (1976a) isolated a rod-shaped virus
    from severely infected cowpeas in Morocco and identified it as a strain
    of CAMV on the basis of its particle length, aphid-transmission host
    range, serology, and physical properties. The Moroccan isolate differed
    from those CAMV isolates previously described (Lovisolo and Conti, 1966;
    Bock, 1973; and Bock and Conti, 197*0 by failing to infect 0cimum
    bas i 1 i cum L. a diagnostic species for CAMV (Bock and Conti, 197**),
    and other plants reported to be systemic hosts for CAMV. Padma and
    Summawar (1973) indicated the value of Chenopodum mrale L. as a good
    indicator host for differentiation, screening and isolation of a rod
    shaped cowpea virus and the icosahedral CPMV. Cytoplasmic inclusions
    were observed in plant cells infected with CAMV (inouye, 1973; and
    Nicolaeseu et al., 1976).
    A virus isolated from cowpea in India (Khatri and Singh, 197*4)
    was reported to be a strain of CPMV. However, the authors reported
    aphid transmission of this virus, so its identification as a strain
    of CPMV is questionable. A filamentous virus approximately 750 nm in
    length isolated from cowpeas in Ghana did not react with antisera
    specific for CAMV, peanut mottle virus, BCMV, and BYMV (Brunt, 197*)).


    Figure 2 Flow diagram outlining the procedure of purification of
    B1CMV using n-butanol as clarifying agent, polyethylene
    glycol (PEG) for virus concentration, CsCl gradient
    centrifugation for separation of virus from host com
    ponents, and differential centrifugation for further
    virus purification. For details, see description in
    materials and methods section.


    Harrison, A. N., and R. T. Gudauskas. 1968b. Effects of some viruses
    on growth and seed production of two cowpea cultivars. Plant
    Dis. Rep. 52:509-511.
    Hiebert, E., and J. G. McDonald. 1973. Characterization of some pro
    teins associated with viruses in the potato Y group. Virology
    56:3^9-361.
    Hiebert, E. and J. G. McDonald. 1976. Capsid protein heterogeneity
    in turnip mosaic virus. Virology 70:144-150.
    Hiebert, E., D. E. Purcifull, R. G. Christie, and S. R. Christie.
    1971- Partial purification of inclusions induced by tobacco etch
    virus and potato virus Y. Virology 43:638-646.
    Hollings, M., and 0. M. Stone. 1965- Studies of Pelargonium leaf
    curl virus. II. Relationships to tomato bushy stunt and other
    viruses. Ann. Appl. Biol. 56:87-98.
    lizuka, N. 1973. Seed transmission of viruses in soybean. Tohoku
    Natl. Agrie. Exp. Stn. Bull. 46:131-141.
    lizuka, N. 1974. Southern bean mosaic virus in soybean. Plant
    Protection 28:471-474
    lizuka, N., and T. Yunoki. 1974. Peanut stunt virus isolated from
    soybean, Glycine max Merr. Tohoku Agrie. Exp. Stn. Bull. 47:
    1-12.
    Inouye, T. 1973. Characteristics of cytoplasmic inclusions induced
    by bean yellow mosaic virus. Nogaku Kenkyu 54:155-171.
    Jones, R. T., and S. Diachun. 1977. Serologically and biologically
    distinct bean yellow mosaic virus strains. Phytopathology 67:
    831-838
    Kahn, R. P. 1956. Seed transmission of the tomato-ring spot virus
    in the Lincoln variety of soybean. Phytopathology 46:295.
    Kaiser, W. J., D. Danesh, M. Okhovat, and G. H. Mossahebi. 1968.
    Diseases of pulse crops (edible legumes) in Iran. Plant Dis.
    Rep. 52:687-691.
    Kaiser, W. J., and G. H. Mossahebi. 1974. Natural infection of mung-
    bean by bean common mosaic virus. Phytopathology 64:1209-1214.
    Kaiser, W. J., and G. H. Mossahebi. 1975. Studies with cowpea aphid-
    borne mosaic virus and its effect on cowpea in Iran. FA0 Plant
    Protection Bull 23:33-39.


    BLACKEYE COWPEA MOSAIC VIRUS: PURIFICATION, PARTIAL CHARACTERIZATION,
    SEROLOGY, AND IMMUNOCHEMICAL AND CYTOLOGlCAL TECHNIQUES
    FOR DETECTION OF VI RUS-INFECTED LEGUME SEEDS
    By
    J. ALBERS 10 A. LIMA
    IN
    A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
    THE UNIVERSITY OF FLORIDA
    PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
    DEGREE OF DOCTOR OF PHILOSOPHY
    UNIVERSITY OF FLORIDA
    1978
    V


    SYSTEMICALLY INFECTED TISSUE
    O.5L KP04 pH 7.5 + 0.5-1.0% Na2S0
    GRIND
    I
    FILTER
    8% RUTANOL
    CsCl GRADIENT CENTRIFUGATION:
    d=1.28g/cc 120000g 18 hr
    COLLECT VIRUS ZONE
    CENTRIFUGATION: 11700g lOnrin
    PELLET
    (Discard)
    SUPERNATANT
    CENTRIFUGATION: 85000g 90min
    PEL
    ET
    0.02M TRIS pH 8.2
    VIRUS
    SUPERNATANT-
    (Discard)


    15
    chopped with a razor blade in 2% PTA, pH 65, containing 0.1% BSA
    on a glass slide and a small quantity of the resulting cell extract
    was deposited on a carbon coated Formvar film supported by 75 x 300
    mesh copper grids. Excess liquid was then removed by touching
    momentarily the edge of the grid with a filter paper and the specimen
    was allowed to air-dry. The purified virus was stained directly on
    the grid. A small drop of virus solution was deposited on the grid.
    After 1 2 min, the virus solution was partially blotted with a piece
    of filter paper and a small drop of 2% PTA solution was added. The
    grid was blotted and allowed to air-dry. The grids were then examined
    in a Philips Model 200 electron microscope. The virus particles were
    observed, photographed and their sizes were estimated by comparing
    projected micrographs to micrographs of a diffraction grating (2160
    lines/mm). Twenty-five virus particles from leaf extracts and 190
    particles from a purified preparation were measured and classified
    according to their length at intervals of 50 and 20 nm.
    Blackeye cowpea mosaic virus-grids prepared according to the
    serologically specific electron microscopic technique (SSEM) developed
    by Derrick and Brlansky (1976) were also used for virus particle measure
    ments. Parlodion film grids sensitized with B1CMV-antiserum (B1CMV-As)
    were treated with cowpea leaf extract containing B1CMV and positively
    stained with 1% uranyl acetate in 50% ethanol. The SSEM technique will
    be described in more detail in Chapter II.
    Stability of Virus in Sap
    Thermal inactivation point (TIP), 1ongevity jn^ v|t££ (LIV) and
    dilution end point (DEP) were determined for B1CMV using C. amarant¡color
    as an assay plant. The TIP was determined by heating crude sap of


    BLACKEYE COWPEA MOSAIC VIRUS: PURIFICATION, PARTIAL CHARACTERIZATION,
    SEROLOGY, AND IMMUNOCHEMICAL AND CYTOLOGlCAL TECHNIQUES
    FOR DETECTION OF VI RUS-INFECTED LEGUME SEEDS
    By
    J. ALBERS 10 A. LIMA
    IN
    A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
    THE UNIVERSITY OF FLORIDA
    PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
    DEGREE OF DOCTOR OF PHILOSOPHY
    UNIVERSITY OF FLORIDA
    1978
    V

    To my wife, Diana, and my son, Roberto, who
    with understanding, friendship, and love
    helped to transform a goal into a reality.

    ACKNOWLEDGEMENTS
    I wish to express my sincere gratitude and appreciation to Dr. Dan
    E. Purcifull, chairman of my supervisory committee, for his invaluable
    counsels, friendship, advice, and constant guidance during the course
    of this investigation.
    Appreciation is extended to other members of my supervisory com
    mittee, Drs. Ernest Hiebert, John R. Edwardson, Raghavan Charudattan,
    Francis W. Zettler, and Daniel A. Roberts for their helpful suggestions
    during the research and their efforts in criticizing the manuscript.
    I also wish to extend my appreciation to Mr. Richard G. Christie for
    his valuable help with the light microscope and for his constant en
    thusiasm for teaching useful cytological techniques for diagnosing
    plant virus diseases. The understanding and cooperation of Mr. S.
    Christie, Mr. W. Crawford, Mrs. J. Hill, and Mrs. D. Miller during
    the laboratory experiments are also greatly appreciated.
    1 further wish to extend my gratitude to Mrs. Maria I. Cruz for
    her understanding and cooperation as the Secretary of the International
    Programs of the University of Florida, Gainesville, and for her time
    spent in typing this dissertation.
    I was supported by funds from the United States Agency for Inter
    national Development (USAID), Universidade Federal do Cear, and Ford
    Foundation, to whom I wish to express my sincere thanks.
    Special recognition is expressed to my wife, Diana, whose patience
    friendship, and love made this work possible.
    i i i

    TABLE OF CONTENTS
    Page
    ACKNOWLEDGEMENTS ¡
    LIST OF TABLES vi
    LIST OF FIGURES vi i
    VIRUS ABBREVIATIONS x
    ABSTRACT xi
    CHAPTER I PURIFICATION, PARTIAL CHARACTERIZATION, AND
    SEROLOGY OF BLACKEYE COWPEA MOSAIC VIRUS I
    Introduction 1
    Literature Review 2
    Materials and Methods 10
    Sources of Virus Isolate 10
    Virus and Inclusion Purification II
    Virus Particle Size Determination 1 4
    Stability of Virus in Sap 15
    Polyacrylamide Gel Electrophoresis of Viral
    and Inclusion Proteins 16
    Sedimentation Coefficient Determination.... 17
    Sero 1 ogy 18
    Antiserum production for virus and
    cytoplasmic inclusions 18
    Serological tests 19
    Serological relationships between B1CMV
    and other potyvi ruses 21
    Light and Electron Microscopy of Virus
    Induced Pinwheel Inclusions.. 22
    Host Range and Screening Cowpea Varieties
    for Resistance 23
    Results 2 4
    Purification and Properties of Blackeye
    Cowpea Mosaic Virus 2b
    Purified Inclusion Preparations b2
    Virus Particle Size and Stability in Sap... 47
    Serology. 52
    Light and Electron Microscopy 64
    Host Range and Resistant Cowpea Varieties.. 65
    Discuss ion 71
    i v

    Page
    CHAPTER II IMMUNOCHEMICAL ANO CYTOLOGICAL TECHNIQUES FOR
    DETECTION OF LEGUME VIRUSES IN INFECTED SEEDS.... 84
    I nt roduct ion., 84
    Literature Review.....,....,,.,, 86
    Seed-Borne Viruses in V i gna spp 87
    Seed-Borne Viruses in Glycine max 90
    Seed-Borne Viruses in Phaseolus vulgaris... 92
    Materials and Methods 94
    Source of Seed and Seed Germination 94
    Preparation of Antigens for Serology 95
    Double Immunodiffusion Tests 95
    Single Radial Immunodiffusion Tests 96
    Serologically Specific Electron Microscopy. 97
    Double Immunodiffusion Tests and SSEM for
    Detection of Other Viruses in Germinated
    Legume Seeds 98
    Serology and Microscopy of Cytoplasmic
    Inclusions Induced by B1CMV and SoyMV
    in Hypocotyls of Germinated Seeds......... 98
    Results,.., 99
    Preparation of Antigens for Serological
    Tests 99
    Double Immunodiffusion Tests 102
    Single Radial Immunodiffusion Tests 107
    Serologically Specific Electron Micro
    scopy. Ill
    Double Immunodiffusion Tests and SSEM
    for Detection of Other Viruses in
    Germinated Legume Seeds...,.., 116
    Serology and Mycroscopy of Cytoplasmic
    Inclusions Induced by BlCMV and SoyMV in
    Hypocotyls of Germinated Seeds 121
    Discuss ion.. 132
    LITERATURE CITED. 138
    BIOGRAPHICAL SKETCH 154
    v

    LIST OF TABLES
    Table Page
    I Symptoms and results of serological assays on
    varieties of cowpea, Vigna unguiculata mechanically
    inoculated with B1CMV, BCMV-S, CMV, and CPMV 70
    II Comparison of immunodiffusion tests with hypocotyl
    discs and growirig-on tests for detection of virus-
    infected seeds 1 08
    v i

    LIST OF FIGURES
    Figure Page
    1 Systemic and localized symptoms induced by blackeye
    cowpea mosaic virus (BICMV) in cowpea, V. ungui culata
    'Knuckle Purple Hull' and C. amarant ¡color 26
    2 Flow diagram outlining the procedure of purification
    of BICMV using n-butanol as clarifying agent 28
    3 Flow diagram outlining the procedure for purification
    of BICMV and its cytoplasmic inclusions, using
    chloroform and carbon tetrachloride as clarifying
    agents . 30
    4 Flow diagram outlining the steps carried out during
    the purification of BICMV and its cytoplasmic inclu
    sions by a combination of the first and second methods
    for purification of virus and inclusions 32
    5 Absorption spectra of purified preparations of BICMV
    in 0.02 M Tris buffer, pH 8,2, and BICMV cytoplasmic
    inclusions in the same buffer ^
    6 Electron microscopy of BICMV in a purified preparation
    and in cowpea leaf extracts ^7
    7 Histograms of lengths of BICMV particles from purified
    preparation negatively stained with phosphotungstate,
    and cowpea leaf extract using the serologically spe
    cific electron microscopy and uranyl acetate as a 39
    positive stain
    8 Histograms of BICMV particle lengths from two different
    electron microscopic preparations to show particle
    length distribution from 600 to 900 nm 41
    9 Schlieren patterns from sedimentation velocity experi
    ment with stored and fresh purified preparations of
    BICMV 44
    10 Electrophoretic analyses of BICMV induced cytoplasmic
    inclusions and BICMV capsid protein in 6% polyacryl
    amide gel 46
    11 Electron micrographs of purified preparations of
    BICMV cytoplasmic inclusions stained with molybdate.. 49
    v 1 1

    Figure Page
    12 Double immunodiffusion tests in agar medium containing
    0.8* Noble agar, 1.0* NaN^, and 0.5% SDS 51
    13 Single radial diffusion tests in agar media containing
    different concentrations of SDS and antisera for BICMV
    and cowpea mosaic virus (CPMV) 54
    1 ^ Single radial diffusion tests with SDS and pyrrolidine
    degraded capsid protein of BICMV and CPMV 56
    15 Reciprocal double immunodiffusion tests with B1CMV
    and other potyviruses in medium containing 0.8* Noble
    agar, 1.0* NaN^, and 0.5% SDS prepared in 0.05 M Tris-
    HC1 buffer, pHj7.2 6l
    16 Immunodiffusion tests with B1CMV, Moroccan isolate of
    cowpea aphid-borne mosaic virus (CAMV) and siratro
    strain of bean common mosaic virus (BCMV-S) in agar
    medium containing 0.8* Noble agar, 1.0* NaN_, and
    0.5% SDS prepared in 0.05 M Tris-HCl buffer, pH 7.2. 63
    17 Photomicrographs of cytoplasmic inclusions in
    epidermal strips of cowpea leaves systemically in
    fected with B1CMV, stained with a combination of
    calcomine orange and luxol brilliant green 67
    18 Electron micrographs of ultrathin sections of cowpea
    leaf cells infected with BICMV showing cross-sections
    and longitudinal sections of pinwheel inclusions 69
    19 Double immunodiffusion tests with extracts from
    different portions of B1CMV-infected and healthy
    4-5-day-old cowpea seedlings 101
    20 Diagram showing methods for assaying legume seeds
    by single and double radial immunodiffusion 104
    21 Double immunodiffusion tests with hypocotyls from
    healthy and B1CMV-infected 4-5-day-old cowpea
    seedlings in medium containing 0.8* Noble agar,
    1.0* NaN^, and 0.5* SDS, prepared in water 106
    22 Single radial immunodiffusion tests with hypocotyl
    extracts from healthy and B1CMV-infected, 4-5-day-
    old cowpea seedlings 110
    23 Electron micrographs of BICMV in hypocotyl extracts
    from the same cowpea seedling using different
    preparat ions 113
    v i i i

    Figure
    Page
    24
    Electron micrographs of serologically specific
    electron microscopy with BlCMV, BCMV-S, and CPMV
    115
    25
    Double immunodiffusion tests with hypocotyls of 4-5-
    day-old bean and soybean seedlings using antiserum
    for BCMV-S and for SoyMV,
    118
    26
    Electron micrographs of serologically specific
    electron microscopy with extracts from BCMV- and
    SoyMV-infected hypocotyls
    120
    27
    Double immunodiffusion tests with hypocotyl extracts
    from 4-5-day-old seedlings of cowpea and soybean
    using antisera for B1CMV, SoyMV, and their cyto
    plasmic inclusions
    123
    28
    Photomicrographs showing different views of cytoplas
    mic inclusions induced by BlCMV in epidermal strips
    of cowpea hypocotyl tissue stained with a combination
    of calcomine orange and luxol brilliant green
    125
    29
    Photomicrographs showing different views of epidermal
    cells of hypocotyls from 4-5-day-old soybean seedlings
    containing cytoplasmic inclusions induced by SoyMV...
    127
    30
    Electron micrographs of ultrathin sections of cells
    from hypocotyls of 4-5-day-old seedlings infected
    with BlCMV
    129
    31
    Electron micrographs of ultrathin sections of
    hypocotyl cells of 4-5~day-o|d soybean seedlings
    grown from SoyMV-infected seeds...
    131
    i X

    VIRUS ABBREVIATIONS
    Virus Names Abbreviation
    Bean common mosaic virus BCMV
    Bean common mosaic vi rus-siratro isolate BCMV-S
    Bean pod mottle virus BPMV
    Bean yellow mosaic virus BYMV
    Bidens mottle virus B i MV
    Blackeye cowpea mosaic virus B1CMV
    Clover yellow vein virus CYVV
    Commel ina mosaic virus CoMV
    Cowpea aphid-borne mosaic virus CAMV
    Cowpea chlorotic mottle virus CCMV
    Cowpea mild mottle virus CMMV
    Cowpea mosaic virus CPMV
    Cowpea ringspot virus CpRV
    Cowpea yellow mosaic virus CYHV
    Cucumber mosaic virus CMV
    Dasheen mosaic virus DMV
    Iris mosa ic virus I MV
    Lettuce mosaic virus LMV
    Pea seed-borne mosaic virus PSMV
    Pepper mottle virus PeMV
    Pepper vein mottle virus PVMV
    Pokeweed mosaic virus..... PWMV
    Potato vi rus X PVX
    Potato vi rus Y PVY
    Southern bean mosaic virus SBMV
    Soybean mosaic virus SoyMV
    Sugarcane mosaic virus SMV
    Tobacco etch virus TEV
    Tobacco mosaic virus TMV
    Tobacco ringspot virus TRSV
    Turnip yellow mosaic virus TuMV
    Watermelon mosaic virus-1 WMV-1
    Watermelon mosaic virus-2 WMV-2
    x

    Abstract of Dissertation Presented to the Graduate Council
    of the University of Florida in Partial Fulfillment of the Requirements
    for the Degree of Doctor of Philosophy
    BLACKEYE COWPEA MOSAIC VIRUS: PURIFICATION, PARTIAL CHARACTERIZATION,
    SEROLOGY, AND IMMUNOCHEMICAL AND CYTOLOGICAL TECHNIQUES
    FOR DETECTION OF VI RUS-INFECTED LEGUME SEEDS
    By
    J. ALBERS 10 A. LIMA
    March, 1978
    Chairman; Dan E. Pureifull
    Major Department: Plant Pathology
    Blackeye cowpea mosaic virus (B1CMV) was increased in cowpea, Vigna
    ungui cu lata (L.) Walp., 'Knuckle Purple Hull1, and infected leaves were
    used for virus and cytoplasmic inclusion purification. Either n-butano]
    or a combination of chloroform and carbon tetrachloride was used in the
    clarification process. Polyacrylamide gel electrophoresis of sodium
    dodecyl sulfate (SDS) dissociated inclusions and virus revealed that
    the inclusions were made of a single protein estimated to have a molecu
    lar weight (MW) around 70,000 daltons whereas freshly purified B1CMV
    consisted of a main protein component with a MW of 3^,000 daltons and
    two smaller proteins with MWs of 29,000 and 27,000 daltons. Purified
    B1CMV had a 260/280 nm absorption ratio of 1.2 and a modal length of
    753 nm. Freshly purified BICMV preparations showed a single sedimenting
    peak with s2157-159 S. The purified BICMV cytoplasmic inclusions
    had absorption spectra characteristic for proteins. Electron micro
    scopy of purified inclusions revealed the presence of tubes showing
    striations with periodicities of approximately 5 nm.

    Antisera reactive in SDS-immunodiffus i on were obtained against
    untreated virions, pyrrolidine degraded coat protein, and untreated BlCMV
    cytoplasmic inclusions. Reciprocal double immunodiffusion tests with
    SDS-treated antigens showed that BlCMV is serologically unrelated to
    seven potyviruses and serologically related to, but distinct from:
    bean common mosaic virus (BCMV) bean yellow mosaic virus (BYMV) cow-
    pea aphid-borne mosaic virus (CAMV), dasheen mosaic virus (DMV) lettuce
    mosaic virus (LMV), potato virus Y (PVY), soybean mosaic virus (SoyMV),
    tobacco etch virus (lEV), and watermelon mosaic virus-2 (WMV-2). The
    intragel cross-absorption technique was also used to demonstrate distinc
    tion between closely related potyviruses. Agar medium impregnated with
    a mixture of antisera was used for serodiagnosis of BlCMV and cowpea
    mosaic virus in cowpea.
    Light and electron microscopy of cytoplasmic inclusions induced
    by BlCMV, siratro (Macropti Iium atropurpureum (D.C.) Urb.) strain of
    BCMV (BCMV-S) and CAMV revealed that they are similar to those induced
    by the potyviruses from Edwardson's subdivision-I. The different reac
    tions induced by BlCMV, BCMV-S, and CAMV in some cowpea varieties in
    dicated that they can also be used as differential hosts for these
    three potyviruses. Sources of resistance for BlCMV were found among
    the cowpea varieties tested. Based on its physical, biological, cyto-
    logical, and immunoctiemica1 properties, BlCMV can be differentiated
    from any other virus that infects cowpea.
    Cytoplasmic inclusions induced by BlCMV in cowpea and by SoyMV
    in soybean were detected by serology, light microscopy, and electron
    microscopy in hypocoiyls of 4-5-day-old seedlings grown from virus-
    infected seeds.
    x i i

    Immunodiffusion tests and serologically specific electron micro
    scopy were used to detect B1CMV in hypocotyls of 4-5-day-old cowpea
    seedlings grown from B1CMV-infected seeds. Discs of individual hypo
    cotyls were embedded into the agar medium 4-5 mm away from the anti
    serum wells. Virus-specific precipitin lines formed between virus-
    infected hypocotyl discs and antiserum wells, whereas no reactions
    were observed with healthy hypocotyls. Precipitin lines were also
    observed with extracts of mixtures from infected (] g) and healthy
    (up to 29 g) tissues These immunochemical techniques were also used
    for detecting BCMV in hypocotyls of infected 4-5-day-old Phaseolus
    vulgaris L. seedlings and for detecting SoyMV in infected Glycine max
    (L.) Herr, seedlings. Single radial immunodiffusion tests with ex
    tracts or discs of cowpea hypocotyls were also useful for detecting
    B1CMV in germinated seeds. The reliability and simplicity of the
    immunodiffusion tests make them suitable for use in routine seed health
    testing program in any laboratory.
    x i i i

    CHAPTER I
    PURIFICATION, PARTIAL CHARACTERIZATION, AND
    SEROLOGY OF BLACKEYE COWPEA MOSAIC VIRUS
    Introduction
    Cowpea, Vigna unguiculata (L.) Walp. (=Vigna sinensis (L.) Endl.),
    is grown as a crop in high-temperature areas of tropical and subtropi
    cal countries. Cowpea seeds constitute a source of good quality pro
    tein and dried seeds are an important part of the diet of many people
    in the tropical and subtropical world, particularly in Africa and the
    rural zone of northeastern Brazil. The fresh seeds and immature pods
    are also eaten and they can be frozen or canned as is sometimes done
    in the United States. Cowpeas are also grown as fodder plants for hay,
    silage or pasture and used as a green manure and cover crop. When
    grown under optimum conditions, cowpea can produce seed yields as high
    as 2,600 Kg/ha. However, several factors limit cowpea yields in most
    fields. Virus diseases are considered as a major limiting factor to
    the production of cowpeas in several countries (Dale, 1949; Wells and
    Deba, 1961; Toler et al., 1963; Brantley et al., 1965; Kuhn et al.,
    1966; Harrison and Gudauskas, 1968a; Harrison and Gudauskas, 1968b;
    Gay and Winstead, 1970; Zettler and Evans, 1972; Bock, 1973; Phatak,
    1974; Haque and Persad, 1975; Kaiser and Mossahebi, 1975; and Lima and
    Nelson, 1977). Several viruses infect cowpea, and many of them can be
    transmitted through seeds from infected cowpea plants. The most im
    portant cowpea seed-borne virus in the southeastern United States is
    1

    2
    an aphid-transmitted, filamentous virus approximately 750 nm long
    (Harrison and Gudauskas, 1968a; 1968b; Gay and Winstead, 1970; Zettler
    and Evans, 1972; and Uyemoto et a 1., 1973). This virus was first
    isolated in Florida by Anderson (1955a), who designated it "blackeye
    cowpea mosaic virus" (BICMV) (Anderson, 1955b), a name that has been
    retained by Zettler and Evans (1972) and Edwardson et al. (1972).
    Because no antiserum specific for BICMV was available, and because
    only sparse information about the virus properties could be found in
    the literature, the first part of this research was undertaken to
    purify and characterize BICMV jn vitro and in vivo. Antisera prepared
    for BICMV and its cytoplasmic inclusions were used for serological
    characterization of the virus. Some methods for virus and inclusion
    purification, as well as certain physical, biological, immunochemical
    and cytological properties of BICMV were described in the present
    investigation. An abstract of part of this research was already
    published (Lima et al., 1976).
    Literature Review
    Several viruses infect cowpea, V. ungu ¡culata, causing different
    types of mosaic. The first report about mosaic of cowpea was published
    in 1921 by Elliot, who reported a high incidence of cowpea virus
    disease in Arkansas (Elliot, 1921). Smith (192A) demonstrated ex
    perimentally that the cowpea virus was transmitted either by rubbing
    the leaves of diseased and healthy plants together or by the bean leaf
    beetle, Ceratoma trifurcata Forst. Subsequently, Gardner (1927) working
    with a cowpea virus, observed that it was transmitted through seeds
    of certain cowpea varieties.

    3
    A widespread mosaic disease was reported on different cowpea
    varieties in Trinidad (Dale, 1949). Dale (1949) observed that the
    virus responsible for the disease was not transmitted by Aphis
    medicaginis Koch, but that the leaf beetle, Ceratoma ruficornis (01iv.)
    was a good vector and was probably responsible for transmitting the
    virus in the field. On the basis of his studies, he concluded that
    the virus was unrelated to those described by McLean (1941), Snyder
    (1942) and Yu (1946), but was more likely the virus studied by Smith
    (1924). Dale (1953) subsequently confirmed that the cowpea mosaic
    virus isolated from Trinidad was efficiently transmitted by ruficornis,
    but not by aphids.
    Lister and Thresh (1955) isolated a virus from cowpea and identi
    fied it as a strain of tobacco mosaic virus (TMV). They observed that
    a purified preparation of the virus contained rod-shaped particles of
    varying lengths, indistinguishable from the particles of TMV, and was
    precipitated specifically with antiserum prepared against TMV. A cow
    pea strain of TMV was also isolated from a range of leguminous hosts
    at Ibadan, Nigeria (Chant, 1959). Chant (1959) also found another
    virus infecting cowpea in Nigeria and as its physical properties
    differed from other cowpea viruses, he proposed the name cowpea yellow
    mosaic virus (CYMV). The virus was purified and an antiserum prepared
    against it. Both TMV and CYMV were transmitted by the beetle Ootheca
    mutabi1 is Sahib. In subsequent work, Chant (i960) studied the influence
    of TMV and CYMV on growth rate and yield of cowpea, and found that
    infection of cowpea with the cowpea strain of TMV did not affect yield
    as much as infection with CYMV. Wells and Deba (1961) tested 116 cow
    pea varieties and 342 indigenous pure lines against CYMV and observed

    4
    that 6 varieties and 16 pure lines were resistant. Robertson (1965)
    screened 79 cowpea varieties for resistance to CYMV in a screened
    greenhouse. Those varieties that showed no local or systemic reactions
    when inoculated with the virus were classified as immune; those that
    developed necrotic lesions but did not become systemically infected
    were classified as resistant; and those that showed systemic infection
    were classified as susceptible. Chant (1962) found that the cowpea
    virus from Trinidad caused local lesions on Chenopodiurn ama rant¡color
    Coste and Reyn., Mucuna atterrina Holland, Petunia hybrida Vilm., and
    P. vulgaris, and that the virus was polyhedral with a mean diameter
    of approximately 25 nm.
    Doub1e-immunodiffusion tests showed that a cowpea virus from
    Arkansas and the Trinidad cowpea mosaic virus were closely related,
    but not identical serologically and that both were anti gen i cal Iy related
    to bean pod mottle virus (BPMV) (Shepherd,1963). Studying other proper
    ties of the virus, Shepherd (1964) confirmed a close similarity of the
    Arkansas virus with the cowpea mosaic virus from Trinidad (Dale, 1949).
    Walters and Barnett (1964), working with a cowpea mosaic virus serologi
    cally identical to the Arkansas isolate, demonstrated also that it was
    efficiently transmitted by the bean leaf beetle, C. trifurcata. A
    detailed study of three cowpea mosaic virus isolates from Surinam
    (South America), along with the previously reported cowpea viruses
    from Trinidad (Dale, 1949) and Nigeria (Chant, 1959, I960, 1962) re
    vealed that they are strains of cowpea mosaic virus (Agrawal, 1964).
    Detailed descriptions of host range, biophysical, biochemical, and
    immunochemical properties of cowpea mosaic virus were reported, and
    the abbreviation CPMV was proposed to eliminate any possible confusion

    5
    with CMV (cucumber mosaic virus). Cowpea mosaic virus (CPMV) has been
    extensively studied in different laboratories and was fully described
    by van Kammen (1971, 1972). It was selected as the type member of the
    comovirus group (Fenner, 1976) and reported from several other parts
    of the world, including Brazil (earner et al., 1969; and Lima and
    Nelson, 1977), Nigeria (Williams, 1975), Venezuela (Debrot and Rojas,
    1967), and Puerto Rico (Perez and Cortes-Mon1 lor, 1970; and Alconero
    and Santiago, 1973).
    Kuhn (1964b) purified and characterized a new virus isolated
    from cowpea in Georgia and named it cowpea chlorotic mottle virus
    (CCMV), which was subsequently described by Bancroft (1971). This
    virus belongs to the bromovirus group (Fenner, 1976) and is physically
    similar to brome mosaic virus (Bancroft, 1970) and broad bean mottle
    vi rus (Gibbs, 1972) neither of which produces symptoms in cowpea
    (Bancroft, 1971).
    Strains of cucumber mosaic virus (CMV) are also known to infect
    cowpea. Cucumber mosaic virus strains have been isolated from naturally
    infected cowpeas showing mosaic symptoms in southeastern United States
    (Anderson, 1955a; Kuhn, 1964a; and Harrison and Gudauskas, 1968a),
    Italy (Vovlas and Avgelis, 1972), Morocco (Fischer and Lockhart, 1976b),
    and South Africa (Klesser, i960). An aphid-transmitted, spherical
    virus, approximately 25 run in diameter, was also reported from India
    by Chenulu et al. (1968). According to their descriptions, the virus
    closely resembles a strain of CMV.
    Shepherd and Fulton (1962) identified a seed-borne virus of cowpea
    as a strain of southern bean mosaic virus (SBMV) (Shepherd, 1971).
    Although a virus isolated from naturally infected cowpea in Arkansas

    6
    had properties somewhat similar to the cowpea strain of SBMV, the two
    viruses were not serologically related (Shepherd, 1963)-
    A carlavirus isolated from cowpea in Ghana was described and
    designated as cowpea mild mottle virus (CMMV) by Brunt and Kenten
    (1973) and Brunt (197*0- Cowpea mild mottle virus is seed-borne in
    cowpeas, is 650 nm in length and is apparently not transmitted by
    aphids.
    A virus with small isometric particles, isolated from Iranian
    cowpea seeds was considered as new and named cowpea ringspot virus
    (CpRV) on the basis of symptomatology and particle morphology, which
    were similar to other ringspot viruses (Phatak, 197*+; and Phatak et
    al., 1976). According to Phatak (197*+) the virus was not transmitted
    by aphids, induced intracellular inclusions in cowpea, had a wide
    experimental host range and was serologically unrelated to AO other
    isometric viruses most of which commonly infect various legumes. Cow
    pea ringspot virus was also transmitted in 15-20% of the seeds of three
    cowpea cultivars (Phatak et al., 1976).
    McLean (1 9*+1) studied some physical and biological properties of
    a cowpea virus and found that it was transmitted by the following
    species of aphids: Macrosiphum solani Ashm., Acynthosiphon pisum
    (Harris), Aphis gossypii Glover, Myzus persi cae (Sulz.) but not by
    the bean leaf hopper (Empoasca fabae Le. B.), the tarnished plant bug
    (Lygus pratensis L.), the Mex¡can bean beetle (Epilachra corrupta Mis.),
    and the striped cucumber beetle (Diabrotica vittata Faba). Snyder
    (19*+2) described a mosaic disease of asparagus bean, Vigna sesquipedal is
    Wight, and also studied some biological and physical properties of
    the causal agent. His positive results obtained with aphid transmission

    7
    indicated that these viruses were not identical to the one described
    by Smith (1924). A cowpea virus similar to those described by McLean
    (1941) and Snyder (1942) was reported from China by Yu (1946). The
    virus which was transmitted by aphids was also seed-borne in cowpea.
    In addition to cowpea, the virus also infected lima bean and adzuki
    bean, Phaseolus angularis (Wi 1 Id.) Wight (=V igna angularis (WiI Id.)
    Ohwi. and Ohshi) (Yu, 1946), Cowpea viruses apparently similar to
    those were also reported from Ceylon (Abrygunawardena and Perera, 1964),
    Germany (Brandes, 1964), India (Nariani and Kandaswany, 1961), and New
    Guinea (van Velsen, 1962).
    An aphid-borne virus isolated from cowpea in northern Italy was
    studied by Lovisolo and Conti (1966), and designated as cowpea aphid-
    borne mosaic virus (CAMV). The virus was a rod, approximately 750 nm
    long, and was seed-borne in cowpea, but appeared to be clearly different
    from BICMV isolated in Florida (Anderson, 1955b), As reported by Lovisolo
    and Conti (1966), the virus was first recorded and described in Italy by
    Vidano (1959) and Rui (i960). The virus was transmitted in a non-persistent
    manner by M. Pers i cae, Aphis fabae Scop., A, medicaginis Koch, A. gossypii,
    and Macros iphum euphorb ? ae (Thomas) (Vidano and Conti, 1965). A similar
    virus was later isolated in East Africa and three strains of this virus
    were differentiated by host range and serology (Bock, 1973). It was
    also observed that CAMV is distantly serologically related to bean common
    mosaic virus (BCMV) (Lovisolo and Conti, 1966; and Bock, 1973), but
    no direct serological relationship was detected with the African
    type strain of CAMV and potato virus Y (PVY) bean yellow mosaic virus
    (BYMV), pea seed-borne mosaic virus (PSMV), clover yellow vein virus
    (CYVV) soybean mosaic virus (SoyMV) sugarcane mosaic virus (SMV) ,

    8
    tobacco severe etch virus (TEV), and iris mosaic virus (I MV) (Bock,
    1973; and Bock and Conti, 197*0- A seed-transmitted virus tentatively
    identified as CAMV was considered to be responsible for the most im
    portant and widespread disease of cowpeas in Iran (Kaiser et al., 1968).
    Additional studies about various properties of the Iranian isolate of
    CAMV indicated its similarity to the Italian and African isolates (Kaiser
    and Mossahebi, 1979). A CAMV isolate was also reported from Japan in
    fecting adzuki bean, P. angularis, under natural conditions (Tsuchizaki
    et al., 1970). Fisher and Lockhart (1976a) isolated a rod-shaped virus
    from severely infected cowpeas in Morocco and identified it as a strain
    of CAMV on the basis of its particle length, aphid-transmission host
    range, serology, and physical properties. The Moroccan isolate differed
    from those CAMV isolates previously described (Lovisolo and Conti, 1966;
    Bock, 1973; and Bock and Conti, 197*0 by failing to infect 0cimum
    bas i 1 i cum L. a diagnostic species for CAMV (Bock and Conti, 197**),
    and other plants reported to be systemic hosts for CAMV. Padma and
    Summawar (1973) indicated the value of Chenopodum mrale L. as a good
    indicator host for differentiation, screening and isolation of a rod
    shaped cowpea virus and the icosahedral CPMV. Cytoplasmic inclusions
    were observed in plant cells infected with CAMV (inouye, 1973; and
    Nicolaeseu et al., 1976).
    A virus isolated from cowpea in India (Khatri and Singh, 197*4)
    was reported to be a strain of CPMV. However, the authors reported
    aphid transmission of this virus, so its identification as a strain
    of CPMV is questionable. A filamentous virus approximately 750 nm in
    length isolated from cowpeas in Ghana did not react with antisera
    specific for CAMV, peanut mottle virus, BCMV, and BYMV (Brunt, 197*)).

    9
    An aphid-transmitted virus was responsible for complete loss of cowpea
    in irrigated areas of northern Nigeria (Raheja and Leleji, 197*0. Based
    on the fact that the virus was neither mechanically transmitted nor
    seed-borne in cowpea, Raheja and Leleji (197**) concluded that it was
    either an atypical strain of CAMV or a new virus not previously described.
    A virus isolated from Crotalaria spectabi 1is Roth in a field at
    Gainesville, Florida, was studied by Anderson ( 1955b) and designated
    blackeye cowpea mosaic virus (B1CMV). Anderson (1955b, 1955c) reported
    that BICMV infected plants of cowpea, Crotalaria and Desmodiurn in the
    field, but considered Crota 1 aria and Desmodiurn as secondary hosts for
    the virus. In a subsequent study, Anderson (1959) observed that BICMV
    was transmitted by M. persi cae but not by the bean leaf beetle,
    £. trifurcata. Corbett (1956) found that BICMV was serologically
    related to BYMV and identified it as a strain of BYMV. Based on Corbett's
    conclusion, several subsequent reports have referred to BICMV as a cow
    pea strain of BYMV (Brierly and Smith, 1962; Kuhn, 196**a; Kuhn et a 1 ,
    1965; and Harrison and Gudauskas, 1968a).
    Light and electron microscopic studies of BICMV and BYMV showed
    marked cytological differences between these two flexuous rod-shaped
    viruses (Edwardson et al., 1972). According to Edwardson et al. (1972),
    the cytoplasmic inclusions induced by BICMV were consistently different
    from those induced by BYMV. In the light microscope, groups of plates
    were observed in cells of BYMV-infected tissues, whereas groups of
    tubes were seen in cells of epidermal strips obtained from B1CMV-infected
    tissue. Electron microscopy of ultrathin sections indicated that
    BICMV-induced cytoplasmic inclusions consisted of pinwheels with scrolls,
    whereas BYMV-induced cytoplasmic inclusions were made of pinwheels and

    10
    laminated aggregates. Light and electron microscopic investigations re
    vealed that B1CMV induces nuclear inclusions in C_. spectabi 1 is (Zettler
    et al., 196?; Edwardson et al., 1972; and Christie and Edwardson, 1977),
    while no such inclusions were observed in cells of spectab i 1 i s
    infected with BYMV. Based on those cytological distinctions, Edwardson
    et al. (1972) concluded that BICMV and BYMV are distinct members of
    the potyvirus group Subsequently, Zettler and Evans (1972) demonstrated
    that BICMV and BYMV had dissimilar host ranges, providing additional
    evidence that they are distinct viruses.
    In host range studies, BICMV was shown to be very similar to BCMV,
    but different from watermelon mosaic virus-2 (WMV-2), (Uyemoto et al.,
    1973). Leaf-dip preparations of B1CMV-infected tissue revealed the
    presence of flexuous rods, 750 nni long, and double immunodiffusion
    tests with BCMV arid WMV-2 antisera indicated that BICMV was serologi
    cally identical to BCMV and related to, but distinct from WMV-2
    (Uyemoto et al., 1973).
    Materials and Methods
    Source of Virus Isolate
    The blackeye cowpea mosaic virus used in this study was isolated
    from infected seeds of cowpea V_. ungu ¡culata 'Knuckle Purple Hull'
    harvested from a field in Gainesville, Florida. The virus was trans
    mitted by aphids from infected cowpea plants grown from infected seeds
    to non-infected 'Knuckle Purple Hull1 plants. Two aphids (M. persicae)
    were used per test plant and each aphid was allowed to have an acqui
    sition period of 30 to 60 sec. A single test plant showing typical
    mosaic was assayed by leaf-dip electron microscopy for the presence of

    rod-shaped virus particles and used as the initial source of inoculum
    for virus propagation. The virus was mechanically transmitted from
    the selected infected plant to healthy 'Knuckle Purple Hull' seedlings,
    where it was increased for virus and inclusion purification, and
    other studies.
    Virus and Inclusion Purificar ion
    Blackeye cowpea mosaic virus was propagated in either V_. unguiculata
    or Ni cot ana benthamiana Domin, and systemically infected leaves were
    used for virus and inclusion purification. Either r^-butanol or a com
    bination of chloroform and carbon tetrachloride was used in the clari
    fication process. The adaxial surface of the primary leaves of 5 to
    7-day old cowpea seedlings were inoculated with BICMV obtained by
    grinding infected lecif tissue in 0.05 M potassium phosphate (KPO^)
    buffer, pH 7-5 (1/2, w/v). The first trifoliolate leaves showing
    typical mosaic were collected 15 to 18 days later and subjected to the
    following purification procedures based on previous works (Hiebert et
    a 1., 1971; Hiebert and McDonald, 1973; and McDonald and Hiebert, 1975).
    n^ Butanol clarification method. Two hundred to 400 g of leaf
    tissue were homogenized in a blender with two parts (w/v) of 0.5 M
    KPO^ buffer, pH 7-5, containing 0.5 to 1.0% sodium sulfite (Na^SO^).
    The resulting extract was filtered through a double layer of cheese
    cloth and enough n-butanol was added to make a final concentration of
    8% (v/v). This mixture was stirred overnight at 4 C and the coagulated
    green debris obtained was removed by a low speed centrifugation at
    11,700 in a Sorvall Centrifuge (Sorvall Superspeed RC2-B Automatic
    Refrigerated Centrifuge) for 10 min. Virions were precipitated from
    the supernantant by the addition of 6 8% (w/v) of polyethylene glycol

    12
    MW 6000 (PEG) followed by stirring for 60 min. The precipitated virions
    were collected by centrifugation at 13,200 g for 10 min. The resulting
    pellet was resuspended in 0.02 M KPO^, pH 8.2, containing 0.1%
    2-mercapthoethanol (2-ME) (v/v) and the virus was separated from the
    host components by eguilibrium density gradient centrifugation (120,000 £
    for 16 18 hr in a Heckman SW 50.1 rotor) in 30% cesium chloride (CsCl)
    prepared in the same buffer. The virus zone, located at 12 to 15 mm
    from the bottom of ihe tube, was collected dropwise through a hole
    punched in the bottom of the tube and diluted with 0.02 M KPO^, pH 8.2,
    containing 0.1% 2-ME. The virus preparation was clarified by centrifuga
    tion at 11,700 g for 10 min and reconcentrated by centrifugation at
    85,000 lj for 90 min. The final pellet was resuspended in 0.02 M Tris
    buffer, pH 8.2, and the virus concentration was determined spectrophoto-
    metrically using an extinction coefficient of 2.4 mg/ml (Purcifull,
    1966). The optical density (0.0.) readings for the virus at wavelengths
    of 260 and 280 nm were corrected for light scattering before estimating
    the 260/280 ratio and concentrations of virus in purified preparations.
    The correction for light scattering was done by plotting the log of
    the optical densities against the wavelengths of 320, 340, and 360 nm
    and extrapolating these values to 230 300 nm range of wavelength.
    Chloroform-carbon tetrachloride clarification method. This
    clarification process was selected when it was desirable to purify
    both the virus and inclusions from the same batch of tissue. System-
    ¡cally infected tissue (200 400 g) were homogenized in a solution
    containing 1.30 ml of 0.5 M KPO^ (pH 7.5), 0.35 ml of chloroform, 0.35
    ml of carbon tetrachloride, and 5.0 mg of Na^SO^ per gram of tissue.

    13
    The homogenized mixture was centrifuged in a Sorvall Centrifuge at
    5,000 rpm for 5 niin and the pellet containing the organic solvents was
    discarded. The aqueous phase was centrifuged at 13,200 g for 15 min
    to precipitate the virus induced inclusions. The supernatant was
    treated as previously described for virus purification and the pellet
    containing the inclusions was resuspended in 0.05 M KPO^, pH 8.2, and
    0.1% 2-ME. The inclusion suspension was homogenized in a Sorvall
    Omni-mixer homogenizer for 2 min and enough Triton X-100 was added to
    make a final concentration of St (v/v). After stirring for one hour
    at 4 C this mixture was subjected to a low speed centrifugation of
    27,000 g for 15 min to precipitate the inclusions. The pellet was
    resuspended in 10 to 20 ml of 0.02 M KPO^, pH 8.2, containing 0.1%
    2-ME, and homogenized for 30 sec. The inclusions were sedimented
    again by centrifugation of 27,000 g for 15 min. The pellet was homogen
    ized for 30 sec and the homogenate was layered on a sucrose step
    gradient made up of 10 ml of 80%, 7 ml of 60%, and 7 ml of 50% (w/v)
    sucrose in 0.02 M KPO^, pH 8.2. The gradient was centrifuged for one
    hour at 27,000 rpm in a Beckman SW 25.1 rotor. The inclusions layered
    on top of the 80% sucrose zone and were collected by droplet from the
    bottom of the tube. To remove the sucrose, the inclusions were diluted
    in 0.02 M KPO^, pH 8 2, and precipitated by a centrifugation at 27,000
    for 15 min. The pellet was resuspended in 0,02 M Tris, pH 8,2, and
    inclusion yield was estimated spectrophotometrica11y after being dis
    rupted in 2% sodium dodecyl sulfate (SDS). The inclusion preparations
    were either immediately used for immunization of rabbits or stored at
    -20 C by either freezing directly or by freeze-drying.

    ]k
    Clarification with n-but.anol and chloroform-carbon tetrachloride.
    Because n-butanol resulted in virus preparations of higher purity, but
    chloroform-carbon tetrachloride was superior for preservation of in
    clusion proteins (Hiebert, unpublished), these solvent systems were
    combined for purification of virus and inclusions from the same batch
    of tissue. Infected tissue was homogenized with two parts (w/v) of
    0.5 H KPO^, pH 7.5, containing 0 5 1.0% Na^SO^. The homogenate was
    filtered through cheesecloth and subjected to centrifugation at 11,700 g
    for 10 min. The supernatant was used for virus purification as de
    scribed previously using rvbutanol for clarification. The pellet was
    resuspended in approximately 2 volumes of 0.5 M KP0(, pH 8.2, 0.5%
    Na2S0^, homogenized with one volume of chloroform-carbon tetrachloride
    (1:1, v/v) and centrifuged at 5,000 rpm for 5 min in a Sorvall Centri
    fuge. The aqueous phase was subjected to a centrifugation at 11,700 g
    for 15 min. The supernatant was collected for additional virus puri
    fication using PEG, equilibrium density-gradient centrifugation and
    differential centrifugation. The pellet was resuspended in 0.05 M
    KP0,, pH 8.2, containing 0.1% 2-ME and treated with 5% Triton X-100.
    The inclusions were then purified by sucrose step gradient centrifuga
    tion as described above.
    Virus Particle Size Determination
    Crude leaf extracts from systemically infected cowpea plants and
    purified virus preparations were negatively stained in 2% potassium
    phosphotungstate (PTA), pH 6.5, containing 0.1% bovine serum albumin
    (BSA) prior to photography in an electron microscope. The procedure
    used was similar to those previously described (Edwardson et al., 1968
    and Purcifull et al., 1970). Small pieces of B1CMV-infected leaf were

    15
    chopped with a razor blade in 2% PTA, pH 65, containing 0.1% BSA
    on a glass slide and a small quantity of the resulting cell extract
    was deposited on a carbon coated Formvar film supported by 75 x 300
    mesh copper grids. Excess liquid was then removed by touching
    momentarily the edge of the grid with a filter paper and the specimen
    was allowed to air-dry. The purified virus was stained directly on
    the grid. A small drop of virus solution was deposited on the grid.
    After 1 2 min, the virus solution was partially blotted with a piece
    of filter paper and a small drop of 2% PTA solution was added. The
    grid was blotted and allowed to air-dry. The grids were then examined
    in a Philips Model 200 electron microscope. The virus particles were
    observed, photographed and their sizes were estimated by comparing
    projected micrographs to micrographs of a diffraction grating (2160
    lines/mm). Twenty-five virus particles from leaf extracts and 190
    particles from a purified preparation were measured and classified
    according to their length at intervals of 50 and 20 nm.
    Blackeye cowpea mosaic virus-grids prepared according to the
    serologically specific electron microscopic technique (SSEM) developed
    by Derrick and Brlansky (1976) were also used for virus particle measure
    ments. Parlodion film grids sensitized with B1CMV-antiserum (B1CMV-As)
    were treated with cowpea leaf extract containing B1CMV and positively
    stained with 1% uranyl acetate in 50% ethanol. The SSEM technique will
    be described in more detail in Chapter II.
    Stability of Virus in Sap
    Thermal inactivation point (TIP), 1ongevity jn^ v|t££ (LIV) and
    dilution end point (DEP) were determined for B1CMV using C. amarant¡color
    as an assay plant. The TIP was determined by heating crude sap of

    16
    B1CMV-infected cowpea leaves to 45, 50, 55, 60, 65, 70, and 75 C for
    10 min. All treated saps as well as unheated sap of B1CMV-infected
    tissue were rubbed on the test plants, which were maintained in green
    house conditions for at least three weeks for observation of symptoms.
    Crude sap of infected leaves obtained in deionized water was
    placed in test tubes and assayed for infectivity after storage at room
    temperature for 0, 8, 16, 24, 48, and 72 hr. For the DEP determination,
    crude juice was extracted from B1CMV-infected leaves, and the extract
    was diluted to 10 10 10 10 10 and 10 ^ with deionized
    water prior to assay.
    Polyacrylamide Gel EIectrophores?s of Viral and Inclusion Proteins
    The polyacrylamide gel electrophoresis studies were performed
    according to the method of Weber and Osborn (1969) as modified by
    Hiebert and McDonald (1973). Running gels of approximately 75 mm in
    height were prepared with 6% acrylamide (7.5 ml sodium phosphate buffer,
    pH 7.2; 15.0 ml water; 0.15 ml 10% SDS; 6.0 ml of 30% acrylamide;
    0.045 ml N, N, N1, N1-tetramethy1ened¡amine (TEMED) and 1.2 ml ammonium
    persulphate 15 mg/ml), and a well-forming gel of 8% acrylamide with one-
    fifth the electrophoresis buffer concentration (1.2 ml buffer; 7.2 ml
    H^O; 0.2 ml 10% SDS; 3.0 ml of 30% acrylamide; 0.04 ml TEMED, and 0.3
    ml ammonium persulphate 15 mg/ml) was cast on top of them. Disasso
    ciated protein solutions, 20 50 pi samples in approximately 20%
    sucrose and one-fifth the electrophoresis buffer concentration, were
    placed into the formed wells. The top of the samples were covered
    with a cap gel of composition similar to the well-forming gel. The
    electrophoresis was performed in a vertical slab electrophoresis
    apparatus, Ortec, Model 4010/4011, Ortec, Incorporated, Oak Ridge, Tenn.,

    17
    for 1.5 to 4.0 hr at 160 V with a pulsed constant power supply at
    300 pulses per second and about 90 mA current.
    Prior to being used for electrophoresis, the protein was disasso
    ciated by mixing 0.2 ml of protein solution with 0.1 ml of 10% SDS and
    10 20 pi 2-ME and heating this mixture in boiling water for 1 to 2
    min. Samples of 20 50 pi of disassociated proteins were added to
    0.1 ml of one-fifth of the electrophoresis buffer concentration, con
    taining 30% sucrose and 0.15% SDS.
    Serum albumin (MW 67,000), glutamate dehydrogenase (MW 53,000),
    ovalbumin (MW 43,000), carbonic anhydrase (MW 29,000), and TMV coat
    protein (MW 17,500) were used as protein markers to estimate the
    molecular weight values for inclusions and virus coat protein subunits.
    After electrophoresis, the gel slabs were stained and fixed
    overnight in a staining solution containing 50% methanol, 10% glacial
    acetic acid, and 0.1% Coomassie brilliant blue R250. Before photog
    raphy, the gels were destained by soaking them for 8 hr in a solution
    made up of 10% methanol and 7-5% acetic acid followed by several
    changes in the solution over a period of several days. The distances
    migrated by the protein subunits into the running gels were measured
    from the photographs of the stained gels.
    Sedimentation Coefficient Determination
    The sedimentation rates of fresh and stored purified B1CMV in
    either 0.02 M Tris buffer, pH 8.2 or 0.05 M borate buffer, pH 8.2
    were measured with a Beckman Model E analytical ultracentrifuge ac
    cording to the method of Markham (i960). After the rotor reached a
    speed of 27,690 rpm photographs were taken at 4 min intervals using

    18
    Schlieren optics. The data were corrected for standard water viscosity
    conditions at 20 C, but not for the effect of virus concentration. The
    virus concentrations used varied from 0.5 to 1.0 mg/ml.
    Serology
    Antiserum product ion for virus and cytoplasmic inclusions. Antisera
    were obtained by injecting a New Zealand white rabbit with untreated
    virions and a second rabbit with pyrrolidine-degraded virus protein.
    All rabbits selected for immunization were first bled to produce normal
    sera. The concentrations of untreated B1CMV in 0.02 M Tris buffer,
    pH 8.2, used in the immunization process varied from 1.0 to 2.0 mg of
    nuc1eoprotein per ml of purified solution. B1CMV used for pyrrolidine
    degradation was suspended in 0.005 M borate buffer, pH 8.2. The virus
    protein was degraded according to the method used by Shepard (1972).
    A virus solution was mixed with an equal volume of 5% pyrrolidine in
    distilled water (v/v). The mixture was then immediately dialyzed
    against two liters of 0.05 M borate buffer, pH 8.2, containing 0.37%
    actual formaldehyde for approximately A8 hr at A C to remove the
    pyrrolidine and fix the protein subunits.
    A series of A to 5 intramuscular injections was given to each
    rabbit with an interval of 10 to 15 days between the injections. Each
    injection consisted of 1.0 to 2.0 ml preparations of virus or degraded
    viral protein vigorously emulsified with equal volume of Freund's com
    plete or incomplete adjuvants (Difco). Booster injections were given
    at intervals of about 2 months.
    The immunized animals were bled every week, starting 10 to 15
    days after the last injection of the initial series of A 5 injections.

    19
    The rabbits were fasted for 4 12 hr prior to each bleeding and 30 -
    50 ml of blood were collected into glass tubes according to the proce
    dure described by Purcifull and Batchelor (1977). Blood samples were
    allowed to clot for approximately 45 min at 37 C in a waterbath. The
    clotted blood was subjected to a centrifugation of 2,000 rpm in a
    Sorvall table centrifuge for 10 min. The antisera were transferred
    with a Pasteur pipette to conica1-bottomed tubes and clarified by a
    second centrifugation at 5,000 rpm for 10 min. Antiserum specificity
    and titer were determined by Ouchterlony (1962) double-diffusion tests
    in SDS-agar plates. The antisera were stored at -20 C by either
    freezing directly or after freeze-drying.
    The B1CMV-induced cytoplasmic inclusions (BICMV-l) used for anti
    serum production were purified from N_, bent ham i ana. Freshly purified
    cylindrical inclusions, which were unreactive with antiviral sera, were
    used for immunization and the foot pad route of immunization (Ziemiecki
    and Wood, 1975) was used. The rabbit received three injections into
    the foot pad, each containing 0.1 ml of purified inclusions (0.1 0.2
    O.D. units/ml at 280 nm) in 0.02 M Tris, pH 8.2, emulsified with an
    equal volume of either Freund's complete or incomplete adjuvants.
    Serological tests. Both double and single immunodiffusion tests
    in agar gel were used in the present study. Most double immunodiffusion
    tests were performed in agar medium containing 0.8% Noble agar (Difco);
    0.5% SDS (Sigma) and 1.0% NaN^ (Sigma) in deionized water (Purcifull
    and Batchelor, 1977), or 0.05 M Tris-HCl buffer pH 7-2. Reactant wells
    were punched in the solidified agar medium with an adjustable gel
    cutting device made by Grafar Corp., Detroit, Mich. Routinely the
    wells (7 mm in diameter) were punched in an hexagonal arrangement

    20
    consisting of a center well with six peripheral wells spaced 4 5 mm
    from the center well as measured from the edges of the wells. Different
    gel patterns were also used in certain tests. Antigens used as reactants
    were prepared either in deionized water or in 1.5% SDS solution, ac
    cording to Purcifull and Batchelor (1977). In the first case, fresh
    tissue was ground with a mortar and pestle in deionized water (1/2,
    w/v) and expressed through cheesecloth. The second method which was
    more commonly used, consisted of grinding fresh tissue in 1.0 ml of
    water per gram of tissue and adding 1.0 ml of 3-0% SDS per gram of
    tissue prior to expressing the sap through cheesecloth. The antigens
    and undiluted antisera were pipetted directly into the appropriate
    wells, and the. plates were incubated in a moist chamber at 2b C for
    24 48 hr. The development of precipitation patterns was observed
    by looking at the plates, which were illuminated from the bottom with
    indirect lighting. The reactants were removed and 15% charcoal (Nor it
    A) in water (w/v) was added into the wells before photographs were
    taken.
    Single radial immunodiffusion tests were conducted in agar media
    containing 0.8% Noble agar, 1.0% NaN^, 0.3 or 0.5% SDS, and 10, 15, or
    20% B1CMV antiserum. Media were prepared either with antiserum obtained
    for untreated B1CMV and antiserum for pyrrolidine degraded B1CMV-
    protein. Each SDS concentration in the media was tested with antigens
    prepared in distilled water or in 1.5% SDS. During medium preparation,
    care was taken to avoid heating the antisera over 50 C and while ex
    posed to SDS, the antisera were maintained at 50 C for less than 2 min.
    Single radial diffusion plates were also prepared with a mixture
    of antisera to BICMV and CPMV. The CPMV-ntiserum was prepared by

    21
    immunizing a rabbit with CPMV degraded by SDS according to a procedure
    described by Purcifull and Batchelor (1977). A lyophilized, purified
    preparation containing approximately 3 mg of CPMV was resuspended in
    1 ml of 1.0% SDS solution containing 2.0% 2-ME, and boiled for ap
    proximately 5 min before emulsification with Freund's adjuvant and
    intramuscular injection into a rabbit. Three similar injections were
    given into the same rabbit with 7~day intervals between injections.
    Serological relationship between B1CMV and other potyviruses. Re
    ciprocal double imiTuinod i f fus ion tests with B1CMV and the following
    potyviruses were conducted in SDS-contain ing media: bean yellow mosaic
    virus (BYMV) bean common mosaic virus (BCMV-BV-1), bean common mosaic
    virus-siratro isolate (BCMV-S), bidens mottle virus (BiMV), dasheen
    mosaic virus (DMV) lettuce mosaic virus (LMV) pepper mottle virus
    (PeMV), potato virus Y (PVY) soybean mosaic virus (SoyMV) tobacco
    etch virus (TEV), turnip mosaic virus (TuMV) watermelon mosaic virus-1
    (WMV-l), and watermelon mosaic virus-2 (WMV-2). The source of each
    antiserum was as follows: BYMV (Jones and Diachun, 1977); BCMV-BV-1
    (j. K. Uyemoto, New York State Agricultural Experiment Station); BCMV-S
    (Lima et al., 1977); DMV (Abo El-Nil et al., 1977); BiMV, LMV, PeMV,
    PVY, SoyMV, TEV, TuMV, WMV-l, and WMV-2 (D. E. Purcifull, University
    of Florida, Gainesville).
    Using BICMV-As, the serological relationship of B1CMV with
    commelina mosaic virus (CoMV) (Morales and Zettler, 1977), a Moroccan
    isolate of CAMV (Fischer and Lockhart, 1976a), pepper veinal mottle
    virus (PVMV) and pokeweed mosaic virus (PWMV) were also studied in
    double diffusion tests with SDS-treated antigens. In all serological
    te3ts, the reactants were arranged so that B1CMV was always placed in

    22
    a well adjacent to the other virus-well. Sap extracts from appropriate
    healthy host tissues were included as controls in all serological tests,
    and all antigens were also tested against normal serum.
    The intragel cross-absorption technique described by van Regenmortel
    (1966) was also used to study the serological relationships of B1CMV
    with BCMV-S and CAMV. Purified preparations of heterologous antigens
    (BCMV -S or CAMV) were placed in the center well and allowed to diffuse
    for approximately 2^ hr. The excess of the antigen preparations were
    then removed and the B1CMV antiserum was added in the same well. At
    the same time, the homologous and the heterologous antigens were po
    sitioned in the outer wells.
    Light and Electron Microscopy of Virus Induced Pinwheel Inclusions
    Epidermal leaf strips obtained from systemically infected cowpea,
    Y_* ungu i culata, were floated on a 5% solution of Triton X-100 for 5
    to 10 min and subsequently stained with a combination of calcomine
    orange and "luxol" brilliant green as described by Christie (1967).
    The stained leaf strips were mounted in euparal on glass slides and
    examined with a light microscope for the presence of cytoplasmic in
    clusions. Similarly, strips from noninocu 1 ated V_. unguiculata were
    also stained and examined in the light microscope as controls.
    Cylindrical inclusions were examined in situ in ultrathin sec
    tions with an electron microscope. Small pieces were taken from
    symptomatic areas of systemically infected cowpea leaves and fixed
    for 2 to 3 hr at room temperature in Karnovsky's forma Idehyde-
    g1utara1dehyde fixative prepared in 0.1 M cacodylate buffer, pH 7.2
    (Karnovsky, 1965). After washing with 0.1 M cacodylate buffer, the
    small leaf pieces were postfixed for 1 to 2 hr at room temperature

    23
    in 2% osmium tetroxide and progressively dehydrated in an increasing
    ethanol solution series. The leaf pieces were maintained for 5 to 15
    min in each ethanol solution at room temperature. The pieces were
    stained overnight at b C in a solution of 75% ethanol containing 2%
    uranyl acetate and subsequently dehydrated in a second series of ethanol
    solutions (75 100%) followed by 100% acetone or propylene oxide.
    They were then embedded in plastic containing Epon 812, Araldite 502,
    and dodecenylsuccinic anhydride. Ultrathin sections were cut with a
    diamond knife in a Sorvall MT-2 ultramicrotome and mounted on copper
    grids with carbon-coated Formvar film. The specimens mounted on the
    grids were poststained with 9% potassium permanganate (2 min), 1%
    uranyl acetate (2 min), and lead citrate (2 min). These sections as
    well as those obtained from non inocu1 a ted cowpea plants were examined
    with a Philips Model 200 electron microscope.
    Purified B1CMV-I preparations were mounted on carbon-coated
    Formvar film supported by copper grids and stained with either 1%
    ammonium molybdate or 2% uranyl acetate, before examination by electron
    microscopy.
    Host Range and Screening Cowpea Varieties for Resistance
    Test plants were inoculated with crude sap from 'Knuckle Purple
    Hull' systemically infected with B1CMV. The inoculum was prepared by
    grinding leaf tissue in 0.05 M KPO^, pH 7-5 (112, w/v). The inocula
    tions were done by rubbing the inoculum on carborundum-dusted leaves
    of the test plants which were maintained in greenhouse conditions for
    at least one month for observation of symptoms. All inoculated plants,
    including those that did not show any symptoms were checked serologically
    for the presence of BICMV.

    24
    The cowpea varieties were also inoculated with CPMV, CAMV, and
    BCMV-S. Crude sap from all inoculated cowpea plants were also tested
    in double immunodiffusion against antisera specific for CPMV, B1CMV,
    and BCMV-S, respectively. Since CAMV was shown to be serologically
    related to B1CMV, the serological tests to detect its presence in the
    inoculated plants were done with BlCMV antiserum.
    Result s
    Purification and Properties of Blackeye Cowpea Mosaic V i rus
    Purified preparations of BlCMV were obtained from systemically in
    fected leaves of either V. unguiculata 'Knuckle Purple Hull' (Fig. 1-A)
    or N^. benthamiana using the purification procedures diagrammed in
    Figures 2, 3, and 4. The best yield with the highest degree of purity
    was obtained using the first method of virus purification (Fig. 2) and
    infected cowpea leaves (Fig. 1-A) as a source of virus. The first
    trifol¡oate cowpea leaves collected 15 to 18 days after inoculations
    gave the highest yield of virus (8 10 mg) per 100 g (fresh weight)
    of infected tissue and n-butanol proved to be the best clarifying agent
    for cowpea tissue. An opalescent,sharp virus-band was usually obtained
    after equilibrium density gradient centrifugation in 30% CsCl. The
    virus zone was located at 12 to 15 nm from the bottom of the tube
    while most of the green host components stayed at the top portion of
    the gradient. The clear pellet obtained after a high speed centrifuga
    tion of virus removed from CsCl gradients confirmed the absence of
    colored host components. The combination of chloroform and carbon
    tetrachlor¡de,a 1 though necessary for inclusion purification, was an
    inferior method of clarification for obtaining virus from cowpea

    Figure I Systemic and localized symptoms induced by blackeye cowpea
    mosaic virus (BICMV) in cowpea, ungu i cu lata 'Knuckle
    Purple Hull' and C. ama rant¡color.
    A) Typical mosaic on secondary trifol¡oate leaf of cow
    pea plant inoculated with BICMV (l), and primary
    trifoliate leaf showing vein clearing (2).
    B) Local lesions on leaf of C. amarant¡color inoculated
    with BICMV.

    26
    \

    Figure 2 Flow diagram outlining the procedure of purification of
    B1CMV using n-butanol as clarifying agent, polyethylene
    glycol (PEG) for virus concentration, CsCl gradient
    centrifugation for separation of virus from host com
    ponents, and differential centrifugation for further
    virus purification. For details, see description in
    materials and methods section.

    SYSTEMICALLY INFECTED TISSUE
    O.5L KP04 pH 7.5 + 0.5-1.0% Na2S0
    GRIND
    I
    FILTER
    8% RUTANOL
    CsCl GRADIENT CENTRIFUGATION:
    d=1.28g/cc 120000g 18 hr
    COLLECT VIRUS ZONE
    CENTRIFUGATION: 11700g lOnrin
    PELLET
    (Discard)
    SUPERNATANT
    CENTRIFUGATION: 85000g 90min
    PEL
    ET
    0.02M TRIS pH 8.2
    VIRUS
    SUPERNATANT-
    (Discard)

    Figure 3 Flow diagram outlining the procedure for purification
    of B1CMV and its cytoplasmic inclusions, using chloroform
    and carbon tetrachloride as clarifying agents. The pro
    cedure is described in the text.

    30
    SUPERNATANT-
    (Discard)
    PELLET
    (Discard)
    SUPERNATANT^
    (Discard)
    SYSTEMICALLY INFECTED TISSUE
    0.5M KP04 pH 7.5 + CHCI3+ CC14+ 1% Na2S03
    CENTR
    PELLET
    (Discard)
    FUGATION: 4,000g 5min
    SUPERNATANT
    CENTR
    SUPERNATANT
    (Virus)
    8% |eG
    STIR : 60min
    FUGATION: lT,700g 15min
    CEN
    PELLET
    (Inclusions)
    0.05M KP04 pH 8.2 + 0.1% 2-ME
    HOMOGENIZATION
    RIFUGATION: 11,700g lOmin
    5%
    CEN
    PELLET
    0.0j!M KP04 pH 8.2 + O.U 2-ME
    RITON-X
    RIFUGATION: 27,000g 15min
    -SUPERNATANT
    (Discard)
    PELLET
    CsCl GRADIENT CENTRIFUGATION:
    d=lj 28g/cc-120000g 18hr
    COLLECT VIRUS ZONE
    CENTRIFUGATION: ll,700g lOmin
    SUCROSE STEP GRADIENT CENTRIFUGATION:
    45,(J00g 60min
    COLLECT INCLUSION ZONE
    SUP
    CEN
    CEN
    RNATANT
    RIFUGATION: 85,000g 90min
    RIFUGATION: 27,000g 15min
    -5UPERNATANT
    (Discard)
    PELLET
    PELLET
    0.02M TRIS pH 8.2
    INCLUSIONS
    0.02M TRIS pH 8.2
    VIRUS
    I

    Figure k Flow diagram outlining the steps carried out during the
    purification of BICMV and its cytoplasmic inclusions by
    a combination of the first (Fig. 2) and second (Fig. 3)
    methods for purification of virus and inclusions.

    32
    INFECTED TISSUE

    33
    tissues. With this method, a clear sap was obtained after the first
    low speed centrifugation but the virus zone in the CsCl gradient was
    not very well separated from the host components.
    Plants of C, amarant ¡color and \/_. unguiculata mechanically
    inoculated with purified preparations of B1CMV showed the first symp
    toms of local lesions and systemic mosaic (Fig. I) ^ and 7 days after
    inoculation, respectively. The ultraviolet absorption curve (Fig, 5)
    obtained for the purified preparations of B1CMV had a maximum between
    260 and 262 nm, and a minimum at 2^ to 2^5 nm. The ratio between
    the absorption at wavelengths of 260 and 280 nm was approximately 1,2
    after correction for light scattering, as would be expected for a
    member of the PVY group. This value is consistent and agrees with those
    of other long flexuous rod-shaped viruses (Shepherd and Purcifull, 1971;
    Tosic et a 1., 197^; and Barnett and Alper, 1977), The virus solutions
    showed strong stream birefringence and electron microscopic examinations
    indicated that 73% of the 190 virus particles examined were between
    700 and 800 nm (Figs, 6, 7, 8). The rods observed in the purified
    preparations (Fig. 6) indicated a low percentage of virus fragmentation
    during the purification processes. As the result of end-to-end virus
    aggregation, a few particles with 1^00 to 1500 nm were also observed.
    Purified virus preparations usually were relatively free of normal plant
    constituents when examined with the electron microscope and in the
    spect rophotometer,
    Sedimentation coefficients determined for the virus at 20 C either
    in 0.02 M Tris buffer, pH 8.2, or in 0.05 M borate buffer, pH 8.2,
    indicated that B1CMV sedimented as a single species with the s^q values
    of 157 159 S. On the other hand, the Schlieren pattern (Fig. 9)

    Figure 5 Absorption spectra of purified preparations of B1CMV
    in 0.02 H Tris buffer, pH 8.2, and B1CMV cytoplasmic
    inclusions in the same buffer.

    Wave Length (nm)
    Absorbance of Purified B1CMV
    0.40

    Figure 6 Electron microscopy of B1CMV in a purified preparation
    and in cowpea leaf extracts.
    A) Purified preparation of BlCMV negatively stained
    with 2% phosphotungstic acid, pH 6.5, containing
    0.]% BSA;
    B) Serologically specific electron microscopy (SSEM)
    of leaf extract from cowpea plants systemically
    infected with BlCMV. Antiserum for BlCMV diluted
    1/1000 in 0,05 M Tris buffer, pH 7.2, was used
    to sensitize the grid and the virus particles
    were positively stained with 1% uranyl acetate.
    Note the considerable increase in virus concentra
    tion compared with the normal leaf-dip preparation
    (0;
    C) Leaf-dip preparation of cowpea leaf tissue
    systemically infected with BlCMV, negatively
    stained with 2% phosphotungstate.


    Figure 7 Histograms of lengths of B1CMV particles from purified
    preparation negatively stained with phosphotungstate
    (A), and cowpea leaf extract using the serologically
    specific electron microscopy and uranyl acetate as a
    positive stain (B). Class interval for both histograms
    50 nm.

    400 600 800 1000 1200 l400 1600
    PARTICLE LENGTH (nm)
    NUMBER
    OF PARTICLES

    Figure 8 Histograms of BICMV particle lengths from two different
    electron microscopic preparations to show particle length
    distribution from 600 to 900 nm. Class interval = 20 nm.
    A) Particle length distribution of BICMV from purified
    preparation negatively stained with phosphotungstate;
    B) Particle length distribution of BICMV from cowpea
    leaf extract prepared on grids sensitized with BICMV
    antiserum and positively stained with uranyl acetate.

    600 700 800 900
    PARTICLE LENGTH (nm)
    NUMBER OF PARTICLES
    o
    PO -Pi
    o o
    O'
    o
    oo
    o
    o
    no -P cn
    o o o
    00
    o
    -F-

    revealed a difference in S values between B1CMV in fresh preparations
    and B1CHV in purified preparations stored at A C for more than 30
    days. Both virus preparations showed a single sedimenting peak, but
    the s2q values for BICMV in fresh preparations and at a concentration
    varying from 0.5 to 1.0 mg/ml ranged from 157 to 159 S while the S2Q
    values for the virus in the stored preparations and at the same con
    centrations ranged from 1 AO to IA2 S (Fig. 9). The lower sedimenta
    tion coefficients obtained for the stored virus suggested that a
    change in virus mass (MW) had occurred. Hiebert and McDonald (1976)
    observed some possible enzymatic degradation of capsid protein of
    purified turnip mosaic virus. Proteolytic degradation of capsid
    protein of stored purified preparations of B1CMV was also observed
    by polyacrylamide gel electrophoresis (PAGE) studies. Polyacrylamide
    gel electrophoresis analysis of SDS-degraded virus of a freshly
    purified preparation of B1CMV revealed a main protein component with
    an estimated molecular weight (MW) of 3^,000 daltons and two smaller
    ones with MWs of 29,000 and 27,000 daltons (Fig. 10). These smaller
    components may have arisen by degradation of the slow moving component
    during storage (Hiebert and McDonald, 1976). Stored BICMV preparations
    contained only the faster moving protein components with MWs of 29,000
    and 27,000 daltons (Fig. 10), presumably derived from 3^,000 daltons
    component.
    Purified Inclusion Preparations
    Using either of the methods outlined in Figs. 3 and A, purified
    cylindrical inclusions induced by BICMV were obtained from the same
    batches of systemically infected leaf tissue of V. unguiculata or

    Figure 9 Schlieren patterns from sedimentation velocity experiment
    with stored (A), and fresh (B) purified preparations
    of B1CMV. Photograph was taken 8 minutes after the rotor
    reached a speed of 27,690 rpm. Sedimentation is from
    left to right.

    44

    gure 10 Electrophoretic analyses of BlCMV induced cytoplasmic inclusions (BICMV-l) and B1CMV
    capsid protein in a 6% polyacrylamide gel slab containing 0.1% sodium dodecyl sulfate
    (SDS) and sodium phosphate buffer, dH 7.2.
    A) Polyacrylamide gel electrophoresis of SDS dissociated proteins of BICMV-l
    (a), viral coat protein of freshly purified preparation of BlCMV (b) viral coat
    protein of purified BlCMV after 30 days of storage at A C, (c), and marker
    proteins (d-h). The migrations of the marker proteins on the gel are repre
    sented by the arrows: (d) bovine serum albumin, (e) glutamate dehydrogenase,
    (f) ovalbumin, (g) carbonic anhydrase, and (h) TMV capsid protein.
    B) Molecular weight determination of SDS dissociated proteins of BICMV-l and capsid
    protein of fresh and old purified. BlCMV preparations in polyacrylamide gel electro
    phoresis using marker proteins. The unnumbered circles along the standard line
    represent the following marker proteins with their molecular weights in paren
    theses from top to bottom: bovine serum albumin (67,000 daltons), glutamate
    dehydrogenase (53,000 daltons), ovalbumin (A3,000 daltons), carbonic anhydrase
    (29,000 daltons), and TMV capsid protein (17,500 daltons). The distances from
    the top, correspond to the distances measured in photograph positioned as in (A).
    The estimated molecular weights for the virus and its cytoplasmic inclusion
    proteins are as follows: (1) 70,000 daltons (BICMV-l), (2) 3A,000 daltons
    (undegraded BlCMV coat protein), (3) 29,000 daltons (degraded BlCMV coat protein),
    and (A) 27,000 daltons (degraded virus protein).

    ( 03 [c]
    MARKERS
    M. W. .
    EL 67000
    EL 53000
    2L 43000
    £ -SL 29000
    EL 17500
    6 8 10 12 14
    DISTANCE FROM TOP (cm)

    N. benthamiana used for virus purification. Electron microscopy of
    purified BlCMV-l negatively stained with molybdate revealed the
    presence of tubular inclusions with only trace amounts of host com
    ponents (Fig. 11). At high magnification, striations of protein sub
    units were observed on individual tubes (Fig. 11-D). These regularly
    spaced striations were estimated to have a periodicity of approximately
    5 nm. Striations with similar periodicity have been observed in
    cytoplasmic inclusions induced by several other potyviruses (Edwardson
    et al., 1968; Hiebert and McDonald, 1973; and Morales and Zettler,
    1977). Few virus particles were observed in the purified preparations
    of BlCMV-l which were not reactive to B1CMV-As (Fig. 12). Purified
    preparations of BlCMV-l with the highest degree of purity were obtained
    from N. benthamiana, with yields of 5 to 20 A^g^ units were usually
    obtained from 100 g of fresh weight of N_. benthamiana or \l_. unguiculata
    tissues. The ultraviolet absorption spectrum obtained for SDS disas
    sociated BlCMV-l was typical of proteins, with a maximum at 277 nm
    and a minimum at 246 248 nm (Fig. 5). Polyacrylamide gel electro
    phoresis of SDS-disrupted inclusion proteins revealed a single subunit
    component estimated to have a MW of 70,000 dal tons (Fig. 10).
    Virus Particle Size and Stabi1ity in Sap
    Electron microscopic examinations of purified preparations of
    BICMV negatively stained with PTA indicated that 73% of 190 virus
    particles measured were between 700 and 800 nm with a modal length
    of 753 nm. Particle measurements of several leaf-dip preparations
    negatively stained with PTA and of grids prepared for SSEM with
    infected cowpea leaf tissue gave modal lengths of 758 and 780 nm,

    Figure II
    - Electron micrographs of purified preparations of B1CMV
    cytoplasmic inclusions stained with molybdate. All
    purified preparations consisted of tubes, most of which
    were fragmented during the purification process. Note
    striations (St) on high magnification (D).

    4g

    Figure 12 Double immunodiffusion tests in agar medium containing 0.8% Noble agar, 1.0% NaN and
    0.5% SDS. 3
    A B) Serological tests to demonstrate specificity of B1CMV antiserum (Vs), and
    antiserum obtained against B1CMV induced cytoplasmic inclusions (Is). The
    peripheral wells were filled with the following antigenic solutions prepared
    in 1.5% SDS: (V) purified B1CMV, (l) purified preparations of B1CMV inclusions,
    (Co) extracts of B1CMV-infected cowpea, and (H) crude extract of healthy cow-
    pea. All antigens were also tested against normal serum (Ns).
    C) Serological relationship between B1CMV and a New York isolate of B1CMV.
    The center wells were filled with: B1CMV antiserum (Bis), and normal serum
    (Ns). The peripheral wells were charged with SDS-treated extracts from:
    (Bl) B1CMV-infected cowpea, (NY) isolate of B1CMV from New York in cowpea,
    and (H) healthy cowpea.
    D) Serological relationship between a strain of bean common mosaic virus isolated
    from siratro (BCMV-S) and the B1CMV isolate from New York. The center wells
    were charged with: (Ss) BCMV-S antiserum, and (Ns) normal serum. The peri
    pheral wells were charged with SDS-treated extracts from: (S) bean leaves
    infected with BCMV-S, (NY and N) cowpea leaves infected with the New York
    isolate of B1CMV, (H-lower) healthy bean,and (H-upper) healthy cowpea.


    52
    respectively, with 30% of the particle lengths ranging from 700 to
    800 nm (Figs. 7 and 8). Some variation was observed with the particle
    size of purified virus stained with PTA and virus particles in leaf
    extracts prepared by SSEM and stained with uranyl acetate (Figs. 7
    and 8). On the other hand, grids with less plant debris and higher
    concentrations of virus particles were obtained with SSEM than with
    the conventional leaf-dip preparation (Fig. 6). Using normal leaf-dip
    preparations at least four grids were prepared and 10 electron micro
    graphs were taken to measure a maximum of 25 virus particles. On the
    other hand, 132 virus particles were measured by examining two micro
    graphs obtained by SSEM.
    In cowpea leaf extracts, BICMV had a TIP of 65 C, LIV of 48 hr,
    -4
    and DEP of 10 Blackeye cowpea mosaic virus was still infectious
    after 10 min at 60 C but not at 65 C and lost its infectivity after
    48 hr at room temperature, but not at 24 hr. Sap of cowpea leaves
    systemically infected with B1CMV lost infectivity when diluted more
    than 10 ^ with distilled water.
    Serology
    Antisera specific for B1CMV were obtained against untreated
    virions and pyrrolidine degraded viral protein. Both antisera reacted
    with SDS- or pyrrolidine-treated BlCMV in purified preparations or
    in plant sap in double and single radial diffusion tests (Figs. 12,
    13, 14). Most bleedings were specific for viral antigens; however,
    some bleedings also reacted with extracts from healthy plants, sug
    gesting the presence of antibodies specific for normal plant components.
    To remove these antibodies the antiserum was absorbed with plant

    Figure 13
    - Single radial diffusion tests in agar media containing
    different concentrations of SDS and antisera for black-
    eye cowpea mosaic virus (BICMV-As) and cowpea mosaic
    vi rus (CPMV-As).
    The media in (A, B, C) contain 0,8% Noble agar,
    1.0% NaN 0.3% SDS, and 10% BICMV-As (A), 15% BICMV-As
    (B), and^20% BICMV-As (c). The media in (D, E, F)
    contain 0.8% Noble agar, 1.0% NaN^, 0.5% SDS, and 10%
    BICMV-As (D), 15% BICMV-As (E), and 20% BlCMV-As (F).
    The wells in (A, B, C, D, E, F) were charged with:
    (l) extracts from 81CMV-infected cowpea prepared in
    1.5% SDS 1/2 (w/v), (2) solution used in "1" diluted
    1/2 with 1.5% SDS, (4) solution used in "1" diluted
    1/A with 1.5% SDS, (8) solution used in "1" diluted
    1/8 with 1.5% SDS, and (H) extract from healthy cowpea
    prepared in 1.5% SDS.
    The media in (G, H) contain 0.8% Noble agar,
    1.0% NaN 0.5% SDS, and 15% BICMV-As + 15% CPMV-As
    (G), and 10% BICMV-As + 10% CPMV-As (H). The media
    in (l, J) contain 0.8% Noble agar, 1.0% NaN3, 0.3%
    SDS, and 10% BlCMV-As + 10% CPMV-As (l), and 8%
    BICMV-As + 8% CPMV-As (j). The wells in (G, H, I, J)
    were charged with SDS-treated extracts from: B1CMV-
    infected cowpea (row no. 1), CPMV-infected cowpea
    (row no. 2), cowpea leaf tissue containing both
    B1CMV and CPMV (row no. 3), and healthy cowpea (row
    no. A).

    0
    >
    §
    o o
    +75

    Figure 1A Single radial diffusion tests with SDS and pyrrolidine
    degraded capsid protein of blackeye cowpea mosaic virus
    (B1CMV) and cowpea mosaic virus (CPMV).
    The media in (A, B, C) contain 0.8% Noble agar,
    1.0% NaN 0.5% SDS, and 1 52 B1CMV-As (A) 152 CPMV-As
    (B), and^10% B1CMV-As + 10% CPMV-As (C). The wells
    were charged with SDS-t.reated extracts from: B1CMV-
    infected cowpea (row l), CPMV-infected cowpea (row 2),
    B1CMV and CPMV in cowpea (row 3), and healthy cowpea
    (row A).
    The media in (D, E, F) contain 0.8% Noble agar,
    0.2% NaN?, 0.85% NaC 1 and 152 B1CMV-As (D) 152
    CPMV-As (E), and 10% B1CMV-As + 10% CPMV-As (F)
    prepared in 0.05 M Tris-HCl buffer, pH 7.2. The
    wells were charged with pyrrolidine-treated extract
    from: B1CMV-infected cowpea (row 1), CPMV-infected
    cowpea (row 2), B1CMV and CPMV in cowpea (row 3),
    and healthy cowpea leaves (row A).

    o

    D O
    Q
    ,
    0.
    o 0 g>

    .N
    L
    .w

    57
    components purified from V. ungu¡culata by high speed centrifugation
    according to the method used by Purcifull et al. (1973)- The high
    specificity of most of the antisera obtained against purified B1CMV
    preparations confirmed the efficiency of the methods used for its
    purification. The titers of antiserum varied depending on the bleeding
    date and on the rabbits, but 32 was the highest antiserum titer es
    timated by SDS-gel double immunodiffusion tests with a series of dilu
    tions (1/2, 1/4, 1/d, 1/16, and 1/32) of B1CMV-infected cowpea tissue
    prepared in 1.5% SDS.
    Antiserum specific for cytoplasmic inclusions induced by B1CMV
    was obtained from a rabbit injected with preparations of BICMV-I
    purified from infected tissue of hL benthamiana. The B1CMV-I antiserum
    reacted specifically with purified preparations of B1CMV-1 and crude
    sap of B1CMV-infected cowpea, but not with either purified preparations
    of B1CMV or crude sap of noninocu1ated plants (Fig. 12-B). The posi
    tive reactions with BICMV-I were more evident after 48 hr of incuba
    tion. The results obtained with B1CMV antiserum also indicated that
    B1CMV was not serologically related to its cytoplasmic inclusions
    (Fig. 12-A). Attempts to obtain specific antiserum by injecting
    rabbits with BICMV-I purified from infected cowpea tissue were unsuc
    cessful. All three rabbits injected with BICMV-I purified from in
    fected cowpea developed high titers of antibodies specific for normal
    plant tissue antigens.
    Single radial immunodiffusion studies in SDS-agar medium im
    pregnated with the virus antiserum indicated that appropriate SDS and
    antiserum concentrations need to be previously selected for highest
    sensitivity and to avoid spurious reactions. The best results were

    58
    observed when the antigens were prepared in 1,5% SDS and the medium
    used had 0.3% SDS and 10% antiserum (Fig. 13-A) or 0.5% SDS and 15%
    antiserum (Fig. 13~E). The same results were consistently observed
    with different batches of plates with the same medium compositions.
    Similar results were also observed with CPMV using antiserum obtained
    for SDS-treated virus. On the other hand, different results were ob
    served in SDS medium containing a mixture of BICMV and CPMV antisera.
    All media containing 0.3% SDS were cloudy with all combinations of
    BICMV and CPMV antisera used (Fig. 13-1, -J), indicating some type of
    interaction between SDS and antiserum proteins. Flowever, even with
    the cloudy appearance, some virus-specific reactions were still ob
    served (Fig. 13-1, -J). Clearer media were obtained with 0.5% SDS
    and 10 or 15% of each antiserum. The best reactions, however, were
    observed when both BICMV and CPMV antisera were used at concentrations
    of 10% in media containing 0.5% SDS (Fig. 13~H). Strong precipitin
    rings were observed around the wells containing BICMV or a combina
    tion of BICMV and CPMV whereas weaker reactions were observed around
    the wells containing only CPMV (Figs. )3*H, 1A-C). Unexpectedly, no
    reactions were observed around the wells containing only CPMV in a
    medium containing 15% of each antiserum and 0.5% of SDS (Fig. 13-G).
    Virus-specific reactions using BICMV and CPMV antisera were also
    obtained with the single radial diffusion method described by Shepard
    (1972). Precipi tin rings around the virus-wells were observed when
    the antigens were prepared in 1.5 or 2.5% pyrrolidine and the medium
    used contained 0.8% Noble agar, 0.2% NaN^, and 10 to 15% virus anti
    sera prepared in 0.05 M Tris-HCl buffer, pH 7.2, containing 0.85%
    NaCl. Sharp, white precipitin rings were formed close to edges of

    59
    the wells containing 61CMV in agar medium prepared with BICMV antiserum,
    whereas whitish halos with greater diameters were formed around the
    wells containing CPMV in agar medium impregnated with CPMV antiserum
    (Fig. 1**-D, -E, -F). The same distinction between these two types of
    precipitin rings was observed when both antisera were added into the
    same medium, so that two concentric rings were formed around the
    wells containing both viruses (Fig. I^-F). The inner ring was the
    result of B1CMV-ant¡body specific reactions and the larger halos re
    sulted from CPMV-speoific reactions. This difference in types of
    precipitin rings could be related to the concentration of the antigens
    placed in the wells and to the reciprocal of antibody concentration
    (Shepard, 1972). St ronger and more compact rings were observed with
    CPMV when the antigens were diluted or the antiserum concentration
    was increased.
    Reciprocal double immunodiffusion tests with SDS-treated antigens
    showed that BICMV is serologically related to, but distinct from, the
    following potyviruses: BCMV-BV-1, BCMV-S, BYMV, DMV, LMV, PVY, SoyMV,
    TEV, and WMV-2 (Fig. 15). No reactions were detected, however, with
    certain potyviruses, including BiMV, PeMV, TuMV, WMV-1, CoMV, PVMV,
    and PWMV. Antiserum for BICMV also reacted specifically with CAMV
    forming a distinct spur which extended past the heterologous reaction
    (Fig. 16-A). In all positive serological relationships observed in
    the reciprocal serological tests, spurs were formed in both directions
    (Fig. 15).
    The serological distinctions observed between BICMV and BCMV-S,
    and CAMV by spur formation were demonstrated by the intragel cross
    absorption technique (Fig. 16-B, -D). The heterologous antigens,

    Figure 15
    - Reciprocal double immunodiffusion tests with B1CMV and
    other potyviruses in medium containing 0.8% Noble agar
    1.0% NaNj, and 0.5% SOS prepared in 0.05 M Tris-HCl
    buffer, pH 7-2. All the antigenic solutions were pre
    pared in 1.5% SOS. The center wells were charged with:
    (l) B1CMV antiserum, (2) PVY antiserum, (3) TEV antiserum,
    (A) WMV-2 antiserum, (5) DMV antiserum, (6) BbMV-BV-l
    antiserum, (7) BCMV-S antiserum, (8) SoyMV antiserum,
    (9) BYMV antiserum, (10) BiMV antiserum, and (ll) l.MV
    antiserum. The top rows of wells in all cases were
    charged with SDS-treated extracts from: (a) BICMV-
    infected cowpea, and (c.) healthy cowpea. The bottom rows
    of wells were charged with SOS-treated extracts from:
    A) PVY-infer ted tobacco (b), and healthy tobacco (d);
    B) TEV-infected tobacco (b), and healthy tobacco (d);
    C) WMV-2-infected pumpkin (b), and healthy pumpkin (d);
    D) DMV-infected dasheen (b), and healthy dasheen (d);
    E) BCMV-BV-1 infected bean (|>),and healthy bean (d) ;
    F) BCMV-S-infected bean (b), and healthy bean (d);
    G) SoyMV-in(ected N. benthamiana (b), and healthy
    N. bent hamiana (d); H] BYMV-infected pea (b) and
    healthy pea Td); l) BiMV-infected Nicotiana hybrid (b) ,
    and healthy Nicot¡ana hybrid (d); and J) LMV-infected
    pea (b), and healthy pea (d).


    Figure 16 Immunodiffusion tests with BICMV, Moroccan isolate of
    CAMV and siratro strain of bean common mosaic virus
    (BCMV-S) in agar medium containing 0.8% Noble agar,
    1.0% NaN,, and 0.5% SOS prepared in 0.05 M Tris-HCl
    buffer, pH 7.2.
    A) Serological tests with B1CMV and CAMV. The center
    wells were charged with: B1CMV antiserum (Bis), and
    normal serum (Ns). The peripheral wells were filled
    with SDS-treated extracts from: (Bl) B1CMV-infected
    cowpea, (Ca) CAMV-infected cowpea, and (H) healthy
    cowpea.
    B) Intragel cross-absorption test with B1CMV and CAMV.
    The center wells were charged with: (l) BICMV anti
    serum, (2) purified CAMV and 20 hr later BICMV anti
    serum. The peripheral wells were filled with SDS-
    treated extracts from: (Bl) BlCMV-infected cowpea,
    (Ca) CAMV-infected cowpea, and (H) healthy cowpea.
    C) Serological tests with BICMV and BCMV-S. The center
    wells were charged with: (Bis) BICMV antiserum, (Ss)
    BCMV-S antiserum. The peripheral wells were filled
    with SDS-treated extracts from: (Bl) B1CMV-infected
    cowpea, (S) BCMV-S-infected bean, (Hb) healthy bean,
    and (He) healthy cowpea. The arrows point to spurs.
    D) Intragel cross-absorption test with BICMV and BCMV-S.
    The center wells were filled with: (1) BICMV anti
    serum, and (2) purified BCMV-S and 20 hr later
    BICMV antiserum. The peripheral wells were charged
    with SDS-treated extracts from: (Bl) BICMV-infected
    cowpea, (S) BCMV-S-infected bean, (Hb) healthy bean,
    and (He) healthy cowpea.
    E) Serological tests with BCMV-S and CAMV using two
    different antisera for BCMV-S. The center wells were
    charged with: (l) BCMV-S antiserum from a rabbit
    inoculated with freshly purified preparations of
    BCMV-S, and (2) BCMV-S antiserum obtained from the
    same rabbit after a booster injection with purified
    BCMV-S stored at k C for 30 days. The peripheral
    wells were charged with SDS-treated extracts from:
    (S) BCMV-S-infected bean, (Ca) CAMV-infected cowpea,
    (He) healthy cowpea, and (Hb) healthy bean.
    F) Serological test with fresh and stored purified
    preparations of BICMV. The center well was charged
    with BICMV antiserum, and the peripheral wells with
    SDS-treated new purified preparation of BICMV (Np),
    old purified BICMV (Op), and healthy cowpea extracts
    (H).

    63

    64
    which were placed in the center well prior to the antiserum, cross-
    reacted with and fully precipitated the cross-reacting antibodies at
    the region of optimal proportions close to the center well.
    Serological distinction was also observed between a freshly
    purified preparation of BICMV and purified B1CMV stored at 4 C for
    more than 30 days (Fig. 16-F). This suggested some enzymatic degrada
    tion of certain BICMV antigenic determinants during the storage period
    Serological relationship studies between CAMV and BCMV-S using
    BCMV-S antisera obtained by different bleedings of the same rabbit
    indicated that the antiserum specificity varied according to the
    immunization program and the conditions of the antigenic solution used
    A highly specific antiserum for BCMV-S was obtained from a rabbit in
    jected with approximately 8 mg of freshly purified preparations of
    BCMV-S. Using this specific antiserum it was possible to show a com
    plete serological distinction between BCMV-S and CAMV (Fig. 16-E).
    About three months after the initial immunization, a booster injection
    with a purified preparation of BCMV-S stored at 4 C for more than
    one month was given to the same rabbit. All antisera obtained 15 days
    or more after the booster injection reacted with CAMV, forming a spur
    between CAMV and BCMV-S when they were placed into adjacent antigen
    wells around the antiserum well (Fig. 1 6-E).
    Light and Electron Microscopy
    Light microscopic observations of epidermal leaf strip prepara
    tions from plants systemically infected with B1CMV revealed the pres
    ence of tubular cytoplasmic inclusions similar to those described by
    Edwardson et al. (1972) and Edwardson (1974) for B1CMV. Side views
    of groups of tubular inclusions were easily observed in B1CMV-infected

    65
    leaves (Fig.17), and at high magnification end views of them could
    be seen as small dots by changing the microscope focus. In ultrathin
    sections of B1CMV-infected tissue, these inclusions consisted of tubes
    attached to a central core, forming pinwheels (Fig. 18), similar to
    those induced by the potyviruses from Edwardson's subdivision-I
    (Edwardson, 197*0- As reported by Edwardson (197*0, the pinwheels
    contained arms with pronounced curvatures and tight scroll-like
    tubular inclusions. Only tubes were observed in purified preparations
    of cytoplasmic inclusions induced by B1CMV (Fig. 11).
    Host Range and Resistant Cowpea Varieties
    Blackeye cowpea mosaic virus was readily transmitted mechanically
    from cowpea 'Knuckle Purple Hull' to the following plants in which it
    was detected serologically and caused the following symptoms:
    Crotalaria spectabilis (mosaic); Glycine max (L.) Mer. (mild mottle
    and chlorotic spots); Macroptilium atropurpureum (DC.) Urb. (mosaic);
    Macroptilium bracteatum (L.) Urb. (mosaic); Nicotiana benthamiana
    (mottle); 0cimum basi 1icum (local lesions); Phaseolus vulgaris 'Black
    Turtle-2' (epinasty, necrosis, yellowing) and 'Bountiful' (chlorotic
    spots on inoculated leaves); Vigna unguiculata 'Black Local' (mosaic),
    'Early Ramshorn' (mottle), 'Knuckle Purple Hull' (mosaic), and 12
    Brazilian cowpea cultivars in which the reactions varied from symptom
    less to mosaic (Table I). Small chlorotic lesions were found on the
    leaves of Chenopodium amarant¡color inoculated with purified prepara
    tions of B1CMV or cowpea sap containing BICMV (Fig. 1-B).
    Based on failure to induce symptoms and on negative serological
    results, BICMV did not infect Arachis hypogaea L. 'Florunner', Capsicum

    Figure 17
    - Photomicrographs (A, B, C, D) of cytoplasmic inclusions
    in epidermal strips of cowpea leaves systemically infected
    with B1CMV, stained with a combination of calcomine orange
    and luxol brilliant green. A) cells with masses of cyto
    plasmic inclusions, B) details of mass of inclusions seen
    in "A", c) general view of the inclusion distribution in
    epidermal cells, and D) phase contrast micrograph of the
    area photographed in "A". (Ci) cytoplasmic inclusions,
    (CW) plant cell wall, (G) guard cells, and (Nu) nucleus.
    f

    67

    Figure 18 Electron micrographs of ultrathin sections of cowpea leaf
    cells infected with BlCMV showing cross-sections (A, B,
    C) and longitudinal sections (D) of pinwheel inclusions.

    69

    Table I. Symptoms and results of serological assays on varieties of cowpea, Vigna unguiculata
    mechanically inoculated with B1CMV, BCMV-S, CAMV, and CPMV.
    Cowpea Varieties
    Symptoms
    (a)
    Serology
    (b)
    BlCMV
    BCMV-S
    CAMV
    CPMV
    B! CMV
    BCMV-S
    CAMV
    CPMV
    Black Local
    Mt
    M
    +
    _
    +
    _
    Bola de Ouro
    -
    -
    Mt
    Mt
    -
    -
    +
    +
    CE-73
    -
    Mt
    Mt
    Mt
    4-
    4-
    4-
    CE-74
    Mt
    -
    Mt
    -
    +
    -
    +
    -
    CE-175
    M
    -
    Mt
    Mt
    +
    -
    +
    +
    CE-89
    M
    -
    Mt
    -
    +
    -
    +
    -
    CE-3-53
    Mt
    -
    M
    -
    +
    -
    +
    -
    Cream 40
    Mt
    -
    M, Ld
    M Ld.De
    +
    -
    +
    +
    Early Ramshorn
    Mt
    M
    M
    Ne, De
    +
    4-
    +
    +
    Crowder Pea
    -
    -
    Mt
    M, Ld
    -
    -
    +
    +
    1 peane V11
    Mt
    -
    Mt
    -
    +
    -
    +
    -
    Jaguaribe
    M
    -
    Mt
    -
    +
    -
    +
    -
    Knuckle Purple Hul1
    M
    -
    M, Ld
    M,Ld,De
    +
    -
    +
    4-
    Pitiuba
    Mt
    -
    M
    -
    +
    -
    +
    -
    Potomac
    M
    -
    Mt
    -
    +
    -
    +
    -
    Ser ido
    -
    -
    Mt
    Mt
    -
    -
    +
    +
    Snapper Long Pod
    -
    -
    M, Ld
    A.
    -
    -
    +
    -U
    Sete Semanas
    -
    -
    Mt
    -
    -
    -
    +
    -
    V-4 Alagoas
    M
    -
    Mt
    Chi
    +
    -
    +
    +
    V-5 Parayba
    M
    M
    Mt
    +
    -
    +
    +
    (a) Chl= systemic chlorosis, De= plant death; Ld= leaf deformation, M= mosaic, Mt= mottle, Ne=
    systemic necrosis, and = no symptoms.
    (b)
    positive serological reaction in double diffusion tests,
    no serological reaction in double diffusion tests.
    -'j
    o
    not tested

    71
    annuurn L. 1 Ear I y Ca I wonder 1 ; Cucumi s sat ¡ yus L. ; Cucurb i ta pepo L.
    'Small Sugar'; Lupnus angustifoljus L. 'Bitter Blue'; Lupinus luteus
    L. 'Sweet Yellow'; Phaseolus vulgaris 'Black Turtle-1', 'Green Northern
    1140', 'Improved Tendergreen', 'Lake Shasta', 'Michelite 62', 'Pink
    Rosa', 'Pink Viva1, 'Puregold Wax', 'Red Mexican U-3 ^' 'Red Mexican
    U-3 51, 'Top-crop* and 'VC 1822'; Pi sum sativum L. 'Alaska', 'Bonneville'
    and 'Ranger'; and Vicia faba L.
    The reactions of cowpea varieties to mechanical inoculations of
    B1CMV, BCMV-S, CAMV, and CPMV are indicated in Table I. All inoculated
    plants were assayed serologically for the presence of the viruses
    (Table l).
    Discuss ion
    Blackeye cowpea mosaic virus and its cytoplasmic inclusions were
    successfully purified from systemically infected cowpea or N^. benthamiana
    leaf tissues with the procedures outlined herein. The first method of
    virus purification (Fig. 2) gave good yields of highly purified B1CMV,
    and the combination of n butanol and chloroform-carbon tetrachloride
    (Fig. 4) was the better procedure for purification of B1CMV and its
    cytoplasmic inclusions from the same batch of tissue. The high degree
    of purity of the B1CMV preparations indicated by spectrophotometry,
    analytical centrifugation and PAGE analyses, as well as serological
    studies and electron microscopic observations, confirmed the efficiency
    of the pur ification procedures.
    Aggregation of virus particles and virus and host components
    during purification appears to be a limiting factor for obtaining high

    72
    yields of viruses in the PVY group (Shepherd and Pound, I960; van
    Oosten, 1972; Hiebert and McDonald, 1973; Uyeda et al., 1975; and
    Barnett and Alper, 1977). Hiebert and McDonald (1973) reported
    aggregation of virus particles after PEG precipitation. The losses
    of BICMV by low speed centrifugation due to aggregation of virus
    particles were reduced by maintaining the virus in KPO^, buffer, pH
    8.2, after precipitation with PEG.
    Another critical aspect on purification of potyviruses for ob
    taining maximum virus yield is the host used for virus increase. In
    order to obtain a good yield of BICMV from the 'Knuckle Purple Hull1
    variety of cowpea the virus was inoculated into the source plants
    at the age of 3 to 4 days after emergence and the systemically in
    fected leaf tissues were harvested 15 to 18 days after inoculation.
    Attempts to purify the virus from plants inoculated later than that
    or from tissue harvested more than 30 days after inoculation resulted
    in very poor yields of virus and cytoplasmic inclusions.
    Electron microscopic examinations of purified preparations of
    BICMV indicated a low percentage of virus fragmentation during the
    purification processes (Fig. 6-A). Particle measurements of purified
    BICMV and of BICMV particles on grids prepared for SSEM with infected
    cowpea leaf tissue gave two modal lengths (Figs. 7, 8) which differed
    by approximately 30 nm. Variations in lengths of virus particles have
    been extensively observed (Edwardson, 1974). As reviewed by Edwardson
    (1974), virus length variations may be attributed to several factors,
    including sample preparation, host influence, virus strain differences,
    and normal fluctuations in the electron microscope magnification. In
    crease of 50 to 100 nm in certain potyvirus particle lengths induced

    73
    by magnesium ions were reported by Govier and Woods (1971). They in
    dicated that in the presence of Mg ions the particles were straight,
    contrasting with the flexuous particles observed in the absence of
    Mg ions. On antiserum-coated grids several antibodies combine with
    a single virus particle and, possibly, increase; its length. Because
    of the specific antigen-antibody reaction the B1CMV particles were so
    strongly attached to the surface of the antiserum-coated grids that
    they could not be removed by repeated washing. On the other hand,
    positive staining of B1CMV particles with ethanolic uranyl acetate
    may have induced some changes in their lengths. Measurements of 25
    B1CMV particles on grids prepared by conventional leaf-dip preparation
    with PTA gave a modal length similar to that estimated for purified
    B1CMV negatively stained with PTA. Milne and Luisoni (1977) emphasized
    that negative staining gives better preservation and better resolution
    of viral capsids than positive staining. Using SSEM with uranyl
    acetate as a negative stain, Milne and Luisoni (1977), observed no
    change in the normal lengths of TMV and a potexvirus. However, leaf-
    dip preparations often contain too few particles to photograph con
    veniently for virus particle measurements, whereas relatively large
    numbers of particles can be photographed by using the serologically
    specific electron microscopic technique. As indicated by Derrick
    and Brlansky (1976), the addition of sucrose in the extracting and
    washing buffers greatly reduced the amount of plant debris on the
    SSEM grids. High amounts of plant debris in electron microscopic
    preparations are frequent problems in establishing the dimensions of
    a virus.

    Polyacrylamide gel electrophoresis of SDS dissociated cytoplasmic
    inclusions and viral coat proteins clearly indicated that ths viral
    coat protein subunit was smaller than the inclusion subunit (Fig. 10).
    The PAGE results revealed that the inclusions were made of a single
    kind of protein with an estimated molecular weight of 70,000 daltons.
    Polyacrylamide gel electrophoresis of cytoplasmic inclusion prepara
    tions conducted by Hiebert and McDonald (1973) showed one protein
    component with molecular weight of 67,000 daltons for PVY; 67,000 for
    PeMV; 69,300 for BiMV; 69,600 for TEV; and 70,300 for TuMV. The PAGE
    studies also indicated that freshly purified B1CMV consisted of a main
    protein component with a molecular weight around 34,000 daltons. Two
    smaller protein components were also revealed by PAGE analysis of SDS
    denatured viral coat protein (Fig. 10). Since only traces of the
    faster moving proteins were observed with fresh purified B1CMV, and
    greater amounts of these proteinaceous components were revealed by
    PAGE analyses of stored purified B1CMV preparations (Fig. 10), it is
    assumed that the smaller components are due to the degradation of the
    slow moving protein during purification and storage. Hiebert and
    McDonald (1976) observed that some possible enzymatic degradation of
    TuMV capsid protein occurred during storage of purified virus prepara
    tions. The lower sedimentation coefficient estimated for stored
    purified B1CMV preparations (Fig. 9) is further evidence of proteolytic
    degradation of viral coat protein during storage at 4 C. According
    to Hiebert and McDonald (1976), it is likely that '^q values reported
    for potyviruses that are near 140 S represent virus with partially
    degraded capsid protein, whereas those near 160 S represent virus with
    intact capsid protein."
    This proteolytic degradation also changes

    75
    the antigenic properties of viral coat protein (Hiebert and McDonald,
    1976; and Purcifull and Batchelor, 1977). Using antiserum obtained
    for freshly purified BICMV, serological distinction was observed
    between freshly purified preparations of BICMV and purified BICMV
    stored at 4 C for more than 30 days (Fig, 16-F). The serological
    distinctions between different antigen preparations of the same virus
    observed herein are of great significance for serological identifica
    tion and characterization of potyviruses as pointed out by Hiebert and
    McDonald (1976) and Purcifull and Batchelor (1977). It is important
    to keep in mind that purification, storage of either purified virus
    preparations or crude sap containing virus, and mailing of virus-
    infected fresh plant tissues may all result in modifications in the
    antigenic properties of viral coat protein. To solve this problem,
    the preservation of plant virus antigens by 1yophi 1ization of crude
    extracts from infected plants (Purcifull et al., 1975) or purified
    virus preparations is recommended. Blackeye cowpea mosaic virus
    has been maintained in lyophilized condition either in crude sap or
    purified preparation over two years during the course of this study
    without any perceptive change in its antigenic properties.
    Another factor that should be considered during serological
    relationship studies between viruses in the PVY group is the specificity
    of antisera. Variations in the degree of cross reactivity exhibited
    by different antisera obtained against the same virus have been
    attributed to differences between individual animals (van Regenmortel
    and von Wechmar, 1970), route and number of injections used in the
    immunization program (Hoi 1ings and Stone, 1965) and time of bleeding

    76
    (Tremaine and Wright, 1967; and Koenig and Givord, 197*0- The results
    of the present study indicated that the immunization program and the
    conditions of the antigenic solution used for rabbit immunization may
    also affect the antiserum specificity. A highly specific antiserum
    for BCMV-S was obtained from a rabbit immunized with freshly purified
    BCMV-S, whereas antiserum with a broader cross-reactive spectrum was
    obtained from the same rabbit after a booster injection with a purified
    preparation of BCMV-S stored at 4 C for more than one month. The use
    of such antisera would make it difficult to distinguish between certain
    plant viruses in SDS immunodiffusion tests. The serological distinc
    tion between BICMV and BCMV-S was impossible to detect in SDS double
    immunodiffusion when the BCMV-S antiserum with a wider cross-react ivity
    was used. On the other hand, an antiserum with a wide spectrum of
    activity should be useful for identification of vi rus-infected tissue
    used for plant propagation and possibly for identification of virus
    at the group level. As any vi rus-infected plant organ is undesirable
    for plant propagation the specific virus identification may not be
    necessary in such cases. For example, the BCMV-S antiserum was suc
    cessfully used to identify cowpea seeds infected with B1CMV.
    Unilateral serological relationships observed between B1CMV and
    SoyMV (Fig. 15G) and with B1CMV and BYMV (Fig. 15-H) showed the ne-
    nessity of reciprocal tests for demonstrating the absence of serologi
    cal relationship between two viruses. According to Matthews (1970)
    "to demonstrate that two viruses are serologically unrelated, reactive
    antisera must be prepared against each of the viruses under test."
    Reciprocal tests are also important, to show distinction between two
    closely related viruses. It was more difficult to observe a spur

    77
    between BICMV and WMV-2 when both viruses were tested against antiserum
    to WMV-2 than when they were tested against BICMV antiserum (Fig. 15-C).
    Similar results were observed with BCMV isolates and BICMV (Fig. I5~E,
    -F) which may explain the identical reaction reported by Uyemoto et al.
    (1973).
    It is noteworthy that BICMV and BYMV are serologically distinct,
    though related. This supports the contention of Edwardson et al. (1972)
    and Zettler and Evans (1972) that BICMV and BYMV should be considered
    distinct viruses.
    Serological differences between closely related viruses are better
    detected with antisera of fairly low titer (Matthews, 1970). On the
    other hand, he also stated that a high titer antiserum is preferable
    for demonstrating distant serological relationship. This can be
    illustrated by the serological tests carried out with BICMV and CAMV
    isolates using a BICMV antiserum with a titer of 32. By diluting the
    antiserum to 1/4, no reaction was observed with the heterologous virus
    (CAMV) whereas a fairly good reaction was still detected with the homolo
    gous antigen. The absence of reaction between BICMV and LMV-antiserum
    (Fig. 15-J) may be a result of the low titer of the antiserum.
    The intragel cross-absorption test was effective for demonstrating
    distinctions between two closely related viruses (Fig. 16-B, -D). This
    is additional evidence that serological distinctions that are undetect
    able in conventional doub1e-immunodiffus ion tests may be clearly revealed
    by intragel cross-absorption. Using this test, Matthews (1970) revealed
    a serological difference between type TMV and a nitrous acid induced
    mutant which showed a reaction of identity when tested against

    78
    unabsorbed TMV antiserum. For a full precipitation of the crossreacting
    antibodies close to the center well, a fairly high concentration of
    the heterologous antigen should be used to fill the antiserum well. This
    is illustrated by the intragel cross-absorption tests with B1CHV anti
    serum shown in Figure 16. A precipitin ring was formed very close to
    the center well when a highly concentrated purified preparation of
    BCMV-S (0.5 1.0 mg/ml) was used to absorb B1CMV antiserum (Fig. 16-D)
    whereas the ring formed approximately 2 mm away from edge of the well
    when B1CMV antiserum was absorbed with a less concentrated preparation
    of CAMV (0.01 0.05 mg/ml) (Fig, 16-B). In both cases, though, the
    intragel cross-absorption test showed serological distinction between
    the viruses. The intensity of the reaction between the homologous
    antigen and the cross absorbed antiserum may give some information
    about the degree of relationship between the viruses. Weaker homologous
    reaction indicates closer serological relationship. Based on this,
    the results of the present study clearly indicate that B1CHV is more
    closely related serologically to BCMV-S than to CAMV (Fig. 16-B, -D).
    The different degrees of serological relationships are also indicated
    by the intensity of the precipitin lines spurring over the heterologous
    virus reactions in straight diffusion tests (Fig. 16-A, -C). Serological
    relationship between different potyviruses has been commonly observed
    (Bercks, I960; Purcifull and Shepherd, 196*4; Purcifull and Gooding,
    1970; (Jyemoto et a I 1972; and Shepard et al., 197*0, and the cross -
    absorption of an antiserum with heterologous viruses has also been
    used to study serological relationship between plant viruses in tube
    precipitin tests (Wetter, 1967; and Alba and Oliveira, 1977), and in
    combination with gel diffusion tests (van Regenmortel, 1966; Wetter,

    79
    1967; Nelson and Knuhtsen, 1973; Shepard et a)., 197*1; and Jones and
    Diachun, 1977)- The intragel cross-absorption technique was also ob
    served to be useful in demonstrating cross-protection between sero
    logically distinct strains of plant viruses (Lima and Nelson, 1975).
    The fact that most of the antisera obtained against purified
    B1CMV preparations did not react with extracts of non infected cowpea
    tissue can be added to confirm the efficiency of the virus purifica
    tion procedures described herein. On the other hand, the high popula
    tion of antibodies for normal plant antigens developed by the rabbits
    injected with B1CMV-I purified from infected cowpea was an indication
    that vi rus-infected cowpea tissue may have a high concentration of
    host antigens, which were difficult to separate from the B1CMV cyto
    plasmic inclusions. However, using N, benthamiana as a source plant
    for B1CHV-I purification, antiserum specific for B1CMV-I was obtained.
    This is an additional indication of the useful application of N^.
    benthamiana in plant virus research. Nicotiana benthamiana has been
    artificially infected with more than 50 plant viruses (Quacquare 11i
    and Avgelis, 1975; and Christie and Crawford, in press), showing its
    great potential for cytological, serological, and physiological studies
    of different viruses in the same biological system.
    The foot-pad route of rabbit immunization (Ziemiecki and Wood,
    1975) used to obtain the antiserum specific for B1CMV-I was an efficient
    procedure. The high yield of antibodies obtained for BCMV-S using the
    same route of immunization (Lima et al., unpublished) is additional
    evidence that a high titer antiserum can be obtained at the expense
    of very little antigen.

    80
    Reciprocal immunodiffus ion tests with antisera specific for
    B1CMV and B1CMV-I (Fig. 12-A, -B) confirmed the findings of Hiebert
    et al. (1971), Purcifull et al. (1973), Batchelor (197*0, and McDonald
    and Hiebert (1975) that the inclusion body proteins are immuno
    chemical ly distinct from viral coat protein and host proteins.
    The results of single radial immunodiffusion tests indicated
    that agar-media impregnated with mono-specific antiserum or with a
    mixture of antisera can be used for serodiagnosis of two morphologically
    distinct legume viruses. Single radial immunodiffusion tests were
    first used in plant virology by Shepard (I9&9) for serodiagnosis of
    potato virus X in potato tuber sprouts. Subsequently the same method
    was successfully used to identify plant tissue infected with carla-
    viruses (Shepard, 1970; and Shepard et al. 1971) potyviruses (Uyemoto
    et al., 1972; and Casper, 197*), a cucumovirus (Richter et al., 1975),
    a hordeivirus (Slack and Shepherd, 1975), and tobamovirus (Granett and
    Shalla, 1970; and Clifford, 1977). Radia1 -immunodiffus ion plates con
    taining a mixture of antisera to two or three filamentous viruses have
    been used for detection of potato viruses X, S, and M (Shepard, 1972).
    This, however, appears to be the first report of a multiple-antisera
    medium for detection of both an isometric and a rod-shaped plant viruses.
    Shepard (1969) observed that single radial immunodiffusion was
    more sensitive than double immunodiffusion for detection of PVX in in
    fected plant tissue, but Richter et al. (1975) obtained better results
    with double diffusion tests than with single diffusion for serological
    detection of CMV in naturally infected herbaceous plants. No attempts
    to compare these two serological techniques were made in the present
    study. Some observations, however, indicated that single radial

    81
    immunodiffusion requires fairly large amounts of antiserum and that
    the proper antiserum concentration needs to be previously determined
    for highest sensitivity and to avoid spurious reactions in SDS-agar
    media. Better results in multi-ant¡sera media were obtained when
    pyrrolidine was used as denaturant of virus coat protein.
    The three serologically related but distinct legume viruses,
    BlCMV, BCMV-S, and CAMV can also be differentiated by some biological
    properties. The CAMV isolate was well adapted to cowpea, infecting
    and causing symptoms in all 20 inoculated cowpea varieties. On the
    other hand, five cowpea varieties showed immunity to BlCMV, and only
    two were infected with BCMV-S, which caused very mild symptoms
    (Table I). The different symptomato 1ogicaI reactions induced by CAMV
    and BlCMV in some of the varieties (Table l) clearly indicate that
    they can be used to distinguish these two potyviruses. It was ob
    served, however, that some of the symptoms induced by the viruses
    varied with temperature, light conditions, and age at which the plants
    were inoculated, but no variation was observed with the immunity of
    any cowpea variety. The cowpea varieties that showed immunity to
    BlCMV (Table I) should be included in a cowpea breeding program or in
    a control program for this virus in the southeastern United States.
    Cowpea lines with resistance to other viruses have been selected in
    different parts of the world (Williams, 1977a; and Beier et al.,
    1977). Virus-resistant lines with resistance to other plant pathogens
    have also been identified (Williams, 1977a and 1977b).
    Attempts to compare BlCMV with the East African type of CAMV were
    impossible because all samples of vi rus-infected leaf-tissue arrived
    in high degree of decomposition with the virus already inactivated.

    82
    Serological studies with such decomposed leaf tissue and B1CMV
    antiserum gave results similar to those obtained with the CAMV isolate
    (Fig. 16-A) obtained originally from Morocco. As the inactivation
    of the virus in the decomposed tissue may have destroyed some of its
    antigenic determinants, no conclusive results about its serological
    relationship with BICMV can be derived from these tests.
    Light and electron microscopy of cowpea and other host cells in
    fected with any one of these three legume potyviruses revealed that
    their cytoplasmic inclusions are morphologically similar. In ultrathin
    sections, their inclusions consisted of pinwheels similar to those
    induced by the potyviruses from Edwardson's subdivision-I (Edwardson,
    197*0. The cytoplasmic inclusions induced either by BCMV-S or CAMV,
    however, failed to react with antiserum for B1CMV induced inclusions.
    The low titer of the inclusion antiserum, however, may be one of the
    reasons for the absence of reactions. Despite the great similarity
    in the ultrastructures of pinwheel inclusions induced by B1CMV and CAMV,
    they showed some difference at the light microscope level. Whereas
    B1CMV induced big masses of cytoplasmic inclusions in 'Knuckle Purple
    Hull1 (Fig. 17), only scattered small bundles of inclusions were ob
    served in the cells of this host infected with CAMV. This is also an
    indication of no direct correlation between the severity of the symp
    toms and abundance of cytoplasmic inclusions induced by these poty-
    viruses, since 'Knuckle Purple Hull' is more susceptible to CAMV than
    to B1CMV (Table I). A similar phenomenon was observed with these two
    viruses in spectabilis. In addition to this, the nuclear inclu
    sions readily observed in cells of £. spectabilis systemically

    83
    infected with BlCMV (Christie and Edwardson, 1977) were not seen in
    leaf tissue of this host infected with CAMV.
    In summary, BlCMV is a potyvirus that belongs to Edwardson's
    subdivision-I (Edwaidson, 197*0 and has a modal length of approximately
    750 nm. The BlCMV particles have a single sedimenting peak with s^q =
    157 159 S and have a main protein component with a MW of 3*t,000
    daltons. Its cytoplasmic inclusions are made of tubes which show
    striations with periodicities of approximately 5 nm and consist of a
    single type of protein estimated to have a MW of 70,000 daltons. The
    virus also induces nuclear inclusions in certain hosts including C.
    spectabi 1is. Blackeye cowpea mosaic virus is serologically unrelated
    to seven potyviruses and serologically related to, but distinct from
    eight other potyviruses in SDS-immunodiffus ion. The virus has a narrow
    host range outside leguminosae, is seed-borne in at least two cowpea
    varieties and is transmitted by aphids in a nonpersistent manner.
    Based on its physical, biological, cytological and immunochemical
    properties, BlCMV can be differentiated from any other virus that
    infects cowpea. The antisera prepared for BlCMV and its cytoplasmic
    inclusions were essential tools for the development of the serological
    techniques for detection of vi rus-infected seeds described in Chapter II.

    CHAPTER I I
    IMMUNOCHEMICAL AND CYTOLOGICAL TECHNIQUES FOR
    DETECTION OF LEGUME VIRUSES IN INFECTED SEEDS
    Introduction
    The transmission of plant viruses through seed of infected host
    plants was first demonstrated by Reddick and Stewart (1919), who
    showed that bean common mosaic virus (BCMV) was transmitted by ap
    proximately 50% of seeds from infected Phaseolus vulgaris. Since
    then, the phenomenon of seed transmission of plant viruses has re
    ceived considerable attention and an appreciable number of viruses
    have been demonstrated to be seed-borne to some extent (Fulton, 1964;
    Bennett, 1966; Shepherd, 1972; and Phatak, 1974). Virus can be intro
    duced into a crop at an early stage of plant development through in
    fected seeds. Thus, the production of virus-free seeds, or seed lots
    with very low virus content may provide a very effective control of
    seed-borne plant viruses. Seed certification programs have been
    developed to test seed lots for the presence of viruses and to select
    virus-free seeds. Barley stripe mosaic virus, which is responsible
    for a serious disease in Montana (Afanasiev, 1956), and lettuce mosaic
    virus(LMV), the causal agent of an important disease of lettuce
    (Grogan et al 1952), are good examples of virus diseases against
    which seed certification programs have been successful (Zink et al.,
    1956; Hamilton, 1965; Phatak, 1974; and Slack and Shepherd, 1975).

    85
    Barley stripe mosaic virus has no known vector, such as insects or
    mites, but it has a high rate of seed transmission, which severely
    reduces crop production. On the other hand, LMV is transmitted by
    a low percentage of seeds, but is regularly spread further in the
    field by aphids, resulting in substantial losses (Zink et al., 1956).
    Several methods (Phatak, 197*0 have been developed to detect the
    presence of infected seeds to control plant, diseases caused by seed-
    borne viruses: Growing-on tests. Seeds are planted in greenhouses
    or under other insect-proof conditions and the first leaves of the
    seedlings are observed for the characteristic symptoms which vary
    according to the host-virus combination This method can fail under
    environmental conditions that adversely affect the symptom development
    and with latent strains of a virus that do not produce visible symp
    toms. Indicat or-inoculation tests. Seeds are ground up with buffer
    solution and mechanically inoculated in the indicator hosts. Although
    this method has been used extensively for LMV (Phatak, 197**), it is
    very time consuming and requires considerable greenhouse space.
    Serological tests Immunochemical tests have also been developed
    for detecting virus in extracts from single seeds (Scott, 1961, and
    Lister, 1977), and individual seed embryos (Hamilton, 1965). However,
    no successful results have been obtained in double immunodiffusion
    systems with long flexuous rod viruses such as those from the potyvirus
    group (Phatak, 197A). Electron microscopy. A serologically specific
    electron microscopic technique developed by Derrick and Brlansky (1976)
    has been successfully used to detect the presence of virus particles
    in extracts of groups of seeds (Brlansky and Derrick, 1976).

    86
    The main purpose of the present investigation was to develop ef
    ficient and rapid serodiagnostic techniques to assay legume seed lots
    for the presence of vi rus-infected seeds. Seeds of cowpea, \l_. unguiculata
    'Knuckle Purple Hull* infected with B1CMV were used as a model host-
    virus combination. Immunochemical techniques for detection of B1CMV,
    BCMV, and SoyMV in hypocotyls of germinated vi rus-infected seeds of
    \l_. unguiculata, P_. vulgaris, and G^. Max, respectively, are described
    in this chapter. Abstracts of portions of this research have already
    been published (Lima and Purcifull, 1977a, 1977b),
    Literature Review
    The phenomenon of seed transmission of plant viruses was first
    demonstrated by Reddick and Stewart (1919), who presented strong
    evidence of seed transmission of BCMV in Phaseolus vulgaris. Since
    then, a large body of information has been accumulated about the
    transmission of numerous plant viruses through the seeds of infected
    host plants (Fulton, 1961*; Bennett, 1966; Shepherd, 1972; Baker, 1972;
    and Phatak, 1971*). Among the 183 plant viruses described in the
    Commonwealth Mycological Institute up to September, 1977, (Doi et al.,
    1977), 51 viruses have been experimentally demonstrated to be seed-
    borne to some extent. Several plant viruses are known to be seed-borne
    in many leguminous host plants, but this review will cover only those
    viruses transmitted through seeds of Vigna spp., Glycine max, and
    Phaseolus vu1 garis.

    87
    Seed-Borne Viruses in Vigna spp.
    Gardner (1927) apparently was the first to report the transmission
    of a cowpea virus through seeds of cowpea. Since then, many viruses
    which naturally infect cowpea have been demonstrated to be seed-borne
    in this host.
    A virus isolated from cowpea in Trinidad was demonstrated to be
    transmitted through 8% of seeds of asparagus-bean (Vigna sesquipedal is)
    obtained from vi rus-infected plants (Dale, 1949). The virus is be
    lieved to be a representative strain of cowpea mosaic virus (Agrawal,
    1964; and van Kammen, 1971, 1972). It seems that the seed transmis-
    sibility of CPMV is erratic and depends on the type of virus isolate
    and the cowpea variety involved. A cowpea mosaic virus isolated from
    cowpea grown in Arkansas was seed-borne in this host (Shepherd, 1964).
    Approximately 620 Blackeye1 cowpea plants grown from seeds harvested
    from plants artificially inoculated with CPMV failed to develop mosaic
    symptoms (Perez and Cortes-Mon11 or, 1970). On the other hand, Haque
    and Persad (1975) observed that the rate of seed transmission of CPMV
    varied from zero to 5-8% depending on the cowpea varieties and selec-
    t ions.
    Anderson (1957) reported the seed transmission of three cowpea
    viruses, including a strain of CMV, which was transmitted through
    4 28% of cowpea seeds from artificially infected plants, A virus
    closely resembling a strain of CMV was seed-borne in cowpea with a
    transmission rate of 5 to 16% (Chenulu et a 1. 1968). On the basis
    of symptoms observed on cowpea plants grown from commercial seeds,
    Gay and Winstead (1970) reported seed transmission of CMV, a cowpea

    strain of southern bean mosaic virus and a virus referred to as a cow-
    pea strain of bean yellow mosaic virus. A strain of CMV isolated from
    cowpeas in Morocco was transmitted through approximately 25% of the
    seeds harvested from artificially inoculated cowpcas (Fischer and
    Lockhart, 1976b). the seed transraissibility of the cowpca strain
    of SBMV was firsc demonstrated by Shepherd and Fulton (1962).
    McLean (1961) observed that a cowpea virus was transmitted through
    approximately 5^ of seeds of highly susceptible cowpea-varietics, but
    slightly susceptible or somewhat resistant varieties produced lower
    percentages of virus-infected seeds. Snyder (1962) observed only 3
    to 61 transmission of a cowpca virus through commercial seeds and 37
    to 6IL transmission of the same virus through seeds obtained from
    virus-infected plants. Yu (1966), however, found no difference In
    the percentages of virus-infected cowpea plants grown from seeds ob
    tained from artificially or naturally infected plants. These three
    viruses were also transmitted by aphids (McLean, 1961; Snyder, 1962;
    and Yu, 1966). Similar aphid-transmitted viruses reported from
    India were also demonstrated to be seed-borne (liarlani and Kandaswamy,
    1961; and Yerma, 1971). A rod-shaped virus Isolated from cowpea in
    northern Italy was designated cowpea aphid-borne mosaic virus (CAMV)
    and studied by Lovisolo and Conti (1966) who demonstrated its seed
    transmissibility in cowpea. The transmissibility of CAMV through
    seeds of cowpea has been confirmed by several studies involving dif
    ferent strains of the virus and varieties of the host (Kaiser et al.,
    1966; Tsuchizaki et al., 1970; Bock, 1973; Sock and Conti, 1976;
    Phatak, 1976; Khatri and Singh, 1976; and Fischer and Lockhart, 1976a).

    89
    The mosr important cowpea virus in the southeastern United States
    was first isolated in Florida by Anderson (1955a) who later designated
    it blackeye cowpea mosaic virus (BlCMV) (Anderson, 1955b) and de
    monstrated i ts seed transmissibi Iity in cowpea (Anderson, 1957). The
    seed transmissibi 1ity of BlCMV was confirmed by subsequent studies at
    the University of Florida, Gainesville, Zettler and Evans (1972) found
    as much as 18% of BICMV-infected seeds in lots of certified cowpea
    seeds, and Uyemoto et al. (1973) reported 28% of seed transmission for
    this virus in cowpea 'Knuckle Purple Hull*.
    Cowpea mild mottle virus, a carlavirus isolated from cowpea in
    Ghana, was demonstrated to be transmitted through seeds of cowpea, bean,
    and soybean in variable but sometimes large proportions (Brunt and
    Kenten, 1973, 1974). Cowpea banding mosaic virus, which was identified
    as a member of the cucumovirus group in India, was transmitted through
    high percentages of seeds from infected cowpea varieties (Sharma and
    Varma, 1975). A new isometric cowpea virus isolated from cowpea seeds
    from Iran and designated cowpea ringspot virus had a seed transmission
    rate of 15 to 20% in three cowpea cultivars (Phatak, 1974; and Phatak
    et al., 1976).
    Thus, among the four filamentous and the six isometric viruses
    known to naturally infect cowpea, only TMV (fi Iamentous)and cow
    pea chlorotic mottle virus (isometric) have not been demonstrated to
    be seed-borne in cowpea. Kuhn (1964b) found no evidence of seed
    transmission of cowpea chlorotic mottle virus (CCMV) in more than
    2,100 seeds harvested from infected cowpea, and Gay (1969) observed
    no virus symptoms in 3,000 cowpea plants grown from seeds harvested

    90
    from CCHV-infected plants. According to Gay (1969), CCHV was not trans
    mitted through seeds of cowpea because of virus inactivation during seed
    maturation.
    Seed-Borne Viruses in Glycine max.
    Soybean mosaic virus (SoyMV) which is probably common wherever
    soybean is cultivated (Bos, 1972) was first demonstrated to be seed-
    borne in soybean by Gardner and Kendrick (1921). Subsequently, they
    observed that the virus could overwinter in the vi rus-infected seeds
    lying in the field (Kendrick and Gardner, 192A). Although Kendrick
    and Gardner (192A) had also observed that mottled seeds were produced
    by both healthy and v i rus-infected plants and that diseased seedlings
    were produced by mottled as well as clean seeds, the association of
    seedcoat mottling and virus infection has been reported (Koshimizu
    and lizuka, 1957; Ross, 1963, 1968, 1969; and Kennedy and Cooper,
    1967). A mechanical selection of nonmottled soybean seeds has been
    suggested as a measure for controlling SoyMV in Brazil (Lima-Neto and
    Costa, 1976). However, Ross (1970) observed that SoyMV was equally
    transmitted through mottled and nonmottled seeds from vi rus-infected
    soybean plants, and concluded that the percentage of seed transmission
    for SoyMV in soybean could not be estimated from the amount of mottled
    seeds. Working with a Brazilian isolate of SoyMV, Porto and Hagedorn
    (1975) reported the production of mottled seeds by supposedly non-
    infected soybean cultivars and observed that SoyMV was not transmitted
    through seeds of vi rus-infected soybean cv, 'Bienville', which pro
    duced seeds with a high percentage of mottling. Bean pod mottle virus,

    91
    which is not seed-borne in soybean, also increased the percentage of
    mottled seeds (Ross, 1963).
    Ghanekar and Schwenk (197**) showed a rate of seed-transmission
    for tobacco streak virus varying from 2.6 to 30.6% according to the
    soybean cultivar.
    Among the nine viruses known to infect and cause diseases in soy
    bean in Japan, SoyMV, soybean stunt virus, soybean mild mosaic virus,
    peanut stunt virus, southern bean mosaic virus, and a strain of alfalfa
    mosaic virus were reported to be transmitted to some extent through
    seeds of soybean varieties (Koshimizu and lizuka, 1963; I izuka and
    Yunoki, 197**; Takahashi et a I. 197**; lizuka, 197**; and Tamada, 1977).
    The transmission of nepoviruses through seeds of their host is
    well documented. Tobacco ringspot virus (TRSV), a nepovirus (Fenner,
    1976) responsible for a disease commonly called bud blight of soybean,
    was first reported to be transmitted through seeds of artificially
    inoculated soybean plants in 195** (Desjardins et al., 195**). These
    results were confirmed by subsequent studies (Kahn, 1956; Owusu et al.,
    1968; Athow and Bancroft, 1959; and Demski and Harris, 197**). Kahn
    (1956) also demonstrated the seed-transmissib i 1 i ty of tomato ringspot
    virus in soybean and observed that soybean seeds from plants infected
    with either tobacco or tomato ringspot virus had a lower percentage
    of emergence than seeds from virus-free plants. Three other nepo
    viruses: arabis mosaic, raspberry ringspot and tomato black ring
    viruses were experimentally demonstrated to be transmitted through
    seeds of soybean plants artificially inoculated with these viruses
    (Lister, 1960; and Lister and Murant, 1967).

    92
    Seed-Borne Viruses in Phased us vulgaris
    A large body of evidence (Bos, 1971) has confirmed the first
    demonstration of seed transmission of BCMV in P. vulgaris by Reddick
    and Stewart (1919). Burkholder and Muller (1926) reported that seeds
    from bean plants showing mosaic gave rise to 50% diseased plants.
    The virus remained viable within bean seeds for a period of at least
    30 years (Pierce and Hungerford, 1929). Fajardo (1930) observed
    that both infected and noninfected bean seeds germinated with equal
    readiness and vigor, and concluded that the viability of the seeds
    was not directly affected by the presence of BCMV. Fajardo (1930) and
    others (Crowley, 1957, 1959; and Schippers, 1963) found that plants
    infected at early vegetative growth produced more vi rus-infected seeds
    than those infected at later stages.
    It has been demonstrated that BCMV is transmitted irregularly
    through seeds harvested from vi rus-infected plants. Fajardo (1930)
    observed that some of the seeds in a single pod from bean plants in
    fected with BCMV were v i rus-infected and some were noninfected. By
    crossing vi rus-infected and healthy bean varieties, Nelson and Down
    (1933) provided evidence that BCMV was equally transmitted by ovule
    or pollen from infected parent plants. Similarly, Medina and Grogan
    (i960 obtained high percentage of seed transmission of BCMV through
    either the pollens or ovules of infected plants, but also observed
    that the pollens usually transmitted the virus to a largernumber of
    progeny plants than the ovules. Cross-pollination experiments per
    formed by Schippers (1963) revealed that the embryo infection
    with BCMV and consequently the seed-transmission might originate from

    93
    an infected egg-cell or an infected pollen grain. Studies to determine
    the distribution of BCMV in developing reproductive tissues suggested
    that successful seed transmission was insured by the presence of in
    fective virus within the embryo itself (Ekpo and Saettler, 197*0.
    A correlation between symptom severity and percentage of seed
    transmission of BCMV has been observed in different bean varieties
    (Smith and Hewitt, 1938). Medina and Grogan (1961) suggested that the
    percentage of seed transmission of BCMV was greatly affected by the
    differences in varietal susceptibility of the beans. However, Zettler
    (1966) found similar percentages of seed transmission of BCMV in 5
    different bean varieties.
    Using the bean cultivar VC 1822 as an indicator host for BCMV,
    Provvidenti and Cobb (1975) demonstrated a rate of seed transmission
    from 7 to 22% for BCMV in tepary bean (Phaseolus acutifolius Gray var.
    1 atifo 1ius Freem.), and observed that the virus was carried in the
    embryo but not in the testa. Two BCMV isolates obtained from bean
    grown in the Netherlands were observed to be transmitted through ap
    proximately 20 to 8t)% of seeds harvested from infected bean plants
    (Drijfhout and Bos, 1977).
    A strain of CMV isolated from bean plants grown in Spain was also
    demonstrated to be seed-borne in one of twelve bean varieties studied
    by Bos and Maat (197*0. As they tested only a few seeds from virus-
    infected plants of each bean variety, they suggested that a low per
    centage of seed transmission could possibly occur in other varieties.
    The transmissib i 1ity of SBMV through seeds of bean was first
    demonstrated by Zaumeyer and Harter (19*+3) Cheo (1955) found a very

    9**
    high content of SBMV in the embryos of immature seeds from virus-
    infected bean plants, but observed that the virus concentration dropped
    to a low level or disappeared as the seed matured and dehydrated.
    Cheo's finding was confirmed by Crowley (1959), who found that ap
    proximately 100% embryo infection occurred when bean plants were
    inoculated with SBMV at any time prior to flowering, but no seed
    transmission of this virus occurred when samples of mature dry seeds
    were sown. These findings were supported by the results of McDonald
    and Hamilton (1972), which confirmed that in mature bean seeds, in
    fectious SBMV is confined to the seed coat.
    An ilarvirus was also reported to be seed-borne in bean. Tobacco
    streak virus, the causal agent of a severe disease outbreak of pinto
    bean in State of Colorado, U.S.A., in 19^7, was demonstrated to be
    transmitted by approximately 26% of seeds from plants grown from
    vi rus-infected seeds (Thomas and Graham, 1951).
    In summary, seed-transmission is an important factor in the
    perpetuation and dissemination of numerous viruses that cause diseases
    in cowpea, soybean, and bean. The following methods for detecting
    selected seedborne viruses in legumes may be useful in virus disease
    control programs and as research tools.
    Materials and Methods
    Source of Seed and Seed Germination
    Seeds of cowpea, V. unguiculata 'Knuckle Purple Hull' and 'Early
    Ramshorn', harvested from B1CMV-infected plants were used in the present
    tests. All seed lots showing B1CMV-transmission were obtained either

    95
    in fields of 'Knuckle Purple Hull* cowpeas in Gainesville, Florida,
    or from Gilvan Pio Ribeiro, University of Georgia, Athens. As controls,
    virus-free 'Knuckle Purple Hull' seeds produced in Texas were obtained
    from a local commercial source.
    Seeds were surface sterilized in 0.5% sodium hypochlorite for
    10 min, rinsed thoroughly with sterile deionized water and placed in
    moistened paper towels to germinate. The towels were rolled up, placed
    upright in 30 ml beakers and the seeds were allowed to germinate for
    3 to 5 days at 25 27 C in an incubator.
    Preparation of Antigens for Serology
    Hypocotyls from germinated seeds, singly or in groups of 5 or 10
    were tested in double or single radial immunodiffusion tests. If in
    formation about the percentage of infected seeds was wanted, I 2 mm
    thick discs from individual hypocotyls were cut with a razor blade
    which was rinsed with 95% ethanol and deionized water after cutting
    each hypocotyl, These hypocotyl discs were tested individually in
    double or single immunodiffusion tests by embedding them directly
    into the agar. Groups of 5 or 10 hypocotyls were ground in water or
    1.5% SOS (1/1 w/v) with a mortar and pestle as described previously
    (Purcifull and Batchelor, 1977) and tested against B1CMV and B1CMV-I
    antisera in double and single radial diffusion. Single or bulked
    hypocotyls were checked by SSEM for the presence of B1CMV particles.
    Double Immunodiffusion Tests
    Individual hypocotyls or groups of hypocotyls from germinated
    cowpea seeds were tested against B1CMV antiserum in double diffusion

    96
    tests in agar medium containing 0.8% Noble agar, 0.5% SDS, and 1.0%
    pH 7.2. Eight to twelve discs of individual hypocotyls were embedded
    in the agar medium, k to 5 mm away from each antiserum well. Undiluted
    antiserum for BlCliV was routinely used in these tests, but dilutions
    of 1/2, 1/4, and 1/8 of the antiserum with either normal serum or
    0,05 M Tris buffer, pH 7.2 were also tested against hypocotyl discs
    and extracts of hypocotyl tissue. As controls each hypocotyl was also
    tested against normal serum, and hypocotyls from noninfected seeds
    were also included in each test. Extracts from groups of hypocotyls
    were tested by double immunodiffusion against B1CMV antiserum. Hypo
    cotyl extracts were pipetted into the antigen wells distributed in an
    hexagonal arrangement around the antiserum well. All plates were in
    cubated in a moist chamber at 2k C for 2k to k8 hr. The sensitivity
    of double immunodiffusion for detection of B1CMV in extracts of bulked
    hypocotyls of germinated 'Knuckle Purple Hull' seeds was tested by
    mixing 1.0 g of infected hypocotyl tissue with different amounts of
    noninfected hypocotyls.
    Single Radial Immunodiffusion Tests
    Discs and extracts of hypocotyls were tested by single radial
    immunodiffusion in medium containing 0.8% Noble agar, 0.5% SDS,
    1.0% NaN^ and 15% antiserum for B1CMV buffered with 0.05 M Tris-HCI,
    pH 7.2. Hypocotyl discs were embedded directly into the solidified
    agar medium with the aid of forceps and the plates were incubated
    in a moist chamber at 2k C for 2k to 72 hr. Precipitin reactions
    were detected by direct observation of the plates on a darkfield

    97
    light box or with a binocular microscope (6 to 15 x) in which the
    plates were illuminated from the bottom. Extracts of groups of 5
    hypocotyls were placed into wells (3 mm in diameter) punched in the
    agar medium in a row arrangement. The wells were spaced 3 to 4 mm
    from each other as measured from the edges of the wells and as many
    as 230 germinated seeds could be tested in a 90 x 15 mm plastic petri
    dish. Precipitin rings around the wells charged with extracts from
    infected tissue could be detected anywhere between one to 24 hr.
    Serologically Specific Electron Microscopy
    Hypocotyl extracts prepared in 0.05 M Tris buffer, pH 7-2, con
    taining 0.15 M NaCl and 0.4 M sucrose were examined for the presence
    of B1CMV by SSEM as described previous1 y (oerrick and Brlansky, 1976).
    Copper grids with Parlodion film coated with carbon were treated with
    B1CMV antiserum diluted to 1/1000 in 0.05 M Tris buffer, pH 7.2. The
    grids were washed with 0.05 M Tris buffer and floated on drops of
    hypocotyl extracts lor 3 to 4 hr at room temperature. After washing
    with approximately 2 ml of the extracting buffer and then with ap
    proximately I ml of deionized water, the grids were positively stained
    with 1.0% uranyl acetate in 50% ethanol. The grids were washed again
    with 50% ethanol, dried and examined in the Philips Model 200 electron
    microscope. The sensitivity of SSEM to detect the presence of B1CMV
    particles in extracts from a mixture of hypocotyls was determined by
    diluting infected 'Knuckle Purple Hull' hypocotyl tissue with non-
    infected tissues up to a dilution of 1/50 (w/w). The different mix
    tures of infected and noninfected hypocotyl tissues were ground in

    98
    extracting buffer ()/l, w/v) with mortars and pestles and small drops
    from these extracts were used to treat the antiserum-sensitized grids.
    Double Immunodiffusion Tests and SSEM for Detection of Other Viruses
    in Germinated Legume Seeds
    Double immunodiffusion tests using discs or extracts of hypocotyls,
    and SSEM using hypocotyl extracts were used to detect the presence
    of BCMV and SoyMV in germinated seeds. A seed lot of bean, P^. vulgaris
    Black Turtle' containing BCMV-infected seeds was obtained from Dr.
    Rosario Provvidenti, New York State Agricultural Experiment Station,
    Geneva. Infected seeds were detected by double immunodiffusion and
    SSEM using antiserum obtained for a severe strain of BCMV (BCMV-S)
    isolated from sirat.ro, Macroptilium atropurpureum, in Florida (Lima
    et al.,1977). Extracts of these germinated bean seeds prepared in
    0.05 M potassium phosphate buffer, pH 7-5, were also inoculated in a
    very sensitive local lesion bean line VC-1822 (Provvidenti and Cobb,
    1975). Antiserum for SoyMV was also used to detect SoyMV-infected
    seeds of soybean, Glycine max.
    Serology and Microscopy of Cytoplasmic Inclusions Induced by B1CMV
    and SoyMV in Hypocotyls of Germinated Seeds
    Germinated seeds of cowpea 'Knuckle Purple Hull' infected with
    B1CMV and germinated seeds of soybean 'Midwest' infected with SoyMV
    were serologically identified by double immunodiffusion tests using
    antiserum for B1CMV and SoyMV, respectively, and hypocotyl discs as
    assay antigens. Hypocotyl extracts from the vi rus-infected A-5-day-
    old seedlings were tested by SDS double immunodiffusion tests against
    B1CMV and SoyMV inclusion antisera.

    99
    Cytoplasmic inclusions were examined in epidermal strips of the
    hypocotyls of germinated cowpea seeds infected with B1CMV, and of
    SoyMV-infected hypocotyls of germinated soybean seeds, after staining
    with a combination of calcomine orange and "luxol" brilliant green
    (Christie, 1967).
    Small pieces of B1CMV-infected and noninfected cowpea hypocotyls
    were prepared for ultrathin sectioning as described previously in
    Chapter I. Similarly, healthy and SoyMV-infected soybean hypocotyls
    were prepared for ultrathin sectioning. Sections were cut with a
    diamond knife and stained with potassium permanganate, uranyl acetate,
    and lead citrate. All specimens were examined with a Philips Model
    200 electron microscope.
    Resu 1ts
    Preparation of Antigens for Serological Tests
    Antigens prepared from hypocotyls of germinated seeds proved to
    be very satisfactory for indexing seeds in double and single immuno
    diffusion tests. Neither small discs nor extracts from hypocotyl
    tissues showed any kind of nonspecific reaction that could interfere
    with the virus-specific reaction in double or in single immunodiffusion
    tests. On the other hand, extracts obtained from whole seedlings,
    including roots, cotyledons and primary leaves showed several types
    of precipitat ion patterns when tested against any serum in double
    diffusion tests (Fig. 19). Such nonspecific precipitations were also
    observed with extracts obtained from cotyledons, primary leaves or
    root tissues. These nonspecific reactions were reduced when the agar

    Figure 19
    - Double immunodiffusion tests with extracts from different
    portions of B1CMV-infected and healthy, 4-5-day-old cowpea
    seedlings. A) medium containing 0.8% Noble agar, 1.0%
    NaN^, and 0.5% SDS prepared in deionized water. B) me
    dium with the same composition except that it was prepared
    in 0.05 M Tris-HCl buffer, pH 7-2 instead of water.
    Center wells were charged with: (As) B1CHV antiserum,
    and (Ns) normal serum. The peripheral wells were filled
    with extracts from: (a) hypocotyl from B1CMV-infected
    cowpea seedlings, (b) hypocotyl from healthy cowpea
    seedlings, (c) cotyledons and primary leaves of B1CMV-
    infected seedlings, and (d) cotyledons and primary leaves
    of healthy cowpea. Note the nonspecific precipitate
    formed with cotyledons and primary leaves of virus-
    infected (c) and healthy (d) seedlings and normal serum
    (Ns) and B1CMV antiserum (As). The virus-specific
    precipitin lines clearly shown with vi rus-infected
    hypocotyls (a) and the antiserum (As) was masked by the
    nonspecific precipitates when extracts from cotyledons
    and primary leaves (c) of infected seedlings were used.

    101

    102
    medium was prepared in 0.05 M Tris buffer, pH 7.2 (Fig. 19~B). Based
    on these results a diagram showing several procedures for preparation
    of antigens from leyume seeds is suggested (Fig. 20).
    Double Immunodiffusion Tests
    Blackeye cowpea mosaic virus was detected in hypocotyl extracts
    from bulked seedlings and in small discs of individual hypocotyls of
    cowpea 'Knuckle Purple Hull' and 'Early Ramshorn' by double immuno
    diffusion tests in SDS-gel plates. Virus specific precipitin lines
    were observed with extracts of mixtures of B1CMV-infected and non-
    infected cowpea 'Knuckle Purple Hull' hypocotyls up to a dilution of
    1/30 (w/w), respectively (Fig. 21 -B). This indicated that germinated
    seeds can be divided into groups of up to 30 seed!ings to be tested
    in double immunodiffusion. Approximately equal amounts of hypocotyl
    tissue (0.1 0.4 g) were cut from each seedling and the extracts ob
    tained from each hypocotyl group were deposited into individual antigen
    wells. Precipitin reactions were observed with those groups in which
    at least one seedling was infected with B1CMV. On the other hand, no
    precipitin reactions were observed with extracts obtained from non-
    infected hypocotyl tissue, nor with any hypocotyl extract and normal
    serum (Figs. 19, 21).
    When information about the infection percentage was wanted, discs
    of individual hypocotyls were used in double immunodiffusion. Virus-
    specific precipitin lines formed between B|CMV-infected hypocotyl discs
    and antiserum wells, whereas no reactions were observed with non-
    infected hypocotyls (Fig. 21 C, -D, -F). Good results were observed
    with undiluted antiserum and with antiserum diluted 1/2 and 1/4 with

    Figure 20 Diagram showing methods for assaying legume seeds by
    single and double radial immunodiffusion: I) seeds
    are placed on moistened paper towels, 2) the towels
    are rolled up and placed upright in beakers, 3) ger
    minated seeds after four to five days, b~S) individual
    seedlings are divided into three parts: roots (r) ,
    hypocotyl (hy) and epicotyl (ep) consisting of
    cotyledons and primary leaves, 6) hypocotyl tissue
    is ground with mortar and pestle, 7) hypocotyl ex
    tracts are rested in double radial immunodiffusion
    (DRD), 8) hypocotyl extracts are also tested in single
    radial immunodiffusion (SRD), 3) small discs are cut
    from each hypocotyl, 10) discs from different hypo-
    cotyls are tested by DRD, and 11) discs from different
    hypocotyls are tested by SRD.

    104

    Figure 21 Double immunodiffusion tests with hypocotyl from healthy
    and Bl CHV-infected, 4-5-day-old cowpea seedlings in medium
    containing 0.8% Noble agar, 1.0% NaN^ and 0.5% SDS, pre
    pared in water.
    A B) Serological tests with hypocotyl extracts prepared
    in water. The center wells were charged with:
    (As) B1CHV antiserum, and (Ns) normal serum. The
    peripheral wells were charged with extracts from:
    (1) B1CHV-infected hypocotyls, (H) healthy hypo-
    cotyls, (5) one gram of B1CHV-infected hypocotyl
    mixed with 4 g of healthy hypocotyls, (10) one
    gram of B1CMV-infected hypocotyl and 9 g of
    healthy hypocotyls, (15) one gram of infected
    hypocotyl and 14 g of healthy hypocotyls, (20)
    one gram of infected hypocotyl and 19 g of healthy
    hypocotyls, (25) one gram of infected hypocotyl
    and 24 g of healthy hypocotyls, and (30) one gram
    of infected hypocotyl and 29 g of healthy hypo
    cotyl s.
    C D) Serological tests with saml1 discs of different
    hypocotyls embedded into the agar medium 4 5 mm
    away from the antiserum well. Ten (c) or twelve
    (d) hypocotyl discs are embedded around the cen
    ter wells and the hypocotyl discs are numbered
    from the top in a clockwise direction (arrows).
    Wells were charged with: (As) B1CMV antiserum, and
    (Ns) normal serum. Note virus-antiserum specific
    reactions with hypocotyls: 1, 2, 8, 11, 14, 16,
    18, 19 (0, and 6, 8, 9, 14, 16, 20, and 23 (D).
    E F) Serological tests with hypocotyl extracts (E) and
    hypocotyl discs (F) from healthy and B1CHV-
    infected cowpea seedlings. The center wells were
    charged with: (A) undiluted BICMV antiserum, (A:2)
    B1CMV antiserum diluted 1/2 with normal serum,
    (A:4) B1CMV antiserum diluted 1/4 with normal
    serum, and (A:8) B1CMV antiserum diluted 1/8 with
    normal serum. The peripheral wells were charged
    with extracts from: (5) one gram of B1CHV-infected
    hypocoty1s,and 4 g of healthy hypocotyls, (10) one
    gram of B1CHV-infected hypocotyls and 9 g of healthy
    hypocotyls, and (H) healthy hypocotyls. Hypocotyl
    discs from healthy (h) and B1CHV-infected (i)
    seedlings were alternated around the antiserum wells
    in F.

    106

    107
    normal serum (Fig. 2 I -F). The percentages of B1CMV-infected seeds in
    four different lots of cowpea 'Knuckle Purple Hull' and one 'Early
    Ramshorn' seed lot were estimated by this simplified double immuno
    diffusion test and by the growing-on test (Table ll). Over a hundred
    seedlings tested by this serological technique were randomly checked
    by SSEM for the presence of BICMV particles and in all cases the results
    corresponded.
    Single Radial Immunodiffusion Tests
    The presence of BICMV in cowpea hypocotyl extracts was detected
    by single radial immunodiffusion tests in agar medium impregnated
    with B1CMV antiserum. Precipitin rings were observed around the
    wells charged with extracts from groups of 5 individual hypocotyls
    in which at least one was infected with BICMV, but not around those
    wells charged with extracts of noninfected hypocotyls (Fig. 22). The
    virus-specific reactions started to appear at approximately one hour
    after the hypocotyl extracts had been added into the wells and became
    very clear and evident 3 to 23 hr later. The reactions were still
    very distinct 24 hr after the hypocotyl extracts had been added into
    the wells.
    When small discs of individual hypocotyls were directly embedded
    into the agar medium containing BICMV antiserum, the virus-specific
    reactions took longer to appear. These specific reactions were re
    cognized as opalescent precipitates around infected hypocotyl discs
    that could be better detected under a binocular microscope. The
    opalescent precipitates which usually were located at the ends of the
    hypocotyl discs, started to appear in 24 hr, but were more evident

    Table II Comparison of immunodiffusion tests with hypocotyl discs and growing-on tests for
    detection of vi rus-infected seeds.
    Seed Source
    V i rus
    Seed
    Assay
    Methods
    Tested
    Sero1ogy
    Growi ng'
    -on Test
    Proportion of
    Infected Seeds
    Percentage of
    Infected Seeds
    Proportion of
    Infected Seeds
    Percentage of
    Infected Seeds
    Cowpea (V. unguiculata)
    Knuck1e Purple Hull
    Lot A
    B1CMV
    72/323*
    22.3
    33/160
    20.6
    Lot B
    B1CMV
    12/160
    7.5
    13/173
    7.5
    Lot C
    B1CMV
    5/115
    4.3
    8/145
    5.5
    Lot D
    B1CMV
    17/160
    10.6
    13/141
    9.2
    Early Ramshorn
    B1CMV
    5/100
    5.0
    34/545**
    6.3**
    Soybean (G. max)
    Jupiter
    SoyMV
    4/176
    2.8
    2/89
    2.2
    Bean (P. vulgaris)
    Black Turtle
    BCMV
    3/79
    3-8
    Not tested
    Not tested
    (*) Number of vi rus-infected seeds over number of seeds tested;
    (**)- Pio-Ribeiro, G. (unpublished).

    Figure 22 Single radial immunodiffusion tests with hypocotyl extracts
    from healthy (H) and B1CMV-infected (l), 4-5-day-old cow-
    pea seedlings in media containing 0.8% Noble agar, 1.0%
    NaN^, 0.5% SOS, and 15% B1CMV antiserum (A, C) and 15%
    normal serum (B). The top row and the two bottom rows
    of wells in C were charged with extracts from healthy
    hypocotyls and the others were randomly filled with ex
    tracts from groups of 5 hypocotyls containing 1 or 2
    B1CMV-infected hypocotyl per group.

    Oil

    at 48 to 72 hr after the test had been set up. Usually after 72 hr,
    a nonspecific precipitate also started to appear throughout the agar
    medium.
    Serological 1y Specific Electron Microscopy
    The SSEM technique developed by Derrick and Brlansky (1976) was
    adapted with great success to identify seed lots of cowpea infected
    with BlCMV (Fig. 23). Virus particles were still observed in extracts
    from hypocotyl tissue which was mixed 1/90 (w/w) with noninfected
    hypocotyls (Fig. 24-B). This indicates that to test several seed
    lots by SSEM for the presence of BlCMV, germinated seeds can be divided
    into groups of up to 50 equal pieces of individual hypocotyls. Each
    group of hypocotyl-pieces is ground in the extracting buffer and a
    drop of the extract is then examined by SSEM using antiserum sensi
    tized grids. Virus particles will be observed in extracts obtained
    from those hypocotyl groups in which at least one hypocotyl is in
    fected with BICMV.
    To illustrate the sensitivity of SSEM for detection of virus
    particles in hypocotyl extracts, the same B1CMV-infected hypocotyl
    was used for four different electron microscopic preparations: a) SSEM;
    b) normal dip preparation on a carbon coated Formvar film supported
    in copper grids and negatively stained with 2% PTA; c) preparation
    similar to SSEM using normal serum instead of B1CMV-antiserum; and
    d) preparation similar to SSEM using grids with Parlodion film coated
    with carbon but treated with 0.05 M Tris buffer instead of antiserum
    (Fig. 23). The results showed a great increase of virus concentration
    on the grids sensitized with BlCMV antiserum when compared with the

    Figure 23 Electron micrographs of B1CMV in hypocotyl extracts from
    the same cowpea seedlings using different preparations,
    (A) serologically specific electron microscopy (SSEM)
    with B1CMV antiserum diluted 1/1000 in 0.05 M Tris-HCl
    buffer, pH 7.2, and positively stained with uranyl
    acetate, (B) dip preparation with 2% phosphotungstic acid
    pH 6.5, containing 0.1% BSA, (C) preparation similar
    to SSEM using normal serum instead of BICMV-ant¡serum,
    and (O) preparation similar to SSEM using Tris buffer
    instead of antiserum. The micrograph A represents a
    typical view of the entire grid whereas micrographs B,
    C, and D are selected areas of the grids showing virus
    part icles (arrows).

    113

    Figure 24 Electron micrographs of serologically specific electron
    microscopy with BICMV, BCMV-S, and CPMV.
    A) BICMV antiserum grid and extracts from a B1CMV-
    infected cowpea hypocotyl ;
    B) BICMV antiserum grid and extracts from one gram of
    BICMV-infected hypocotyl and 49 g of healthy cowpea
    hypocotyls. Arrows point to virus particles;
    C) BCMV-S antiserum grid and leaf extract from BCMV-S
    infected bean;
    D) CPMV antiserum grid and leaf extract from CPMV-
    infected cowpea.

    115
    t .
    *
    j
    l
    * s*
    Ji
    t,
    r
    f

    1000 rfhv ,
    t :
    I :
    ;vv
    -O^rv^
    y \ .
    r'^ .
    v

    116
    other grid preparations (Fig. 23). As the antiserum sensitized grids
    were washed several times during their preparation, a great reduction
    in the amount of plant debris on the grids was observed. On the other
    hand, when grids not previously treated with the antiserum were washed,
    the virus particles were also removed (Fig. 23-C, -D).
    The SSEM seemed to be also adequate to assay seeds for polyhedral
    viruses since it was successfully used to observe CPMV particles in
    cowpea leaf extracts using grids treated with antiserum specific for
    CPMV (Fig. 24-D).
    Double Immunodiffusion Tests and SSEM for Detection of Other Viruses
    in Germinated Legume Seeds
    The double immunodiffusion tests and SSEM used to detect B1CMV
    in germinated cowpea seeds were also useful for detecting BCMV in in
    fected germinated bean seeds and SoyMV in hypocotyls of germinated
    soybean seeds infected with SoyMV. Both legume viruses were detected
    by double immunodiffusion tests using hypocotyl extracts from groups
    of 5 seedlings and small discs of individual hypocotyls as assay
    antigens (Fig. 25). The results obtained in the simplified double
    immunodiffusion tests with hypocotyl discs were confirmed by SSEM,
    which also proved to be a very good technique to assay bean and soy
    bean seed lots for the presence of BCMV (Fig. 26-A) and SoyMV (Fig.
    26-B), respectively.
    The results obtained from the inoculation of the hypersensitive
    bean line VC-1822 with extracts from germinated bean seeds were also
    in agreement with those obtained by the immunochemical tests for BCMV.
    The percentages of BCMV-infected bean 'Black Turtle' seed lot and of

    Figure 25 Double immunodiffusion tests with hypocotyls of A-5-day-
    old bean and soybean seedlings using antiserum for
    BCMV-S and for SoyMV.
    A B) Serological tests with hypocotyl discs (A) and
    hypocotyl extracts (B) from healthy and BCMV-
    BV-I infected bean seedlings. The center wells
    were charged with: (As) BCMV-S antiserum, and
    (Ns) normal serum. The peripheral wells were
    filled with extracts from: (l) BCMV-BV-1-
    infected hypocotyls, and (H) healthy bean
    hypocotyls. The hypocotyl discs were embedded
    directly into the agar medium and numbered
    from the top in a clockwise direction (arrows).
    Note a virus-antiserum specific reaction with
    hypocotyl "l" in A.
    C D) Serological tests with hypocotyl discs (c) and
    hypocotyl extracts (D) from healthy and SoyMV-
    infected soybean seedlings. The center wells
    were charged with: (As) SoyMV antiserum, and
    (Ns) normal serum. The peripheral wells were
    filled with extracts from: (l) SoyMV-infected
    hypocotyls, (2) SoyMV-infected Nicotiana
    benrhamiana leaves, (3) healthy N^. benthamiana
    leaves, and (A) healthy soybean hypocotyls.
    Note a positive reaction with hypocotyl "IA"
    in C.

    811

    f
    Figure 26 Electron micrographs of serologically specific electron
    microscopy with extracts from BCMV- and SoyMV-infected
    hypocotyIs.
    A) BCMV-S antiserum-sensitized grid and extract from
    bean hypocoty! infected with BCMV-BV-I;
    B) SoyMV antiserum-sensitized grid and extract from
    SoyMV-infected soybean hypocotyl.

    120

    121
    SoyMV-infected soybean Jupiter1 seed lot were also estimated by the
    simplified double immunodiffusion test using hypocotyl discs (Table ll).
    Serology and Microscopy of Cytoplasmic Inclusions Induced by B1CMV
    and SoyMV in HypocotyIs of Germinated Seeds
    Cytoplasmic inclusions induced by B1CMV in cowpea and by SoyMV
    in soybean were detected by serology, light microscopy and electron
    microscopy in hypocotyls of 4-5-day-old seedlings. In double immuno
    diffusion tests, specific precipitin lines were observed with the in
    clusion antisera and extracts of vi rus-infected hypocotyls but not
    with extracts from healthy hypocotyls (Fig. 27).
    Light microscopic observations of epidermal strips of hypocotyls
    from B1 CMV-infected cowpea and SoyMV-infected soybean seedlings
    readily revealed the presence of cytoplasmic inclusions (Figs. 28, 29).
    Groups of inclusions induced by B1CMV and by SoyMV were abundant in
    cells of virus-infected hypocotyls (Figs. 28, 29), but were not ob
    served in cells of noninfected hypocotyls. Pinwheels with scrolls
    were observed in ultrathin sections of hypocotyl tissues infected
    either with BICMV or SoyMV (Figs. 30, 31), A large number of longi
    tudinal sections of pinwheel inclusions in hypocotyl tissues showed
    that they were abutted to the cell wall close to the plasmodesmata
    (Fig. 31-C, -D). S imilarly, tubes were observed in molybdate-treated
    extracts from hypocotyls infected with either virus.

    Figure 27 Double immunodiffusion tests with hypocotyl extracts from
    ^-5-day-old seedlings of cowpea (A) and soybean (B) using
    antisera for BICMV, SoyMV, and their cytoplasmic inclusions.
    A) Serological test for detecting BICMV cytoplasmic
    inclusions (BICMV-l) in hypocotyls of ^-S-day-old
    cowpea seedlings grown from B1CMV-infected seeds.
    The center wells were charged with: (is) B1CMV- I
    antiserum, (Vs) BICMV antiserum, and (Ns) normal
    serum. The peripheral wells were charged with
    SDS-treated extracts from: (l) B1CMV-infected
    hypocotyls, and (H) healthy hypocotyls.
    B) Serological test for detection of SoyMV cyto
    plasmic inclusions (SoyMV-l) in hypocotyls of
    ^-S-day-old soybean seedlings grown from SoyMV-
    infected seeds. The center wells were charged
    with: (is) SoyMV-l antiserum, (Vs) SoyMV anti
    serum, and (Ns) normal serum. The peripheral
    wells were charged with SDS-treated extracts from:
    (l) SoyMV-infected hypocotyls, and (H) healthy
    soybean hypocotyls.

    123

    Figure 28 Photomicrographs showing different views (A, B, C, D) of
    cytoplasmic inclusions (arrows) induced by B1CMV in epi
    dermal strips of cowpea hypocotyl tissue stained with a
    combination of calcomine orange and luxol brilliant green.
    (Cl) cytoplasmic inclusions, (CW) plant cell wall, and
    (Nu) nucleus.

    szi

    Figure 29 Photomicrographs showing different views (A, B, C, D) of
    epidermal cells of hypocotyls from ^5_day-old soybean
    seedlings containing cytoplasmic inclusions (arrows)
    induced by SoyMV. The hypocotyl epidermal strips were
    stained with a combination of calcomine orange and luxol
    brilliant green. (Cl) cytoplasmic inclusions, (CW)
    plant cell wall, and (Nu) nucleus.

    127

    Figure 30 Electron micrographs of ultrathin sections of cells from
    hypocotyls of t-S-day-old cowpea seedlings infected with
    BlCMV. Note cross-sections (A, B, C) and longitudinal
    sections (D) of pinwheel inclusions induced by BlCMV.
    (CW) plant cell wall, (IS) intercellular space, (m) mito
    chondrion, and (pw) pinwheel inclusions.

    631

    Figure 31
    - Electron micrographs of ultrathin sections of hypocotyl
    cells of ^-5ciay-o 1 d soybean seedlings grown from SoyMV-
    infected seeds. Note cross-sections (A, B) of the pin-
    wheels and longitudinal views of the inclusions abutted
    to the plant cell wall at the piasmodesmata (C, D).
    (CW) plant cell wall, (pi) plasmodesma, and (pw) pinwheel
    inc1 usions.

    131

    132
    Discussion
    Seed-transmission of plant viruses is of agricultural importance
    because efficient virus transmission in space and time can be provided
    through seeds of infected host plants. The international exchange of
    several legume seeds is probably partly responsible for the worldwide
    distribution of important virus diseases of legumes. The occurrence
    of SoyMV in soybean and BCMV in bean in most countries where these
    crops have been tested for viruses (Bos, 1971, 1972) are good examples
    to illustrate this point. According to Phatak (197*0, several legumes
    are propagated in association with their seed-borne viruses on a wide
    scale in certain subtropical and tropical regions that lack organized
    seed certification and breeding programs. Brunt and Kenten (1973) ex
    plained the prevalence of cowpea mild mottle virus in cowpea in Ghana
    by the high rate of seed transmission in cowpeas and by the traditional
    practice of sowing seeds from crops previoysly grown in the same region.
    The control of virus diseases of legumes through the production
    of virus-free seeds or seed lots with very low percentage of infected
    seeds has been advocated frequently (Fajardo, 1930; Thomas and Graham,
    1951; Zettler and Evans, 1972; Baker, 1972 ; Phatak, 197**; Sinclair and
    Shurtleff, 1975; and Williams, 1975). The increase in percentage of
    infected seeds produced in crops with low initial infection and the
    efficient secondary spread of certain viruses into the growing crop
    from a small amount of primary inoculum randomly located throughout
    the planting, emphasize the importance of maintaining a low tolerance
    level in seed certification programs.

    133
    Several techniques have been developed for diagnosing seed-borne
    viruses in routine seed health testing laboratories. Phatak (197*0
    grouped those techniques into dry examination (visual inspection of
    dry seeds), biological tests (growing-on and infectivity tests), bio
    chemical tests (colorimetric, histochemica1, and serological tests),
    and biophysical tests (electron microscopy). According to Phatak
    (197**) the highly specific serological tests are the best among the
    tests available to assay seed lots for the presence of vi rus-infected
    seeds. Several serological tests have been used for detecting virus-
    infected seeds (Scott, 1961; Hamilton, 1965; Phatak, 197**; Slack and
    Shepherd, 1975; Lundsgaard, 1976; and Lister, 1977). However, the
    present investigation contains the first successful results with
    double immunodiffusion tests for detecting potyviruses in germinated
    seeds.
    The use of hypoc.otyl tissue of germinated legume seeds eliminated
    the problem of nonspecific reaction commonly observed with extracts
    from seeds per se or germinated seeds, including roots, cotyledons,
    and primary leaves in immunodiffusion tests (Fig. 19). Cockbain et
    al. (1976) reported that when embryos of Vicia faba L. minor seeds
    were tested by double immunodiffusion, the agar became clouded,
    obscuring the virus-specific precipitation lines. The nonspecific
    precipitates that interfered with the vi rus-spec ific reactions (Fig. 19)
    may be related to the high concentration of haemagg1utin ins (lectins)
    reported to be present in legume seeds (Toms and Turner, 1965; Moreira
    and Perrone, 1977; and Fountain and Yang, 1977). It has been demon
    strated that lectins bind specifically to mono and polysaccharides as

    13*
    well as to globulins of normal serum and to virus coat protein. Marshall
    and Nor ins (1965) showed that extracts of P. vulgaris seeds containing
    lectins precipitated a and 3 globulins of normal rabbit serum, Gumpf
    and Shannon (1977) demonstrated that a barley lectin interacted with
    purified BSMV vut_rx> and formed insoluble aggregates that greatly
    reduced the virus infectivity. Phatak (197*) assumed that the high
    content of lectin in soybean seeds was responsible for the unsatisfactory
    results of the passive haemagglutination test with SoyMV in seed ex
    tracts. Thus, the absence of nonspecific reactions with extracts of
    hypocotyl tissue in contrast to the nonspecific precipitates observed
    with extracts of root or cotyledons and primary leaves (Fig. 19) of
    *-5-day-old legume seedlings may be an indication that a very low
    content of lectin is present in the hypocotyl.
    In support of the use of germinated seed for serology, instead of
    seed per se, is the fact that if any seed certification program or
    seed producers will assay for the presence of vi rus-infected seeds,
    they will also test some other seed properties such as percentage of
    seed germination. Consequently, the germinated seeds could be used
    in a concomitant serological indexing program.
    The sensitivity of the double immunodiffusion test indicates that
    germinated seeds can be divided into groups of 5 to 30 seedlings and
    precipitin reactions will be observed with those groups containing at
    least one infected seedling (Fig. 2|). Immunochemical tests with
    higher sensitivity have been also used for indexing vi rus-infected
    seeds. The extremely sensitive enzyme-1inked immunosorbent assay
    (ELISA) (Voller et a I., 1976; and Clark and Adams, 1977) was used to

    135
    detect TRSV and SoyMV in individual soybean seeds (Lister, 1977)- The
    high sensitivity of SSEM for detection of particles of B1CMV, BCMV,
    and SoyHV in infected hypocotyI extracts as well as of other seed-borne
    viruses in seed extracts (Brlansky and Derrick, 1976) make it suitable
    for identification of vi rus-infected seeds when an electron microscope
    is available. The ELISA and SSEM techniques, however, are more complex
    than the double immunodiffusion tests and/or require the use of sophis
    ticated equipment such as an electron microscope.
    A close correlation was observed between the percentages of virus-
    infected seeds estimated by the simplified double immunodiffusion tech
    nique with discs of individual hypocotyls and the growing-on test in
    different legume seed lots (Table ll). The advantages of the hypocotyl
    disc-double immunodiffusion technique over the growing-on test are
    that: a) it is faster and more specific, b) it does not require green
    house space, and c) it does not depend on environmental conditions for
    symptom development. The growing-on test is largely used, sometimes
    in combination with serology, for indexing seed lots for the presence
    of v i rus-infected seeds. The growing-on test, however, requires
    suitable temperature and light under insect-proof conditions for a
    period varying from 2 to 6 weeks. Kaiser and Mossahebi (197*0 reported
    that at times, virus symptoms did not appear until the second or third
    trifoliolate leaf showed up on bean plants grown from seeds infected
    with BCMV. According to Sharma and Varma (1975), the symptoms of cow-
    pea banding mosaic virus appeared only on the first trifoliolate or
    the subsequent leaves of cowpea under low temperature conditions. Tosic
    and Pesie (1975) observed that the greatest number of alfalfa seedlings

    136
    infected with alfalfa mosaic virus could be detected only 1^ days after
    germination and symptom expression was not reliable enough for the es
    timation of percentage of vi rus-infected seeds. Phatak et al. (1976)
    used the indicator-inoculation test in association with the growing-on
    test to estimate the percentage of cowpea seeds infected with cowpea
    ringspot virus because infected seedlings were either symptomless or
    the symptoms were very mild. In assessing the proportion of V. faba
    minor seeds infected with broad bean stain virus, Cockbain et al.
    (1976) found that under high temperature the symptoms were often mild
    and evanescent, and vi rus-infected seedlings remained without obvious
    symptoms for more than 6 weeks. Thus, the quantitative estimation of
    vi rus-infected seeds of leguminous crops by the growing-on test may
    often be difficult due to symptomless infections. On the other hand,
    the reliability and simplicity of the hypocotyl-disc-double immuno
    diffusion test make it highly suitable for commercial certification
    programs. Observations during the course of this research indicated
    that a trained person can set up approximately 50-60 hypocotyl discs
    per hour in an agar plate to be tested by this technique. This sim
    plified double immunodiffusion technique does not require tissue
    grinding, antigen wells, and chemical treatment of the antigen prior
    to incorporation into the agar matrix.
    The single radial immunodiffusion test also proved to be satis
    factory for detection of B1CMV in cowpea hypocotyls. These results
    provide another option for a routine seed health testing program. Sin
    gle radial immunodiffusion in multiple-antisera media could be used
    for indexing legume seeds for more than one virus. As shown in the

    137
    first part of this research, an agar medium impregnated with a mixture
    of antisera was used for serodiagnosis of two morphologically distinct
    viruses in cowpea (Figs, 13, 1*0-
    The presence of cytoplasmic inclusions in tissues of germinated
    seeds is also useful for detection of vi rus infected seeds by light
    microscopy and serology. Cytoplasmic inclusions induced by B1CMV in
    cowpea and by SoyMV in soybean were detected by serology, light micros
    copy and electron microscopy in hypocotyls of germinated seeds. Al
    though the cytoplasmic inclusions induced by potyviruses were found to
    be widespread in systemically infected plants (Sheffield, 19^1; Hampton
    et al., 1973; and Tu, 1973), no previous report has indicated their
    presence in either infected seed per se or in germinated vi rus-infected
    seeds (Sheffield, 19^1; Camargo et al,, 1968; Edwardson, 197^; Andrews
    and Shalla, 197t; Tu, 1975; Martel 1 i and Russo, 1977; and Christie and
    Edwardson, 1977).
    In summary, the following techniques have been demonstrated to be
    useful for detecting potyviruses and/or their cytoplasmic inclusions
    in hypocotyl tissues of A-5-day-old seedlings grown from vi rus-infected
    legume seeds: a) conventional double immunodiffusion, b) hypocotyl-disc-
    double immunodiffusion, c) conventional single radial immunodiffusion,
    d) hypocotyl-disc-single radial immunodiffusion, e) SSEM of virus par
    ticles; and g) light microscopy of cytoplasmic inclusions. The hypo
    cotyl -disc-doub1e immunodiffusion technique is the simplest method to
    use when it is necessary to determine the percentage of vi rus-infected
    seeds.

    LITERATURE CITED
    Abo El-Nil, M. M., F. W Zettler, and E, Hiebert. 1977. Purification,
    serology, and some physical properties of dasheen mosaic virus.
    Phytopathology 67: 1445-1450.
    Abrygunawardena, D. V. W., and S. M D. Perera. 1964 Virus diseases
    affecting cowpea in Ceylon. Trop, Agrie. Mag. Ceylon Agrie. Soc.
    120:181-204
    Afanasiev, M. M. 1956. Occurrence of barley stripe mosaic in Montana.
    Plant Dis. Rep. 40:142.
    Agrawal, H. 0. 1964 Identification of cowpea mosaic virus isolates.
    Mededel. van de Landbouth Wageningen, Nederland 64:1-53.
    Alba, A. P. C., and A. R. Oliveira. 1977. Serological studies on
    viruses of the potato virus V group occurring in Sao Paulo. Summa
    PhytopathoIog ica 2:178-186
    Alconero, R., and A. Santiago. 1973. PhaseoI us lathyroides as a
    reservoir of cowpea virus in Puerto Rico. Phytopathology 63:120-
    123.
    Anderson, C. W. 1955a. Vigna and Crotalaria viruses in Florida. I.
    Preliminary report on a strain of cucumber mosaic virus obtained
    from cowpea plants. Plant Dis. Rep. 39:346-348.
    Anderson, C. W. 1955b. Vigna and Crotalaria viruses in Florida. II.
    Notations concerning cowpea mosaic virus (Marmor Vignae). Plant
    Dis. Rep. 39:349-352.
    Anderson, C. W. 1955c. Vigna and Crotalaria viruses in Florida. III.
    Notations concerning identification difficulties, indicator plants,
    possible vector relationships, and virus maintenance. Plant Dis.
    Rep. 36:354-357
    Anderson, C. W. 1957- Seed transmission of three viruses in cowpea.
    Phytopathology 47:515 (Abstr ).
    Anderson, C. W. 1959. Vigna and Crotalaria viruses in Florida. V.
    Comparative transmission tests with aphids and beetles. Phyto
    pathology 49:117-118
    Andrews, J. H., and T A, Shalla 1974. The origin, development,
    and conformation of amorphous inclusion body components in tobacco
    etch v i ius infected cells. Phytopathology 64:1234-1243.
    138

    139
    Athow, K. L,. and J, B. Bancroft. 1959- Development and transmission
    of tobacco ringspot virus in soybean, Phytopathology 49:697-701.
    Baker, K. F. 1972. Seed pathology. Pages 317-416 j'r^ T. T. Kozlowski,
    ed. Seed Biology Vol, II. Academic Press, New York, 447.
    Bancroft, J, B 1970. Brome mosaic virus. No, 3 j_n_ Descr¡ptions of
    plant viruses. Commonw. Mycol, Inst., Assoc. Appl. Biol, Kew
    Surrey, England. 4 p,
    Bancroft, J. B. 1971. Cowpea chlorotic mottle virus. No. 49 j_n^ De
    scriptions of plant viruses. Commonw. Mycol, Inst,, Assoc, Appl.
    Biol,, Kew, Surrey, England. 4 p.
    Barnett, 0. W., and M. Alper. 1977, Characterization of Iris fulva
    virus. Phytopathology 67:448-454,
    Batchelor, D. L. 1974. Immunogenicity of sodium dodecyl sulfate
    denatured plant viruses and paint viral inclusions, Ph.D. Dis
    sertation, University of Florida, Gainesville, 8] p,
    Beier, H., D J, Siler, M. L. Russell, and G, Bruening. 1977. Survey
    of susceptibility to cowpea mosaic virus among protoplasts and
    intact plants from V i gna sinensis lines. Phytopathology 67:917-921,
    Bennett, C. W. 1966 Seed transmission of plant virus. Pages 221-
    261 jm K, M. Smith and M, A Lauffer, eds. Advances in virus
    research, Vol. 14. Academic Press, New York. 350 p.
    Bercks, R. I960. Serological relationships between beet mosaic virus,
    potato virus Y, and bean yellow mosaic virus. Virology 12:311-313.
    Bock, K. R. 1973. East African strains of cowpea aphid-borne mosaic
    virus. Ann. Appl. Biol, 74:75-83.
    Bock, K, R,, and M. Conti, 1974. Cowpea aphid-borne mosaic virus,
    No, 134 j_n_ Descr i pt ions of plant viruses. Commonw, Mycol, Inst.,
    Assoc, Appl. Biol,, Kew, Surrey, England, 4 p.
    Bos, L 1971. Bean common mosaic virus. No, 73 in Descriptions of
    plant viruses. Commonw. Mycol, Inst., Assoc. Appl. Biol,, Kew,
    Surrey, England, 4 p.
    Bos, L. 1972, Soybean mosaic virus. No. 93 _ij^ Descriptions of plant
    viruses. Commonw. Mycol. Inst., Assoc, Appl. Biol,, Kew, Surrey
    England, 4 p.
    Bos, L., and D. Z. Maat, 1974. A strain of cucumber mosaic virus,
    seed-transmitied in beans, Neth, J, PI. Pathol, 80:113-123.
    Brandes, J. 1964. I dentif i zierung von gestreckten pflanzenpathogenen
    viren auf morphologischer Grundlage. Mitt. biol. BundAnst. Ld u.
    Forstw. 110:1-130.

    Brantley, B. B., C. W. Kuhn, and G. Sowell. 1965. Effect of cucumber
    mosaic virus on southern pea (Vigna sinensis). Proc. Am. Soc.
    Hortic. Sci. 87:355-358.
    Brierley, P., and F. F. Smith. 1962. Three cowpea mosaic viruses from
    gladiolus. Plant Dis. Rep. 46:335-337.
    Brlansky, R. H., and K. S. Derrick. 1976. Detection of seed-borne
    plant viruses using serologically specific electron microscopy.
    Proc. Am. Phytopathol. Soc. 3:334-335 (Abstr.).
    Brunt, A. A. 1974_ Tropical leguminous crops. Glasshouse Crop Re
    search Institute. Annual Report. 1974 (Abstr.).
    Brunt, A. A., and R. H. Kenten. 1973. Cowpea mild mottle, a newly
    recognized virus infecting cowpeas (Vigna ungu i cu lata) in Ghana
    Ann. Appl. Biol. 74:67-74.
    Brunt, A. A., and R. H. Kenten. 1974. Cowpea mild mottle virus. No.
    140 Descript ions of plant viruses. Commonw. Mycol Inst.,
    Assoc. Appl. Biol., Kew, Surrey, England. 4 p.
    Burkholder, W. H., and A. S. Muller. 1926. Hereditary abnormalities
    resembling certain infectious diseases of beans. Phytopathology
    16:731-737.
    Camargo, I. J. B., E. W. Kitajima, and A. S. Costa, 1968. Estudo ao
    microscopio electrnico de tecidos de plantas ¡nfetadas pelo
    virus do mosaico comum e mosaico amarelo do feijoeiro. Bragantia
    27:409-415 -
    Carner, J., K. S i 1berschmidt, and E. Flores. 1969. Ocorroncia do
    virus do mosaico da Vigna no Estado de Sio Paulo. 0 Bioloqico
    35:13-16.
    Casper, R. 1974. Serodiagnosis of plum pox virus. Acta Horticulturae
    44:171-172.
    Chant, S. R. 1959- Virus of cowpea, Vigna ungu¡culata L. (Walp.)
    in Nigeria. Arm. Appl. Biol. 47 :565-572.
    Chant, S. R. I960. The effect of infection with tobacco mosaic and
    yellow mosaic viruses on the growth rate and yield of cowpea in
    Nigeria. Empire J. Exp. Agrie. 28:114-120.
    Chant, S. R. 1962. Further studies on the host range and properties
    of Trinidad cowpea mosaic virus. Ann, Appl. Biol. 50:159-162.
    Chenulu, V. V.s J. Sachchidananda, and S. C. Mehta. 1968. Studies on
    a mosaic disease of cowpea from India. Phytopathol. Z. 63:381-
    387.

    141
    Cheo, P. C. 1955- Effect of seed maturation on inhibition of southern
    bean mosaic virus in bean. Phytopathology 45:17-21.
    Christie, R G. 1967. Rapid staining procedures for differentiating
    plant virus inclusions in epidermal strips. Virology 31:268-271.
    Christie, R G., and J. R. Edwardson. 1977. Light and electron micros
    copy of plant virus inclusions. Florida Agrie. Exp. Stn. Monogr.
    Ser. 9, 150 p
    Christie, S. R., and W E. Crawford. 1977. Plant virus range of
    Nicotiana benthamiana. Plant Dis. Rep. (in press).
    Clark, M. F., and A N. Adams. 1977. Characteristics of the microplate
    method of enzyme-1 inked immunosorbent assay for the detection of
    plant viruses. J. Gen. Virol. 34:475-483
    Clifford, H. T. 1977. Immunodiffusion techniques for the detection of
    the Cymbidium mosaic and Odontoglossum ringspot viruses of orchids.
    M. S. Thesis, University of Florida, Gainesville. 63 p.
    Cockbain, A. J., R. Bowen, and S. Vorra-Urai, 1976. Seed transmission
    of broad bean stain virus and Echtes ackerbohnenmosaik-vi rus in
    field bean (Vicia faba). Ann. Appl. Biol 84:321-332.
    Corbett, M. K. 1956. Serological and morphological relationships of
    plant viruses. Florida Univ. Agrie. Exp. Stn. Annu. Rep., p. 117-
    118.
    Crowley, N. C. 1957. Studies on the seed transmission of plant virus
    diseases. Aust. J. Biol. Sci. 10:449-464.
    Crowley, N. C. 1959. Studies on the time of embryo infection by seed-
    transmitted viruses. Virology 8:116-123.
    Dale, W. T. 1949- Observations on a virus disease of cowpea in Tri
    nidad. Ann. App. Biol. 36:327-333.
    Dale, W. T. 1953. The transmission of plant viruses by biting insects,
    with particular reference to cowpea mosaic. Ann. Appl. Biol. 40:
    385-392.
    Debrot, A. E., and C. E. Benitez de Rojas, 1967. Identificacin del
    virus del mosaico de la soya en Venezuela. Agron. Trop. 17:75-86.
    Demski, J. W., and H. B. Harris. 1974. Seed transmission of viruses
    in soybean. Crop Sci. 14:888-890.
    Derrick, K. S., and R. H. Brlansky. 1976. Assay for virus and myco-
    plasmas using serologically specific electron microscopy. Phyto
    pathology 66:815-820.

    142
    Desjardins, F. R,, R. L. Latterell, and J. E. Mitchell. 1954. Seed
    transmission of tobacco-ringspot virus in Lincoln variety of
    soybean Phytopathology 44 ; 86.
    Doi, Y., M. U. Chang, and K. Yora. 1977- Orchid fleck virus. No. 183
    in Descriptions of plant viruses. Commonw. Mycol. Inst., Assoc.
    Appl. Biol., Kew, Surrey, England. 3 p>
    Drijfhout, E and L Bos. 1977. The identification of two new strains
    of bean common mosaic virus, Neth. J PI Pathol, 83:13_25-
    Edwardson, J. R. 1974. Some properties of the potato virus-Y group.
    Fla. Agrie. Exp Stn, Monogr. Ser, 4. 398 p.
    Edwardson, J, R., D E, Purcifull, and R. G. Christie. 1968. Structure
    of cytoplasmic inclusions in plants infected with rod-shaped viruses
    Virology 34:250-263.
    Edwardson, J. R., F, W. Zettler, R. G. Christie, and I. R. Evans. 1972.
    A cytological comparison of inclusions as a basis for distinguishing
    two filamentous legume viruses. J, Gen. Virol. I 5:113 ~118.
    Ekpo, E. I. A., and A W Saettler. 1974 Distribution pattern of bean
    common mosaic virus in developing bean seed. Phytopathology 64:269
    270.
    Elliot, J. A. 1921 A mosaic of sweet and red clovers. Phytopathology
    11:146-148
    Fajardo, T. G 1930. Studies on the mosaic disease of the bean.
    Phytopathology 2 0:4 69-494
    Fenner, F. 1976. Classification and nomenclature of viruses. Inter-
    virology 7:4-115.
    Fischer, H 0 and B E. Lockhart. 1976a. A strain of cowpea aphid-
    borne mosaic virus isolated from cowpeas in Morocco. Phytopathol.
    Z 85:43-48.
    Fischer, H. U., and B. E. Lockhart. 1976b. A strain of cucumber mosaic
    virus isolated from cowpeas in Morocco. Phytopathol. Z. 85:132-138
    Fountain, D. W,, and W Yang. 1977. Isolectins from soybean (Glycine
    max) Biochfm. Biophys. Acta 492:176-185
    Fulton, R. W 1964. Transmission of plant viruses by grafting, dodder,
    seed, and mechanical inoculation. Pages 39-67 hi M, K. Corbett,
    and H. D. Sfsler, eds. Plant Virology. Univ. Florida Press, Gaines
    ville, 527 p,

    1A3
    Gardner, M. W. 1927 Seed transmission of cowpea mosaic. Proc. Ind.
    Acad. Sc i. A3.
    Gardner, M. W., and J. B. Kendrick. 1921. Soybean mosaic. J. Agrie.
    Res. 22:111-1 IA
    Gay, J. 0. 1969. Effect of seed maturation on the infectivity of
    cowpea chlorotic mottle virus. Phytopathology 59:802-8oA.
    Gay, J. D., and E. E. Winstead. 1970. Seed-borne viruses and fungi
    from southern pea seed grown in eight states. Plant Dis. Rep.
    5A:2A3-2A5.
    Ghanekar, A. M., and F. W. Schwenk. 197A. Seed transmission and dis
    tribution of tobacco streak virus in six cultivars of soybeans.
    Phytopathology 6A:112-11 A
    Gibbs, A. J. 1972. Broad bean mottle virus. No. 101 in Description
    of plant viruses. Commonw. Mycol. Inst., Assoc. Appl. Biol.,
    Kew, Surrey, England. A p.
    Govier, D. A., and R. D. Woods, 1971 Changes induced by magnesium
    ions in the morphology of some plant viruses with filamentous
    particles. J. Gen. Viol. 13:127-132.
    Granett, A. L., and T. A. Shalla. 1970. The relation of tobacco mosaic
    virus X-protein to amorphous cellular inclusions (X-bodies).
    Phytopathology 60:A19-A2A.
    Grogan, R. G., J. E. Welch, and R. Bardin. 1952. Common lettuce mosaic
    and its control by the use of mosaic-free seed. Phytopathology
    A2:573-578.
    Gumpf, D. J., and L M. Shannon. 1977. Interaction of barley stripe
    mosaic virus and barley lectin. Proc. Am. Phytopathol. Soc. A
    (i n press) (Abst r ).
    Hamilton, R. I. 1965. An embryo test for detecting seed-borne barley
    stripe mosaic virus in barley. Phytopathology 55:798-799.
    Hampton, R. 0., S. Phillips, J E. Knesek, and G. I. Mink. 1973.
    011rastructuraI cytology of pea leaves and roots infected by pea
    seed-borne mosaic virus. Arch. Ges. Virusforsch. A2:2A2-253.
    Haque, S. Q., and G. C. Persad. 1975. Some observations on the seed-
    transmission of beetle-transmitted cowpea mosaic virus. Pages
    119-121 jji J. Bird, and K. Maramorosh eds. Tropical Diseases of
    Legumes. Academic Press, New York, 171 p.
    Harrison, A. N., and R. T. Gudauskas. 1968a. Identification of
    viruses isolated from cowpeas in Alabama. Plant Dis, Rep. 52:
    3A-36.

    Harrison, A. N., and R. T. Gudauskas. 1968b. Effects of some viruses
    on growth and seed production of two cowpea cultivars. Plant
    Dis. Rep. 52:509-511.
    Hiebert, E., and J. G. McDonald. 1973. Characterization of some pro
    teins associated with viruses in the potato Y group. Virology
    56:3^9-361.
    Hiebert, E. and J. G. McDonald. 1976. Capsid protein heterogeneity
    in turnip mosaic virus. Virology 70:144-150.
    Hiebert, E., D. E. Purcifull, R. G. Christie, and S. R. Christie.
    1971- Partial purification of inclusions induced by tobacco etch
    virus and potato virus Y. Virology 43:638-646.
    Hollings, M., and 0. M. Stone. 1965- Studies of Pelargonium leaf
    curl virus. II. Relationships to tomato bushy stunt and other
    viruses. Ann. Appl. Biol. 56:87-98.
    lizuka, N. 1973. Seed transmission of viruses in soybean. Tohoku
    Natl. Agrie. Exp. Stn. Bull. 46:131-141.
    lizuka, N. 1974. Southern bean mosaic virus in soybean. Plant
    Protection 28:471-474
    lizuka, N., and T. Yunoki. 1974. Peanut stunt virus isolated from
    soybean, Glycine max Merr. Tohoku Agrie. Exp. Stn. Bull. 47:
    1-12.
    Inouye, T. 1973. Characteristics of cytoplasmic inclusions induced
    by bean yellow mosaic virus. Nogaku Kenkyu 54:155-171.
    Jones, R. T., and S. Diachun. 1977. Serologically and biologically
    distinct bean yellow mosaic virus strains. Phytopathology 67:
    831-838
    Kahn, R. P. 1956. Seed transmission of the tomato-ring spot virus
    in the Lincoln variety of soybean. Phytopathology 46:295.
    Kaiser, W. J., D. Danesh, M. Okhovat, and G. H. Mossahebi. 1968.
    Diseases of pulse crops (edible legumes) in Iran. Plant Dis.
    Rep. 52:687-691.
    Kaiser, W. J., and G. H. Mossahebi. 1974. Natural infection of mung-
    bean by bean common mosaic virus. Phytopathology 64:1209-1214.
    Kaiser, W. J., and G. H. Mossahebi. 1975. Studies with cowpea aphid-
    borne mosaic virus and its effect on cowpea in Iran. FA0 Plant
    Protection Bull 23:33-39.

    145
    Karnovsky, M. J. 1965. A formaldehyde-glutaraldehyde fixative of high
    osmolality for the use in electron microscopy. J. Cell Biol.
    27:137A.
    Kendrick, J. B., and M. W. Gardner. 1924, Soybean mosaic: seed
    transmission and effect on yield. J. Agr. Res. 27:91-98.
    Kennedy, B. W., and R. L. Cooper. 1967. Association of virus infection
    with mottling of soybean seed coats. Phytopathology 57:35-37.
    Khatri, H. L., and L. Singh. 1974. Studies on a mosaic disease of
    cowpea. J. Res. 11:289-294,
    Klesser, P. J. I960. Viruses diseases of cowpea. Bothalia 7:233-251.
    Koenig, R., and L. Givord. 1974. Serological interrelationships in
    the turnip yellow mosaic virus group. Virology 58:119-125.
    Koshimizu, S., and T. lizuka. 1957. Relationship between the brown
    speck of soybean seed and soybean mosaic. Ann. Phytopathol. Soc.
    Japan 22:18 (Abstr.).
    Koshimizu, Y., and N. lizuka. 1963- Studies on soybean virus diseases
    in Japan. Tohoku Natl. Agrie. Expt. Stn. Bull. 27:1-103.
    Kuhn, C. W. 1964a. Separation of cowpea virus mixtures. Phytopa
    thology 54:739-740.
    Kuhn, C. W. 1964b. Purification, serology, and properties of a new
    cowpea virus. Phytopathology 54:853-857.
    Kuhn, C. W., B. B. Brantley, and G. Sowell. 1965. Immunity to bean
    yellow mosaic virus in cowpea. Plant Dis. Rep. 49:879-881.
    Kuhn, C. W., B. B. Brantley, and G. Sowell. 1966. Southern pea
    viruses: identification, symptomatology, and sources of resis
    tance. Univ. Georgia Agrie. Exp. Stn. Bull. 157. 22 p.
    Lima, J. A. A., and M. R. Nelson. 1975- Squash mosaic virus variabil
    ity: nonreciproca1 cross-protection between strains. Phytopa
    thology 65:837-8^0.
    Lima, J. A. A., and M. R. Nelson. 1977. Etiology and epidemiology of
    mosaic of cowpea in Cear, Brazil. Plant Dis. Rep. 61:864-867.
    Lima, J. A. A., and D. E. Purcifull. 1977a. Immunochemical tests
    for detection of blackeye cowpea mosaic virus in infected seeds.
    Proc. Am. Phytopathol. Soc. 4 (in press) (Abstr.).
    Lima, J. A. A., and D. E. Purcifull. 1977b. Serology and microscopy
    of cylindrical inclusions induced by blackeye cowpea mosaic and
    soybean mosaic viruses in hypocotyls of germinated seeds. Proc.
    Am. Phytopathol. Soc. ** (in press) (Abstr.).

    Lima, J. A. A., D. E. Purcifull, and E. Hiebert. 1976. Purification
    and serology of blackeye cowpea mosaic virus. Proc. Am. Phytopathol.
    Soc. 3:248 (Absrr.).
    Lima, J. A. A., D. E. Purcifull, and R. Sonoda. 1977. Some properties
    of a potyvirus isolated from siratro in Florida. Proc. Am. Phyto
    pathol. Soc. 4 (in press) (Abstr.).
    Lima-Neto, V. C., and A. S. Costa. 1976. Transmissao comparative do
    virus do mosaico comn da soja por sementes com manchas-caf e
    nao manchadas. Fitopatologa 11:20 (Abstr.).
    Lister, R. M. I960. Transmission of soil-borne viruses through seed.
    Virology 10:547-549-
    Lister, R. M. 1977- Detection of viruses in soybean seed by enzyme-
    linked immunosoibant assay. Proc. Am. Phytopathol. Soc. 4 (in
    press) (Abstr.)
    Lister, R. M., and A F. Murant. 1967- Seed-transmission of nematode-
    borne viruses. Ann. Appl. Biol. 59:49-62.
    Lister, R. H., and J M. Thresh. 1955. A mosaic disease of leguminous
    plants caused by a strain of tobacco mosaic virus. Nature 175:
    1047-1048.
    Lovisolo, 0., and M. Conti. 1966. Identification of an aphid-
    transmitted cowpea mosaic virus. Neth. J. PI. Pathol. 72:265-269.
    Lundsgaard, T. 1976 Routine seed health testing for barley stripe
    mosaic virus in barley seeds using the latex-test. J. Plant Dis.
    Protection 83:2/8-283.
    Markham, R. I960. A graphical method for rapid determination of
    sedimentation coefficients. Biochem. J. 77:516-519.
    Marshall, W. H and L. C. Norins. 1965- Antigenic properties of
    extract of Phaseolus vulgaris seeds (phytohaemagg1utinin) routinely
    used in leucocyte cultures. Aust. J. Exp. Biol. Med. Sci. 43:
    213-228.
    Martelli, G. P., and M. Russo. 1977- Plant virus inclusion bodies.
    Pages 175-266 jm M. A. Lauffer, F. B. Bang, K. Maramorosch, and
    K. M. Smith, eds. Advances in virus research, Vol. 21. Academic
    Press, New York. 409 p.
    Matthews, R. E. F. 1970. Plant Virology. Academic Press, New York.
    778 p.
    McDonald, J. G., and R. I. Hamilton. 1972. Distribution of southern
    bean mosaic virus in the seed of Phaseolus vulgaris. Phyto
    pathology 62:387-389.

    147
    McDonald, J. G., and E. Hiebert. 1975. Characterization of the capsid
    and cylindrical inclusion proteins of three strains of turnip
    mosaic virus. Virology 63:295-303.
    McLean, D. M. 1941. Studies on mosaic of cowpea Vigna sinensis. Phyto
    pathology 31:420-430.
    Medina, A. C., and R. G. Grogan. 1961. Seed transmission of bean mosaic
    viruses. Phytopathology 51:452-456.
    Milne, R. G., and E. Luisoni. 1977- Rapid immune electron microscopy
    of virus preparations. Pages 265-281 [r^ K. Maramorosch, and H.
    Kiprowski, eds. Methods in Virology, Vol. VI. Academic Press, New
    York. 542 p.
    Morales, F. J., and F. W. Zettler. 1977. Characterization and electron
    microscopy of a potyvirus infecting Commelina diffusa. Phyto
    pathology 67:839-843.
    Moreira, R. de A., and J. C. Perrone. 1977. Purification and partial
    characterization of a lectin from Phaseolus vulgaris. Plant
    Physiol. 59:783-787.
    Nariani, T. K., and T. K. Kandaswany. 1961. Studies on a mosaic
    diseaseof cowpea (Vigna sinensis Savi). Indian Phytopathol. 10:
    77-82.
    Nelson, R., and E. E Down. 1933. Influence of pollen and ovule in
    fection in seed transmission of bean mosaic. Phytopathology 23:
    25 (Abstr.).
    Nelson, M. R., and H. K. Knuhtsen. 1973. Squash mosaic virus vari
    ability: review and serological comparison of six biotypes. Phyto
    pathology 63:920-926.
    Nicolaescu, M., H. Titu, and M. Paraschiv. 1976. Electron-microscopical
    investigations on the pinwheel structures detected in soybean mosaic-
    virus infected plants. Rev. Roum. Biol. Biol. Veg. 21:67-69-
    Ouchterlony, 0. 1962. Diffus ion-in-gel methods for immunological
    analysis II. Prog. Allergy 6:30-154.
    Owusu, G. K., N. C. Crowley, and R. I. B. Francki. 1968. Studies of
    the seed-transmission of tobacco rinqspot virus. Ann. Appl. Biol.
    61 : 195-202.
    Padma, R., and A. S. Summanwar. 1973. Chenopodium mrale a differ
    ential host for cowpea mosaic virus. Curr. Sci. 42:620.
    Perez, J. E.( and A. Cortes-Mon11 or. 1970. A mosaic virus of cowpea
    from Puerto Rico. Plant Dis. Rep. 54:212-216.

    148
    Phatak, H. C. 1974. Seed-borne plant virus identification and
    diagnosis in seed health testing. Seed Sci. Technol. 2:3~155.
    Phatak, H. C., J. R. Diaz-Ruiz, and R. Hull. 1976. Cowpea ringspot
    virus: a seed transmitted cucumovirus. Phytopathol. Z. 87:132-
    142.
    Pierce, W. H., and C. W. Hungerford. 1929. A note on the longevity
    of the bean mosaic virus. Phytopathology 19:605-606.
    Porto, M. D. M., and D. 1. Hagedorn. 1975. Seed transmission of a
    Brazilian isolate of soybean mosaic virus. Phytopathology 65:
    713-716.
    Provvidenti, R., and E. D. Cobb. 1975- Seed transmission of bean
    common mosaic virus in tepary bean. Plant Dis. Rep. 59:966-969.
    Purcifull, D. E. 1966. Some properties of tobacco etch virus and its
    alkaline degradation products. Virology 29:8-14.
    Purcifull, D. E., and D. L. Batchelor. 1977. Immunodiffusion tests
    with sodium dodecyl sulfate (SDS)-treated plant viruses and plant
    viral inclusions. Univ. Florida Agrie. Exp. Stn. Bull. No. 788
    (Tech.). 39 p
    Purcifull, D. E., S. R. Christie, and D, L. Batchelor. 1975. Preser
    vation of plant virus antigens by freeze-drying. Phytopathology
    65:1202-1205.
    Purcifull, D. E., J. R. Edwardson, and S. R. Christie. 1970. A morpho
    logical comparison of inclusions induced by tobacco etch and
    potato Y viruses. Phytopathology 60:779~803.
    Purcifull, D. E., and G. V. Gooding. 1970. Immunodiffusion tests
    for potato Y and tobacco etch viruses. Phytopathology 60:1036-
    1039.
    Purcifull, D. E., E. Hiebert, and J. G. McDonald, 1973- Immunochemical
    specificity of cytoplasmic inclusions induced by viruses in the
    potato Y group. Virology 55 = 275~279-
    Purcifull, D. E., and R. J. Shepherd. 1964. Preparation of the protein
    fragments of several rod-shaped plant viruses and their use in
    agar-gel diffusion tests. Phytopathology 54:1102-1108.
    Quacquarel 1 i A., and A. Avgelis. 1975. Ni cot ana bent hamiana Domin,
    as host for plant viruses. Phytopathol. Medit. 14:36-39-
    Raheja, A. K., and 0. I. Leleji. 1974. An aphid-borne virus disease
    of irrigated cowpea in Northern Nigeria. Plant Dis. Rep. 58:1080-
    1084.

    149
    Reddick, D., and V. B. Stewart. 1919. Transmission of virus of bean
    mosaic in seed and observations on thermal death-point of seed and
    virus. Phytopathology 9:445-450.
    Richter, J., J. Polak, and E. Proll. 1975. Ausarbeitung von Verfahren
    zum Serologischen sche1lnachweis des Gurkenmosaik-vi rus in naturlich
    infizierten krautigen Pflanzen. Arch. Phytopathol. v. Pfanzenschutz
    11:307-318.
    Robertson, D. G. 1965- The local lesion reaction for recognizing cowpea
    varieties immune from and resistant to cowpea yellow mosaic virus.
    Phytopathology 55:923-925.
    Ross, J. P. 1963. Interaction of the soybean mosaic and bean pod mottle
    viruses infecting soybeans. Phytopathology 53:887 (Abstr.).
    Ross, J. P. 1968. Effect of single and double infections of soybeans
    with soybean mosaic and bean pod mottle viruses on yields and seed
    characters. Plant Dis. Rep. 52:344-348.
    Ross, J. P. 1969. Effect on time and sequence of inoculation of soy
    beans with soybean mosaic and bean pod mottle viruses on yields
    and seed characters. Phytopathology 59:1404-1408.
    Ross, J. P. 1970. Effect of temperature on mottling of soybean seed
    caused by soybean mosaic virus. Phytopathology 60:1798-1800.
    Rui, D. I960. Apporto alie conoscenze sulle virosi dei vegetal i. In-
    formatore Agrac., Veroma 23:15-17.
    Schippers, B. 1963. Transmission of bean common mosaic virus by seed
    of Phaseolus vulgaris L. cultivar Beka. Acta Bot. Neerl. 12:433-
    497.
    Scott, H. A. 1961. Serological detection of barley stripe mosaic
    virus in seeds and in dehydrated leaf tissue. Phytopathology 51:
    200-201 .
    Sharma, S. R., and A. Varma. 1975- Cure of seed transmitted cowpea
    banding mosaic viruses. Phytopathol. Z. 83:144-151.
    Sheffield, F. M. L. 1941. II. The cytoplasmic and nuclear inclusions
    associated with severe etch virus. J. Roy. Hicroscop. Soc. 61:
    30-45.
    Shepard, J. F. 1969. Serodiagnosis of PVX in potato tuber sprouts.
    Plant Dis. Rep. 53:845-848.
    Shepard, J. F. 1970. A radial immunodiffusion test for the simulta
    neous diagnosis of potato viruses S and X. Phytopathology 60:
    1669-1671.

    150
    Shepard, J. F. 1972. Gel-d¡ffus ion methods for the serological detec
    tion of potato viruses X, S, and M. Montana Agrie. Exp. Stn. Bull.
    No. 662. 22 p.
    Shepard, J. F., J. W. Jutila, J. E. Catlin, F. S. Newman, and W. H.
    Hawkins. 1971. Immunodiffusion assay for potato virus M infection
    Phytopathology 61:873-874.
    Shepard, J. F., G. A. Secor, and D. E. Purcifull. 1974. Immunochemical
    cross-reactivity between the dissociated capsid proteins of PVY
    group plant viruses. Virology 58 : 464-475.
    Shepherd, R. J. 1963. Serological relationship between bean pod mottle
    virus and cowpea mosaic viruses from Arkansas and Trinidad. Phyto
    pathology 53:865-866.
    Shepherd, R. J. 1964. Properties of a mosaic virus of cowpea and its
    relationship to the bean pod mottle virus. Phytopathology 54:
    466-473.
    Shepherd, R. J. 1971. Southern bean mosaic virus. No. 57 |n Descrip
    tions of plant viruses. Commonw. Mycol. Inst., Assoc. Appl. Biol.
    Kew, Surrey, England. 4 p.
    Shepherd, R. J. 19/2. Transmission of viruses through seed and pollen.
    Pages 267-292 m C. Kado, and H. 0. Agrawal,eds. Principles and
    Techniques in Plant Virology. Van Nostrand Reinhold Company, New
    York. 688 p.
    Shepherd, R. J., and R. W. Fulton. 1962. Identity of a seed-borne
    virus of cowpea. Phytopathology 52:489-493.
    Shepherd, R. J., and G. S. Pound. I960. Purification of turnip mosaic
    virus. Phytopathology 50:797-803.
    Shepherd, R. J., and D. E. Purcifull. 1971. Tobacco etch virus. No.
    55 jjt Descriptions of plant viruses. Commonw. Micol. Inst., Assoc.
    Appl. Biol., Kew, Surrey, England. 4 p.
    Sinclair, J. B., and M. C. Shurtleff. 1975. Compendium of soybean
    diseases. Am. Phytopathol. Soc., St. Paul, Minnesota. 69 p.
    Slack, S. A., and R. J. Shepherd. 1975. Serological detection of
    seed-borne barley stripe mosaic virus by a simplified radial-
    diffusion technique. Phytopathology 65:948-955.
    Smith, C. E. 1924. Transmission of cowpea mosaic by bean leaf-beetle
    Science 60:268.
    Smith, F. L., and W. B. Hewitt. 1938. Varietal susceptibility of
    common bean mosaic and transmission through seed. Calif. Agrie.
    Exp. Stn. Bull. 612:3-18.

    151
    Snyder, W. C. 19^2. A seed-borne mosaic of asparagus bean, Viftna
    sesqui peda lis. Phytopathology 32;5l8-523.
    Takahashi, K,, Y. Tanaka, and Y. Tsuda, 197^, Soybean mild mosaic
    virus. Ann. Phytopathol. Soc. Japan. AO:103-105-
    Tamada, T. 1977, The virus diseases of soybean in Japan, Food
    Fertilizer Technology Center. Tech, Bull. 33. II p,
    Thomas. W, 0., Jr,, and R. W. Graham. 1951, Seed transmission of
    red node in Pinto bean. Phytopathology 4 I : 959-962.
    Toler, R. W, S, S. Thompson, and J. M, Barber. 1963, Cowpea (southern
    pea) diseases in Georgia, 1961-62, Plant D i s Rep, **7 : 7^6 7^+ 7,
    Toms, G, C,, and T, D. Turner, 1965, The seed haemagg1utinins of
    some Phaseolus vulgaris L. cultivars. J. Pharm. Pharmacol. 17:
    1 18 S -12 5 S .
    Tosic, M., R E. Ford, H. E. Moline, and D, E, Mayhew, 197^, Comparison
    of techniques for purification of maize dwarf and sugarcane mosaic
    viruses. Phytopathology 6A;439"iti*2
    Tosic, M., and Z. Pesie, 1975, Investigation of alfalfa mosaic virus
    transmission through alfalfa seed. Phytopathol, Z. 83:320-327,
    Tremaine, J, H., and N. S. Wright. 1967- Cross-reactive antibodies
    in antisera to strains of southern bean mosaic virus. Virology
    31 : A81 -A88.
    Tsuchizaki, T,, K, Yora, and H. Asuyama, 1970, The viruses causing
    mosaic of cowpeas and adzuki beans, and their transmissibi 1ity
    through seeds. Ann. Phytopathol. Soc, Japan 36:112-120.
    Tu, J. C, 1973, Electron microscopy of soybean root and nodules
    infected with soybean mosaic virus. Phytopathology 63:1011-1017,
    Tu, J. C, 1975, Localizations of infectious soybean mosaic virus in
    mottled soybean seeds. Microbios 1b:151-156,
    Uyeda, I., M. Kojima, and D, Murayama. 1975, Purification and serology
    of bean yellow mosaic virus, Ann. Phytopathol. Soc. Japan 4l ;
    192-203.
    Uyemoto, J. K., R. Provvidenti, and D. E. Purcifull. 1973. Host range
    and serological properties of a seed-borne cowpea virus. Phyto
    pathology 63:208-209, (Abs t r-),
    yemoto, J. K,, R. Provvidenti, and W. T. Schroeder, 1972. Serological
    relationship and detection of bean common mosaic and bean yellow
    mosaic viruses in agar gel, Ann. Appl, Biol, 7I:235-2A2.

    152
    van Kammen, A. 1971. Cowpea mosaic virus. No. 47 in Descriptions of
    plant viruses. Commonw. Mycol. Inst., Assoc. Appl. Biol., Kew,
    Surrey, England, A p.
    van Kammen, A. 1972. Plant virus with a divided genome. Annu. Rev.
    Phytopathol. 10:125-151.
    van Oosten, H. J. 1972. Purification of plum pox (sharka) virus with
    the use of Triton X-100. Neth. J, PI. Pathol. 78:33-44.
    van Regenmortel, M. H. V. 1966. Plant virus serology. Pages 207-271
    j_r^ K. M. Smith, and M. A. Lauffer, eds. Advances in virus research
    Vol. 12. Academic Press, New York. 394 p.
    van Regenmortel, M. H. V., and M. B. von Wechmar. 1970. A reexamina
    tion of the serological relationship between tobacco mosaic virus
    and cucumber virus 4. Virology 41:330-338.
    van Velsen, R. J. 1962. Cowpea mosaic, a virus disease of Vigna
    s i nensis in New Guinea. Papua New Gn. Agrie. J. 14:153-161.
    Verma, V. S., 1971. Effect of heat on seed transmission of mosaic
    disease of cowpea (V igna s i nensis Savi). Acta Microbiologica
    Polonica 3:163-166.
    Vidano, C. 1959. Indagini sopra un deperimento della V igna sinens i s
    Endlicher in cultura italiana. Boll. Zool. Agr. Bachic. 3:1-97.
    Vidano, C., and M. Conti, 1965. Transmission i con afidi di un "cowpea
    mosaic virus" isolato da Vigna sinensis Endl. in Italia. Atti
    Accad. Sci. 99:1041-1050.
    Voller, A., A. Bartlett, D. E. Bidwell, M. F. Clark, and A. N, Adams.
    1976. The detection of viruses by enzyme-1 inked immunosorbent
    assay (ELISA). J. Gen. Virol. 33:165-167.
    Vovlas, C,, and A. Avgelis. 1972. Le virosi delle piante ortensi in
    Puglia, VIII. Un mosaico del Fagiolino dall'occhio. Phytopathol.
    Medit. 11:117-118.
    Walters, H. J., and 0. W. Barnett, Jr. 1964. Bean leaf beetle
    transmission of Arkansas cowpea mosaic virus. Phytopathology
    54:911 (Abstr.).
    Weber, K., and M, Osborn. 1969. The reliability of molecular weight
    determinations by dodecyl sulfate-poly-acrylamide gel electro
    phoresis. J. Biol. Chem. 244:4406-4412.
    Wells, D. G., and R. Deba. 1961. Sources of resistance to the cowpea
    yellow mosaic virus. Plant Dis. Rep. 45:878-881.

    153
    Wetter, C. 1967- Immunodiffusion of tobacco mosaic virus and its
    interaction with agar. Virology 31:^98-507.
    Williams, R. J. 1975. Diseases of cowpea (Vigna unguiculata (L.)
    Walp.) in Nigeria. PANS 21:253-267.
    Williams, R. J. 1977a. Identification of multiple disease resistance
    in cowpea. Trop. Agrie. 5^-53-59-
    Williams, R. J. 19771>. Identification of resistance to cowpea (yellow)
    mosaic virus, lrop. Agrie. 5^:61-67.
    Yu, T. F. 19^+6. A mosaic disease of cowpea (Vigna sinensis End 1.) .
    Ann. Appl. Biol. 33:450-^5**-
    Zaumeyer, W. J., and L. L. Harter. 19^3- Two new virus diseases of
    beans. J. Agrie. Res. 67 305~327-
    Zettler, F. W. 1966. Aphid populations in central New York as related
    to epiphytology of bean common mosaic virus. Ph.D. Dissertation.
    Cornell University, Ithaca, New York. 120 p.
    Zettler, F. W. R. G. Christie, and J. R. Edwardson. 1967. Aphid
    transmission of virus from leaf sectors correlated with intra
    cellular inclusions. Virology 33:5^9-552.
    Zettler, F. W., I. R. Evans. 1972. Blackeye cowpea mosaic virus in
    Florida: Host range and incidence in certified cowpea seed.
    Proc. Fla. State Hortic. Soc. 85:99-101.
    Ziemiecki, A., and K. R. Wood. 1975. Serological demonstration of
    virus-specific proteins associated with cucumber mosaic virus
    infection of cucumber cotyledons. Physiol. Plant Pathol. 7:171-
    177.
    Zink, F. W., R. G. Grogan, and J. E. Welch. 1956. The effect of
    percentage of seed transmission upon subsequent spread of lettuce
    mosaic virus. Phytopathology ^+6 :662-66^4.

    BIOGRAPHICAL SKETCH
    Jos Albersio ele Araujo Lima was born July 12, 19*40, in Santana
    do Cariri, Cear, Brazil, He graduated from high school in December,
    1959, at the Colegio Estadual do Cear in Fortaleza, Cear, Brazil.
    He joined the Army in December, 1958, and was honorably dismissed as
    an Army Reserve Officer in December, I960. He attended the Universidade
    Federal do Cear, receiving the Bachelor of Science degree in Agronomy,
    along with a diploma of Agronomic Engineering, in December, 1966. In
    March, 1967, he was hired as an Auxiliary Professor of Plant Pathology
    by the Universidade Federal do Cear, and in 1973, he was promoted to
    Assistant Professor, a position that he has held up to the present
    time. He started a graduate program in Plant Pathology at the Univer
    sity of Arizona, Tucson, in September, 1970, and received his Master
    of Science degree in 1972, In September, 197*4, he entered the Univer
    sity of Florida, receiving the Doctor of Philosophy degree in March,
    1978. He is the author or co-author of approximately ten publications
    of researches in Plant Pathology.
    J, Albersio A. Lima is married to the former Diana Mara de
    Gurgel Caracas, and he is the father of a son, Roberto. He is a mem
    ber of the American PhytopathoIogica1 Society, the Brazilian Phyto-
    pathological Society, the Association of Agronomic Engineering from
    Cear, the Association of University Professors from Cear, and the
    Latinoamerican Association of Plant Pathology.
    15*4

    I certify that I have read this study and that in my opinion it
    conforms to acceptable standards of scholarly presentation and is
    fully adequate, in scope and quality, as a dissertation for the
    degree of Doctor of Philosophy.
    Dan E. Pure i fu 11 tha i rman
    Professor of Plant Pathology
    I certify that I have read this study and that in my opinion it
    conforms to acceptable standards of scholarly presentation and is
    fully adequate, in scope and quality, as a dissertation for the
    degree of Doctor of Philosophy.
    ?
    Charudattan
    Assistant Professor of Plant Pathology
    I certify that I have read this study and that in my opinion*it
    conforms to acceptable standards of scholarly presentation and is
    fully adequate, in scope and quality, as a dissertation for the
    degree of Doctor of Philosophy.
    J^hn R. Edwardson
    Professor of Agronomy
    I certify that I have read this study and that in my opinion it
    conforms to acceptable standards of scholarly presentation and is
    fully adequate, in scope and quality, as a dissertation for the
    degree of Doctor of Philosophy.
    Ernest Hiebert
    Associate Professor of Plant Pathology

    I certify that I have read this study and that in my opinion it
    conforms to acceptable standards of scholarly presentation and is
    fully adequate, in scope and quality, as a dissertation for the
    degree of Doctor of Philosophy.
    /7__
    Daniel A. Roberts
    Professor of Plant Pathology
    I certify that I have read this study and that in my opinion it
    conforms to acceptable standards of scholarly presentation and is
    This dissertation was submitted to the Graduate Faculty of the College
    of Agriculture and to the Graduate Council, and was accepted as partial
    fulfillment of the requirements for the degree of Doctor of Philosophy.
    March 1978
    Dean, College of Agriculture
    Dean, Graduate School



    Figure 16 Immunodiffusion tests with BICMV, Moroccan isolate of
    CAMV and siratro strain of bean common mosaic virus
    (BCMV-S) in agar medium containing 0.8% Noble agar,
    1.0% NaN,, and 0.5% SOS prepared in 0.05 M Tris-HCl
    buffer, pH 7.2.
    A) Serological tests with B1CMV and CAMV. The center
    wells were charged with: B1CMV antiserum (Bis), and
    normal serum (Ns). The peripheral wells were filled
    with SDS-treated extracts from: (Bl) B1CMV-infected
    cowpea, (Ca) CAMV-infected cowpea, and (H) healthy
    cowpea.
    B) Intragel cross-absorption test with B1CMV and CAMV.
    The center wells were charged with: (l) BICMV anti
    serum, (2) purified CAMV and 20 hr later BICMV anti
    serum. The peripheral wells were filled with SDS-
    treated extracts from: (Bl) BlCMV-infected cowpea,
    (Ca) CAMV-infected cowpea, and (H) healthy cowpea.
    C) Serological tests with BICMV and BCMV-S. The center
    wells were charged with: (Bis) BICMV antiserum, (Ss)
    BCMV-S antiserum. The peripheral wells were filled
    with SDS-treated extracts from: (Bl) B1CMV-infected
    cowpea, (S) BCMV-S-infected bean, (Hb) healthy bean,
    and (He) healthy cowpea. The arrows point to spurs.
    D) Intragel cross-absorption test with BICMV and BCMV-S.
    The center wells were filled with: (1) BICMV anti
    serum, and (2) purified BCMV-S and 20 hr later
    BICMV antiserum. The peripheral wells were charged
    with SDS-treated extracts from: (Bl) BICMV-infected
    cowpea, (S) BCMV-S-infected bean, (Hb) healthy bean,
    and (He) healthy cowpea.
    E) Serological tests with BCMV-S and CAMV using two
    different antisera for BCMV-S. The center wells were
    charged with: (l) BCMV-S antiserum from a rabbit
    inoculated with freshly purified preparations of
    BCMV-S, and (2) BCMV-S antiserum obtained from the
    same rabbit after a booster injection with purified
    BCMV-S stored at k C for 30 days. The peripheral
    wells were charged with SDS-treated extracts from:
    (S) BCMV-S-infected bean, (Ca) CAMV-infected cowpea,
    (He) healthy cowpea, and (Hb) healthy bean.
    F) Serological test with fresh and stored purified
    preparations of BICMV. The center well was charged
    with BICMV antiserum, and the peripheral wells with
    SDS-treated new purified preparation of BICMV (Np),
    old purified BICMV (Op), and healthy cowpea extracts
    (H).


    12
    MW 6000 (PEG) followed by stirring for 60 min. The precipitated virions
    were collected by centrifugation at 13,200 g for 10 min. The resulting
    pellet was resuspended in 0.02 M KPO^, pH 8.2, containing 0.1%
    2-mercapthoethanol (2-ME) (v/v) and the virus was separated from the
    host components by eguilibrium density gradient centrifugation (120,000 £
    for 16 18 hr in a Heckman SW 50.1 rotor) in 30% cesium chloride (CsCl)
    prepared in the same buffer. The virus zone, located at 12 to 15 mm
    from the bottom of ihe tube, was collected dropwise through a hole
    punched in the bottom of the tube and diluted with 0.02 M KPO^, pH 8.2,
    containing 0.1% 2-ME. The virus preparation was clarified by centrifuga
    tion at 11,700 g for 10 min and reconcentrated by centrifugation at
    85,000 lj for 90 min. The final pellet was resuspended in 0.02 M Tris
    buffer, pH 8.2, and the virus concentration was determined spectrophoto-
    metrically using an extinction coefficient of 2.4 mg/ml (Purcifull,
    1966). The optical density (0.0.) readings for the virus at wavelengths
    of 260 and 280 nm were corrected for light scattering before estimating
    the 260/280 ratio and concentrations of virus in purified preparations.
    The correction for light scattering was done by plotting the log of
    the optical densities against the wavelengths of 320, 340, and 360 nm
    and extrapolating these values to 230 300 nm range of wavelength.
    Chloroform-carbon tetrachloride clarification method. This
    clarification process was selected when it was desirable to purify
    both the virus and inclusions from the same batch of tissue. System-
    ¡cally infected tissue (200 400 g) were homogenized in a solution
    containing 1.30 ml of 0.5 M KPO^ (pH 7.5), 0.35 ml of chloroform, 0.35
    ml of carbon tetrachloride, and 5.0 mg of Na^SO^ per gram of tissue.


    9**
    high content of SBMV in the embryos of immature seeds from virus-
    infected bean plants, but observed that the virus concentration dropped
    to a low level or disappeared as the seed matured and dehydrated.
    Cheo's finding was confirmed by Crowley (1959), who found that ap
    proximately 100% embryo infection occurred when bean plants were
    inoculated with SBMV at any time prior to flowering, but no seed
    transmission of this virus occurred when samples of mature dry seeds
    were sown. These findings were supported by the results of McDonald
    and Hamilton (1972), which confirmed that in mature bean seeds, in
    fectious SBMV is confined to the seed coat.
    An ilarvirus was also reported to be seed-borne in bean. Tobacco
    streak virus, the causal agent of a severe disease outbreak of pinto
    bean in State of Colorado, U.S.A., in 19^7, was demonstrated to be
    transmitted by approximately 26% of seeds from plants grown from
    vi rus-infected seeds (Thomas and Graham, 1951).
    In summary, seed-transmission is an important factor in the
    perpetuation and dissemination of numerous viruses that cause diseases
    in cowpea, soybean, and bean. The following methods for detecting
    selected seedborne viruses in legumes may be useful in virus disease
    control programs and as research tools.
    Materials and Methods
    Source of Seed and Seed Germination
    Seeds of cowpea, V. unguiculata 'Knuckle Purple Hull' and 'Early
    Ramshorn', harvested from B1CMV-infected plants were used in the present
    tests. All seed lots showing B1CMV-transmission were obtained either


    104


    30
    SUPERNATANT-
    (Discard)
    PELLET
    (Discard)
    SUPERNATANT^
    (Discard)
    SYSTEMICALLY INFECTED TISSUE
    0.5M KP04 pH 7.5 + CHCI3+ CC14+ 1% Na2S03
    CENTR
    PELLET
    (Discard)
    FUGATION: 4,000g 5min
    SUPERNATANT
    CENTR
    SUPERNATANT
    (Virus)
    8% |eG
    STIR : 60min
    FUGATION: lT,700g 15min
    CEN
    PELLET
    (Inclusions)
    0.05M KP04 pH 8.2 + 0.1% 2-ME
    HOMOGENIZATION
    RIFUGATION: 11,700g lOmin
    5%
    CEN
    PELLET
    0.0j!M KP04 pH 8.2 + O.U 2-ME
    RITON-X
    RIFUGATION: 27,000g 15min
    -SUPERNATANT
    (Discard)
    PELLET
    CsCl GRADIENT CENTRIFUGATION:
    d=lj 28g/cc-120000g 18hr
    COLLECT VIRUS ZONE
    CENTRIFUGATION: ll,700g lOmin
    SUCROSE STEP GRADIENT CENTRIFUGATION:
    45,(J00g 60min
    COLLECT INCLUSION ZONE
    SUP
    CEN
    CEN
    RNATANT
    RIFUGATION: 85,000g 90min
    RIFUGATION: 27,000g 15min
    -5UPERNATANT
    (Discard)
    PELLET
    PELLET
    0.02M TRIS pH 8.2
    INCLUSIONS
    0.02M TRIS pH 8.2
    VIRUS
    I


    90
    from CCHV-infected plants. According to Gay (1969), CCHV was not trans
    mitted through seeds of cowpea because of virus inactivation during seed
    maturation.
    Seed-Borne Viruses in Glycine max.
    Soybean mosaic virus (SoyMV) which is probably common wherever
    soybean is cultivated (Bos, 1972) was first demonstrated to be seed-
    borne in soybean by Gardner and Kendrick (1921). Subsequently, they
    observed that the virus could overwinter in the vi rus-infected seeds
    lying in the field (Kendrick and Gardner, 192A). Although Kendrick
    and Gardner (192A) had also observed that mottled seeds were produced
    by both healthy and v i rus-infected plants and that diseased seedlings
    were produced by mottled as well as clean seeds, the association of
    seedcoat mottling and virus infection has been reported (Koshimizu
    and lizuka, 1957; Ross, 1963, 1968, 1969; and Kennedy and Cooper,
    1967). A mechanical selection of nonmottled soybean seeds has been
    suggested as a measure for controlling SoyMV in Brazil (Lima-Neto and
    Costa, 1976). However, Ross (1970) observed that SoyMV was equally
    transmitted through mottled and nonmottled seeds from vi rus-infected
    soybean plants, and concluded that the percentage of seed transmission
    for SoyMV in soybean could not be estimated from the amount of mottled
    seeds. Working with a Brazilian isolate of SoyMV, Porto and Hagedorn
    (1975) reported the production of mottled seeds by supposedly non-
    infected soybean cultivars and observed that SoyMV was not transmitted
    through seeds of vi rus-infected soybean cv, 'Bienville', which pro
    duced seeds with a high percentage of mottling. Bean pod mottle virus,


    102
    medium was prepared in 0.05 M Tris buffer, pH 7.2 (Fig. 19~B). Based
    on these results a diagram showing several procedures for preparation
    of antigens from leyume seeds is suggested (Fig. 20).
    Double Immunodiffusion Tests
    Blackeye cowpea mosaic virus was detected in hypocotyl extracts
    from bulked seedlings and in small discs of individual hypocotyls of
    cowpea 'Knuckle Purple Hull' and 'Early Ramshorn' by double immuno
    diffusion tests in SDS-gel plates. Virus specific precipitin lines
    were observed with extracts of mixtures of B1CMV-infected and non-
    infected cowpea 'Knuckle Purple Hull' hypocotyls up to a dilution of
    1/30 (w/w), respectively (Fig. 21 -B). This indicated that germinated
    seeds can be divided into groups of up to 30 seed!ings to be tested
    in double immunodiffusion. Approximately equal amounts of hypocotyl
    tissue (0.1 0.4 g) were cut from each seedling and the extracts ob
    tained from each hypocotyl group were deposited into individual antigen
    wells. Precipitin reactions were observed with those groups in which
    at least one seedling was infected with B1CMV. On the other hand, no
    precipitin reactions were observed with extracts obtained from non-
    infected hypocotyl tissue, nor with any hypocotyl extract and normal
    serum (Figs. 19, 21).
    When information about the infection percentage was wanted, discs
    of individual hypocotyls were used in double immunodiffusion. Virus-
    specific precipitin lines formed between B|CMV-infected hypocotyl discs
    and antiserum wells, whereas no reactions were observed with non-
    infected hypocotyls (Fig. 21 C, -D, -F). Good results were observed
    with undiluted antiserum and with antiserum diluted 1/2 and 1/4 with


    75
    the antigenic properties of viral coat protein (Hiebert and McDonald,
    1976; and Purcifull and Batchelor, 1977). Using antiserum obtained
    for freshly purified BICMV, serological distinction was observed
    between freshly purified preparations of BICMV and purified BICMV
    stored at 4 C for more than 30 days (Fig, 16-F). The serological
    distinctions between different antigen preparations of the same virus
    observed herein are of great significance for serological identifica
    tion and characterization of potyviruses as pointed out by Hiebert and
    McDonald (1976) and Purcifull and Batchelor (1977). It is important
    to keep in mind that purification, storage of either purified virus
    preparations or crude sap containing virus, and mailing of virus-
    infected fresh plant tissues may all result in modifications in the
    antigenic properties of viral coat protein. To solve this problem,
    the preservation of plant virus antigens by 1yophi 1ization of crude
    extracts from infected plants (Purcifull et al., 1975) or purified
    virus preparations is recommended. Blackeye cowpea mosaic virus
    has been maintained in lyophilized condition either in crude sap or
    purified preparation over two years during the course of this study
    without any perceptive change in its antigenic properties.
    Another factor that should be considered during serological
    relationship studies between viruses in the PVY group is the specificity
    of antisera. Variations in the degree of cross reactivity exhibited
    by different antisera obtained against the same virus have been
    attributed to differences between individual animals (van Regenmortel
    and von Wechmar, 1970), route and number of injections used in the
    immunization program (Hoi 1ings and Stone, 1965) and time of bleeding


    rod-shaped virus particles and used as the initial source of inoculum
    for virus propagation. The virus was mechanically transmitted from
    the selected infected plant to healthy 'Knuckle Purple Hull' seedlings,
    where it was increased for virus and inclusion purification, and
    other studies.
    Virus and Inclusion Purificar ion
    Blackeye cowpea mosaic virus was propagated in either V_. unguiculata
    or Ni cot ana benthamiana Domin, and systemically infected leaves were
    used for virus and inclusion purification. Either r^-butanol or a com
    bination of chloroform and carbon tetrachloride was used in the clari
    fication process. The adaxial surface of the primary leaves of 5 to
    7-day old cowpea seedlings were inoculated with BICMV obtained by
    grinding infected lecif tissue in 0.05 M potassium phosphate (KPO^)
    buffer, pH 7-5 (1/2, w/v). The first trifoliolate leaves showing
    typical mosaic were collected 15 to 18 days later and subjected to the
    following purification procedures based on previous works (Hiebert et
    a 1., 1971; Hiebert and McDonald, 1973; and McDonald and Hiebert, 1975).
    n^ Butanol clarification method. Two hundred to 400 g of leaf
    tissue were homogenized in a blender with two parts (w/v) of 0.5 M
    KPO^ buffer, pH 7-5, containing 0.5 to 1.0% sodium sulfite (Na^SO^).
    The resulting extract was filtered through a double layer of cheese
    cloth and enough n-butanol was added to make a final concentration of
    8% (v/v). This mixture was stirred overnight at 4 C and the coagulated
    green debris obtained was removed by a low speed centrifugation at
    11,700 in a Sorvall Centrifuge (Sorvall Superspeed RC2-B Automatic
    Refrigerated Centrifuge) for 10 min. Virions were precipitated from
    the supernantant by the addition of 6 8% (w/v) of polyethylene glycol


    16
    B1CMV-infected cowpea leaves to 45, 50, 55, 60, 65, 70, and 75 C for
    10 min. All treated saps as well as unheated sap of B1CMV-infected
    tissue were rubbed on the test plants, which were maintained in green
    house conditions for at least three weeks for observation of symptoms.
    Crude sap of infected leaves obtained in deionized water was
    placed in test tubes and assayed for infectivity after storage at room
    temperature for 0, 8, 16, 24, 48, and 72 hr. For the DEP determination,
    crude juice was extracted from B1CMV-infected leaves, and the extract
    was diluted to 10 10 10 10 10 and 10 ^ with deionized
    water prior to assay.
    Polyacrylamide Gel EIectrophores?s of Viral and Inclusion Proteins
    The polyacrylamide gel electrophoresis studies were performed
    according to the method of Weber and Osborn (1969) as modified by
    Hiebert and McDonald (1973). Running gels of approximately 75 mm in
    height were prepared with 6% acrylamide (7.5 ml sodium phosphate buffer,
    pH 7.2; 15.0 ml water; 0.15 ml 10% SDS; 6.0 ml of 30% acrylamide;
    0.045 ml N, N, N1, N1-tetramethy1ened¡amine (TEMED) and 1.2 ml ammonium
    persulphate 15 mg/ml), and a well-forming gel of 8% acrylamide with one-
    fifth the electrophoresis buffer concentration (1.2 ml buffer; 7.2 ml
    H^O; 0.2 ml 10% SDS; 3.0 ml of 30% acrylamide; 0.04 ml TEMED, and 0.3
    ml ammonium persulphate 15 mg/ml) was cast on top of them. Disasso
    ciated protein solutions, 20 50 pi samples in approximately 20%
    sucrose and one-fifth the electrophoresis buffer concentration, were
    placed into the formed wells. The top of the samples were covered
    with a cap gel of composition similar to the well-forming gel. The
    electrophoresis was performed in a vertical slab electrophoresis
    apparatus, Ortec, Model 4010/4011, Ortec, Incorporated, Oak Ridge, Tenn.,


    811


    Figure 19
    - Double immunodiffusion tests with extracts from different
    portions of B1CMV-infected and healthy, 4-5-day-old cowpea
    seedlings. A) medium containing 0.8% Noble agar, 1.0%
    NaN^, and 0.5% SDS prepared in deionized water. B) me
    dium with the same composition except that it was prepared
    in 0.05 M Tris-HCl buffer, pH 7-2 instead of water.
    Center wells were charged with: (As) B1CHV antiserum,
    and (Ns) normal serum. The peripheral wells were filled
    with extracts from: (a) hypocotyl from B1CMV-infected
    cowpea seedlings, (b) hypocotyl from healthy cowpea
    seedlings, (c) cotyledons and primary leaves of B1CMV-
    infected seedlings, and (d) cotyledons and primary leaves
    of healthy cowpea. Note the nonspecific precipitate
    formed with cotyledons and primary leaves of virus-
    infected (c) and healthy (d) seedlings and normal serum
    (Ns) and B1CMV antiserum (As). The virus-specific
    precipitin lines clearly shown with vi rus-infected
    hypocotyls (a) and the antiserum (As) was masked by the
    nonspecific precipitates when extracts from cotyledons
    and primary leaves (c) of infected seedlings were used.


    22
    a well adjacent to the other virus-well. Sap extracts from appropriate
    healthy host tissues were included as controls in all serological tests,
    and all antigens were also tested against normal serum.
    The intragel cross-absorption technique described by van Regenmortel
    (1966) was also used to study the serological relationships of B1CMV
    with BCMV-S and CAMV. Purified preparations of heterologous antigens
    (BCMV -S or CAMV) were placed in the center well and allowed to diffuse
    for approximately 2^ hr. The excess of the antigen preparations were
    then removed and the B1CMV antiserum was added in the same well. At
    the same time, the homologous and the heterologous antigens were po
    sitioned in the outer wells.
    Light and Electron Microscopy of Virus Induced Pinwheel Inclusions
    Epidermal leaf strips obtained from systemically infected cowpea,
    Y_* ungu i culata, were floated on a 5% solution of Triton X-100 for 5
    to 10 min and subsequently stained with a combination of calcomine
    orange and "luxol" brilliant green as described by Christie (1967).
    The stained leaf strips were mounted in euparal on glass slides and
    examined with a light microscope for the presence of cytoplasmic in
    clusions. Similarly, strips from noninocu 1 ated V_. unguiculata were
    also stained and examined in the light microscope as controls.
    Cylindrical inclusions were examined in situ in ultrathin sec
    tions with an electron microscope. Small pieces were taken from
    symptomatic areas of systemically infected cowpea leaves and fixed
    for 2 to 3 hr at room temperature in Karnovsky's forma Idehyde-
    g1utara1dehyde fixative prepared in 0.1 M cacodylate buffer, pH 7.2
    (Karnovsky, 1965). After washing with 0.1 M cacodylate buffer, the
    small leaf pieces were postfixed for 1 to 2 hr at room temperature


    44


    23
    in 2% osmium tetroxide and progressively dehydrated in an increasing
    ethanol solution series. The leaf pieces were maintained for 5 to 15
    min in each ethanol solution at room temperature. The pieces were
    stained overnight at b C in a solution of 75% ethanol containing 2%
    uranyl acetate and subsequently dehydrated in a second series of ethanol
    solutions (75 100%) followed by 100% acetone or propylene oxide.
    They were then embedded in plastic containing Epon 812, Araldite 502,
    and dodecenylsuccinic anhydride. Ultrathin sections were cut with a
    diamond knife in a Sorvall MT-2 ultramicrotome and mounted on copper
    grids with carbon-coated Formvar film. The specimens mounted on the
    grids were poststained with 9% potassium permanganate (2 min), 1%
    uranyl acetate (2 min), and lead citrate (2 min). These sections as
    well as those obtained from non inocu1 a ted cowpea plants were examined
    with a Philips Model 200 electron microscope.
    Purified B1CMV-I preparations were mounted on carbon-coated
    Formvar film supported by copper grids and stained with either 1%
    ammonium molybdate or 2% uranyl acetate, before examination by electron
    microscopy.
    Host Range and Screening Cowpea Varieties for Resistance
    Test plants were inoculated with crude sap from 'Knuckle Purple
    Hull' systemically infected with B1CMV. The inoculum was prepared by
    grinding leaf tissue in 0.05 M KPO^, pH 7-5 (112, w/v). The inocula
    tions were done by rubbing the inoculum on carborundum-dusted leaves
    of the test plants which were maintained in greenhouse conditions for
    at least one month for observation of symptoms. All inoculated plants,
    including those that did not show any symptoms were checked serologically
    for the presence of BICMV.


    147
    McDonald, J. G., and E. Hiebert. 1975. Characterization of the capsid
    and cylindrical inclusion proteins of three strains of turnip
    mosaic virus. Virology 63:295-303.
    McLean, D. M. 1941. Studies on mosaic of cowpea Vigna sinensis. Phyto
    pathology 31:420-430.
    Medina, A. C., and R. G. Grogan. 1961. Seed transmission of bean mosaic
    viruses. Phytopathology 51:452-456.
    Milne, R. G., and E. Luisoni. 1977- Rapid immune electron microscopy
    of virus preparations. Pages 265-281 [r^ K. Maramorosch, and H.
    Kiprowski, eds. Methods in Virology, Vol. VI. Academic Press, New
    York. 542 p.
    Morales, F. J., and F. W. Zettler. 1977. Characterization and electron
    microscopy of a potyvirus infecting Commelina diffusa. Phyto
    pathology 67:839-843.
    Moreira, R. de A., and J. C. Perrone. 1977. Purification and partial
    characterization of a lectin from Phaseolus vulgaris. Plant
    Physiol. 59:783-787.
    Nariani, T. K., and T. K. Kandaswany. 1961. Studies on a mosaic
    diseaseof cowpea (Vigna sinensis Savi). Indian Phytopathol. 10:
    77-82.
    Nelson, R., and E. E Down. 1933. Influence of pollen and ovule in
    fection in seed transmission of bean mosaic. Phytopathology 23:
    25 (Abstr.).
    Nelson, M. R., and H. K. Knuhtsen. 1973. Squash mosaic virus vari
    ability: review and serological comparison of six biotypes. Phyto
    pathology 63:920-926.
    Nicolaescu, M., H. Titu, and M. Paraschiv. 1976. Electron-microscopical
    investigations on the pinwheel structures detected in soybean mosaic-
    virus infected plants. Rev. Roum. Biol. Biol. Veg. 21:67-69-
    Ouchterlony, 0. 1962. Diffus ion-in-gel methods for immunological
    analysis II. Prog. Allergy 6:30-154.
    Owusu, G. K., N. C. Crowley, and R. I. B. Francki. 1968. Studies of
    the seed-transmission of tobacco rinqspot virus. Ann. Appl. Biol.
    61 : 195-202.
    Padma, R., and A. S. Summanwar. 1973. Chenopodium mrale a differ
    ential host for cowpea mosaic virus. Curr. Sci. 42:620.
    Perez, J. E.( and A. Cortes-Mon11 or. 1970. A mosaic virus of cowpea
    from Puerto Rico. Plant Dis. Rep. 54:212-216.


    Figure 7 Histograms of lengths of B1CMV particles from purified
    preparation negatively stained with phosphotungstate
    (A), and cowpea leaf extract using the serologically
    specific electron microscopy and uranyl acetate as a
    positive stain (B). Class interval for both histograms
    50 nm.


    Antisera reactive in SDS-immunodiffus i on were obtained against
    untreated virions, pyrrolidine degraded coat protein, and untreated BlCMV
    cytoplasmic inclusions. Reciprocal double immunodiffusion tests with
    SDS-treated antigens showed that BlCMV is serologically unrelated to
    seven potyviruses and serologically related to, but distinct from:
    bean common mosaic virus (BCMV) bean yellow mosaic virus (BYMV) cow-
    pea aphid-borne mosaic virus (CAMV), dasheen mosaic virus (DMV) lettuce
    mosaic virus (LMV), potato virus Y (PVY), soybean mosaic virus (SoyMV),
    tobacco etch virus (lEV), and watermelon mosaic virus-2 (WMV-2). The
    intragel cross-absorption technique was also used to demonstrate distinc
    tion between closely related potyviruses. Agar medium impregnated with
    a mixture of antisera was used for serodiagnosis of BlCMV and cowpea
    mosaic virus in cowpea.
    Light and electron microscopy of cytoplasmic inclusions induced
    by BlCMV, siratro (Macropti Iium atropurpureum (D.C.) Urb.) strain of
    BCMV (BCMV-S) and CAMV revealed that they are similar to those induced
    by the potyviruses from Edwardson's subdivision-I. The different reac
    tions induced by BlCMV, BCMV-S, and CAMV in some cowpea varieties in
    dicated that they can also be used as differential hosts for these
    three potyviruses. Sources of resistance for BlCMV were found among
    the cowpea varieties tested. Based on its physical, biological, cyto-
    logical, and immunoctiemica1 properties, BlCMV can be differentiated
    from any other virus that infects cowpea.
    Cytoplasmic inclusions induced by BlCMV in cowpea and by SoyMV
    in soybean were detected by serology, light microscopy, and electron
    microscopy in hypocoiyls of 4-5-day-old seedlings grown from virus-
    infected seeds.
    x i i


    LITERATURE CITED
    Abo El-Nil, M. M., F. W Zettler, and E, Hiebert. 1977. Purification,
    serology, and some physical properties of dasheen mosaic virus.
    Phytopathology 67: 1445-1450.
    Abrygunawardena, D. V. W., and S. M D. Perera. 1964 Virus diseases
    affecting cowpea in Ceylon. Trop, Agrie. Mag. Ceylon Agrie. Soc.
    120:181-204
    Afanasiev, M. M. 1956. Occurrence of barley stripe mosaic in Montana.
    Plant Dis. Rep. 40:142.
    Agrawal, H. 0. 1964 Identification of cowpea mosaic virus isolates.
    Mededel. van de Landbouth Wageningen, Nederland 64:1-53.
    Alba, A. P. C., and A. R. Oliveira. 1977. Serological studies on
    viruses of the potato virus V group occurring in Sao Paulo. Summa
    PhytopathoIog ica 2:178-186
    Alconero, R., and A. Santiago. 1973. PhaseoI us lathyroides as a
    reservoir of cowpea virus in Puerto Rico. Phytopathology 63:120-
    123.
    Anderson, C. W. 1955a. Vigna and Crotalaria viruses in Florida. I.
    Preliminary report on a strain of cucumber mosaic virus obtained
    from cowpea plants. Plant Dis. Rep. 39:346-348.
    Anderson, C. W. 1955b. Vigna and Crotalaria viruses in Florida. II.
    Notations concerning cowpea mosaic virus (Marmor Vignae). Plant
    Dis. Rep. 39:349-352.
    Anderson, C. W. 1955c. Vigna and Crotalaria viruses in Florida. III.
    Notations concerning identification difficulties, indicator plants,
    possible vector relationships, and virus maintenance. Plant Dis.
    Rep. 36:354-357
    Anderson, C. W. 1957- Seed transmission of three viruses in cowpea.
    Phytopathology 47:515 (Abstr ).
    Anderson, C. W. 1959. Vigna and Crotalaria viruses in Florida. V.
    Comparative transmission tests with aphids and beetles. Phyto
    pathology 49:117-118
    Andrews, J. H., and T A, Shalla 1974. The origin, development,
    and conformation of amorphous inclusion body components in tobacco
    etch v i ius infected cells. Phytopathology 64:1234-1243.
    138


    127


    Figure 1A Single radial diffusion tests with SDS and pyrrolidine
    degraded capsid protein of blackeye cowpea mosaic virus
    (B1CMV) and cowpea mosaic virus (CPMV).
    The media in (A, B, C) contain 0.8% Noble agar,
    1.0% NaN 0.5% SDS, and 1 52 B1CMV-As (A) 152 CPMV-As
    (B), and^10% B1CMV-As + 10% CPMV-As (C). The wells
    were charged with SDS-t.reated extracts from: B1CMV-
    infected cowpea (row l), CPMV-infected cowpea (row 2),
    B1CMV and CPMV in cowpea (row 3), and healthy cowpea
    (row A).
    The media in (D, E, F) contain 0.8% Noble agar,
    0.2% NaN?, 0.85% NaC 1 and 152 B1CMV-As (D) 152
    CPMV-As (E), and 10% B1CMV-As + 10% CPMV-As (F)
    prepared in 0.05 M Tris-HCl buffer, pH 7.2. The
    wells were charged with pyrrolidine-treated extract
    from: B1CMV-infected cowpea (row 1), CPMV-infected
    cowpea (row 2), B1CMV and CPMV in cowpea (row 3),
    and healthy cowpea leaves (row A).


    f
    Figure 26 Electron micrographs of serologically specific electron
    microscopy with extracts from BCMV- and SoyMV-infected
    hypocotyIs.
    A) BCMV-S antiserum-sensitized grid and extract from
    bean hypocoty! infected with BCMV-BV-I;
    B) SoyMV antiserum-sensitized grid and extract from
    SoyMV-infected soybean hypocotyl.


    77
    between BICMV and WMV-2 when both viruses were tested against antiserum
    to WMV-2 than when they were tested against BICMV antiserum (Fig. 15-C).
    Similar results were observed with BCMV isolates and BICMV (Fig. I5~E,
    -F) which may explain the identical reaction reported by Uyemoto et al.
    (1973).
    It is noteworthy that BICMV and BYMV are serologically distinct,
    though related. This supports the contention of Edwardson et al. (1972)
    and Zettler and Evans (1972) that BICMV and BYMV should be considered
    distinct viruses.
    Serological differences between closely related viruses are better
    detected with antisera of fairly low titer (Matthews, 1970). On the
    other hand, he also stated that a high titer antiserum is preferable
    for demonstrating distant serological relationship. This can be
    illustrated by the serological tests carried out with BICMV and CAMV
    isolates using a BICMV antiserum with a titer of 32. By diluting the
    antiserum to 1/4, no reaction was observed with the heterologous virus
    (CAMV) whereas a fairly good reaction was still detected with the homolo
    gous antigen. The absence of reaction between BICMV and LMV-antiserum
    (Fig. 15-J) may be a result of the low titer of the antiserum.
    The intragel cross-absorption test was effective for demonstrating
    distinctions between two closely related viruses (Fig. 16-B, -D). This
    is additional evidence that serological distinctions that are undetect
    able in conventional doub1e-immunodiffus ion tests may be clearly revealed
    by intragel cross-absorption. Using this test, Matthews (1970) revealed
    a serological difference between type TMV and a nitrous acid induced
    mutant which showed a reaction of identity when tested against




    CHAPTER I I
    IMMUNOCHEMICAL AND CYTOLOGICAL TECHNIQUES FOR
    DETECTION OF LEGUME VIRUSES IN INFECTED SEEDS
    Introduction
    The transmission of plant viruses through seed of infected host
    plants was first demonstrated by Reddick and Stewart (1919), who
    showed that bean common mosaic virus (BCMV) was transmitted by ap
    proximately 50% of seeds from infected Phaseolus vulgaris. Since
    then, the phenomenon of seed transmission of plant viruses has re
    ceived considerable attention and an appreciable number of viruses
    have been demonstrated to be seed-borne to some extent (Fulton, 1964;
    Bennett, 1966; Shepherd, 1972; and Phatak, 1974). Virus can be intro
    duced into a crop at an early stage of plant development through in
    fected seeds. Thus, the production of virus-free seeds, or seed lots
    with very low virus content may provide a very effective control of
    seed-borne plant viruses. Seed certification programs have been
    developed to test seed lots for the presence of viruses and to select
    virus-free seeds. Barley stripe mosaic virus, which is responsible
    for a serious disease in Montana (Afanasiev, 1956), and lettuce mosaic
    virus(LMV), the causal agent of an important disease of lettuce
    (Grogan et al 1952), are good examples of virus diseases against
    which seed certification programs have been successful (Zink et al.,
    1956; Hamilton, 1965; Phatak, 1974; and Slack and Shepherd, 1975).