Comparative disc electrophoretic protein analyses and serological relationships of selected species of Meloidogyne and s...

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Comparative disc electrophoretic protein analyses and serological relationships of selected species of Meloidogyne and some host plants
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Root-knot nematodes   ( lcsh )
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Root-knot   ( fast )
Root-knot nematodes   ( fast )
Entomology and Nematology thesis Ph. D
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Thesis--University of Florida.
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Includes bibliographical references (57-63).
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by Franklin Hon-Ching Chow.
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COMPARATIVE DISC ELECTROPHORETIC PROTEIN
ANALYSES AND SEROLOGICAL RELATIONSHIPS OF SELECTED SPECIES
OF Meloidogyne AND SOME HOST PLANTS









By

FRANKLIN HON-CHING CHOW














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

1977















ACKNOWLEDGEMENTS


The author expresses his sincere appreciation to the

chairman of his supervisory committee, Dr. Vernon G. Perry,

for his generous assistance and enthusiastic encouragement

throughout this study, and to the cochairman of his super-

visory committee, Dr. S. G. Zam, for his advice during the

periods of research.

The author also expresses his gratitude to Dr. D. W.

Dickson and Dr. G. C. Smart, Jr. for serving as members of

the supervisory committee and for their valuable help.

The technical assistance of Mr. R. W. Patrick is also

gratefully acknowledged, and the typing of the dissertation

by Ms. Inez Butler and Mrs. Grace Beal is especially appre-

ciated.

Finally, heartfelt thanks to my wife, Marjorie, for

her patience and assistance during the long period of this

study.








ii
















TABLE OF CONTENTS Page


ACKNOWLEDGEMENTS . . . . . . . . .. ii

LIST OF TABLES . . . . . . . . .. v

LIST OF FIGURES . . . . . . . . . vi

ABSTRACT . . . . . . . . . . . viii

INTRODUCTION . . . . . . . . . . 1

CHAPTER I

DIFFERENTIAL HOST TEST . . . . . . 4

Materials and Methods . . . . . . 5
Results . . . . . . . . . 6
Discussion . . . . . . . . 6

CHAPTER II

DISC ELECTROPHORESIS

Introduction . . . . . . . . 8
Literature Review . . . . . . . 9
Materials and Methods . . . . . .. 11
Results . . . . . . . . . 16
Discussion . . . . . . . . . 30

CHAPTER III

SEROLOGY

Introduction . . . . . . . . 32
Literature Review . . . . . . .. 32
Materials and Methods . . . . . .. 35
Results . . . . . . . . . 36
Discussion . . . . . . . . 51







i ii









Table of Contents Cont'd.


Page
CHAPTER IV

AFFINITY CHROMATOGRAPHY . ... . ...... 52

Introduction . . . . . . . . 52
Materials and Methods . . . . . .. 53
Results . . . . . . . .. . 54
Discussion . . . . . . .. . . 54

CHAPTER V

CONCLUSIONS . . . . . . . . . . 55

LITERATURE CITED .. . . . . . . . . . 57

BIOGRAPHICAL SKETCH . . . . .. . . 64



































iv
















LIST OF TABLES


Table Page

1. Species and populations of Meloidogyne . ... . .. 3

2. Meloidogyne population test on host differentials .. 7

3. Disc electrophoretic analyses of buffer soluble
protein extracts of adult females of Meloidogyne spp 18

4. Disc electrophoretic analyses of buffer soluble
protein extracts of eggs of Meloidogyne spp ... .21

5. Disc electrophoretic analyses of glycoprotein,
mucoprotein, and lipoprotein from buffer soluble
protein extracts of Meloidogyne spp . . . ... 29



























v















LIST OF FIGURES


Figureage

1. Total protein patterns of coomassie blue stained
gels and corresponding densitometer tracings of
stained gels of adult females of Meloidogyne spp . 17

2. Total protein patterns of coomassie blue stained
gels and corresponding densitometer tracings of
stained gels of eggs of Meloidogyne spp . . .. 20

3. Diagrammatic representation of glycoprotein pat-
terns of Meloidogyne spp. adult females and eggs
stained with alcian blue . . . . . . .. 25

4. Diagrammatic representation of mucoprotein pat-
terns of eggs of Meloidogyne spp. stained with
toluidine blue . . . . . . . . . . 26

5. Diagrammatic representation of lipoprotein pat-
terns of adult females and eggs of Meloidogyne
spp. stained with Sudan Black B . . . . .. 28

6. Immunodiffusion patterns of different popula-
tions of adult females of Meloidogyne spp . . .. 38

7. Immunodiffusion patterns of different popula-
tions of eggs of Meloidogyne spp. . . . . ... 39

8. Immunodiffusion patterns of antigens of adult fe-
malcsofMeloidogyne spp. against their own antisera
and against antisera of their own eggs . . ... .41

9. Immunodiffusion patterns of antigens of eggs of
Meloidogyne spp. against their own antisera and
against antisera of their own adult females . .. 42

10. Immunodiffusion pattern of antiserum of adult
female M. arenaria against plant root extracts . . 43

11. Diagrammaticrepresentation of the immunoelectrophore-
tic patterns of different populations of adult females
of Meloidogyne spp.. . . . . . . . . 45



vi









Figure Page

12. Diagrammatic representation of the immuneolectropho-
retic patterns of different populations of eggs of
Meloidogyne spp . . . . . . . . .. 48

13. Diagrammatic representation of the immunoelectropho-
retic patterns of adult females compared to eggs of
Meloidogyne spp . . . . . . . . .. 49

14. Diagrammatic representation of the immunoelectropho-
retic patterns of antisera of adult females of
Meloidogyne spp. against root extracts of 'Porto
Rico' sweetpotato. . . . . . . . . .. 50







































vii









Abstract of Dissertation Prc"; inted to the Graduate Council
of the University of Florida Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


COMPARATIVE DISC ELECTROPHORETIC PROTEIN
ANALYSES AND SEROLOGICAL RELATIONSHIPS OF SELECTED SPECIES
OF Meloidogyne AND SOME HOST PLANTS

By

Franklin Hon-Ching Chow

June, 1977


Chairman: Vernon G. Perry
Cochairman: Stephen G. Zam
Major Department: Entomology and Nematology

Disc electrophoresis and serological techniques were

used to distinguish between Meloidogyne arenaria, M. javanica,

and two populations of M. inconita. The buffer soluble pro-

tein extracts obtained from adult females and eggs of each of

the populations were used in this study. Root extracts of

selected host plants were serologically tested with the antibo-

dies of the nematodes to determine their relationships to the

host range of the nematodes.

Total protein, glycoprotein, mucoprotein, and lipoprotein

contents of nematode populations were analyzed by disc electro-

phoresis. Total protein electrophoresed in 7 percent poly-

acrylamide gel stained by coomassie blue gave the best protein

separation both between and within species. The glycoprotein,

mucoprotein, and lipoprotein patterns where observed were

identical in all populations studied. One qlycoprotein band

and one lipoprotein band were observed in extracts of both

adult females and egqs; however, no mucoprotein band was


vi 1







demonstrated in adult female extracts, but two were shown in

egg extracts.

The four populations of Meloidogyne could be separated

both by immunodiffusion and immunoelectrophoresis. Several

attempts to obtain purified adult female and egg population-

specific antibodies by affinity chromatography were unsuccess-

ful.

Although there were immunoprecipitin bands of root

extracts on 'Porto Rico' sweetpotato when applied against

nematode antisera, no evidence of the relationship between

the nematodes and their host range was shown by serological

techniques.





























ix













INTRODUCTION


The root-knot nematodes of the genus Meloidogyne are

damaging parasites of most agricultural crops grown in the

world.. Some 35 species were recognized by Esser et al (27).

Certain of these species are similar in morphology and host

responses so that identification has proven to be difficult

even when attempted by experienced nematologists. The char-

acters used for species differentiation are variable even from

a population produced on an individual plant. Yet identifica-

tion is of the utmost importance if integrated pest management

procedures are to be successfully employed.

At the present time, identification of the root-knot

nematodes is based partly on the morphology of adult males,

females, and second stage larvae, and some nematologists use

host responses to parasitism as an aid to identification.

Many researchers, however, have found that these criteria are

often confusing and hence not satisfactory.

Modern technology in cytology (75, 76), enzyme and pro-

tein analyses (21, 22, 42), and serology (39, 40, 42, 50, 54)

has proved very helpful in identifying certain nematodes.

Some of the above authors reported that techniques such as

disc electrophoresis, immunodiffusion, and immunoelectrophoresis

offer substantial promise for identification of root-knot

nematodes.


1




2


The objectives of the research reported herein were to

further determine if disc fl-cfrophoresis and serological tech-

niques could be used as aids in identifying populations of

Meloidogyne; to determine if purification of species-specific

antibodies by affinity chromotograph can be utilized and; to

determine if relationships could be demonstrated between host

ranges and serological reactions. In order to accomplish these

objectives, one population each of Meloidogyne arenaria (Neal)

and M. javanica (Treub), and two populations of M. incognita

(Kofoid and White) were selected for investigation (Table 1).

All populations were derived from single egg mass isolates and

were maintained on tomato plants, Lycopersicon esculentum Mill

'Rutgers' in a greenhouse.









Table 1. Species and populations of Meloidogyne.


Population Original Date
Species No. Place Collected 'Host Collected

M. arenaria 000 Greece Supplied by Dr. J.N. Unknown Unknown
Sasser (N.C. State University)

M. incognita (S) 118 St. Johns County, Florida Irish Potato March 1971
Solanum
tuberosum L.

M. incognita (A) 122 Alachua County, Florida Irish Potato May 1971
Solanum
tuberosum L.

M. javanica 129 Suwannee County, Florida Tobacco Augu- -971
Nicotiana
tabacum L.

















LO













CHAPTER I

DIFFERENTIAL HOST TEST


Allen (2) in 1952 demonstrated the intraspecific varia-

tions of four populations of M. incognita acrita on 12 hosts.

Sasser (62, 63) described a method to identify root-knot nema-

todes by host reaction and identified an unknown population by

his methods. He emphasized, however, that some populations may

not be identifiable by this method.

During a two-year tobacco rotation research program Sasser

and Nusbaum (65) reported the existence of strains or races

within M. incognita acrita that differ in their ability to

attack crops. Van der Linde (78) identified a root-knot nema-

tode as M. incognita acrita by host responses despite morpho-

logical evidence which showed the nematode to be more similar

to M. incognita.

Biotypes of root-knot nematodes were further demonstrated

by Goplen et al (33) within M. incognita acrita, M. hapla,

and M. javanica in tests with alfalfa varieties.

In my investigations each of the four root-knot nematode

populations was identified by examination of 10 female perineal

patterns and by morphometrical measurements of 15 larvae.

Host differential tests were conducted in an attempt to con-

firm the identifications and also to seek possible relation-

ships between host ranges and serological reactions.


4




5

Materials and Methods


Nine differential host plants were used to observe the

host responses to different populations of root-knot nematodes.

They were corn, Zea mays L., 'Minnesota A 401'; cotton,

Gossypium hirsutum L., 'Deltapine 16'; peanut, Arachis hypogaea

L., 'Florunner'; pepper, Capsicum fruitescens L., 'California

Wonder'; strawberry, Fragaria ananassa Duch., 'Albritton';

sweetpotato, Ipomoea batatas (L.) Lam., 'Allgold' and 'Porto

Rico'; tobacco, Nicotiana tabacum L., 'NC-95'; and watermelon,

Citrulus vulgaris Schard., 'Charleston Grey.' 'Rutgers' tomato

was used as an indicator of inoculum potential.

All plants were rooted before inoculation. During trans-

plantation into a 15-cm clay pot, each plant was inoculated

with 10,000 eggs and then maintained in a greenhouse with a

temperature range of 25-30 C. There were 5 replicates per

host plant. After 60 days, the plants were removed, the roots

washed and examined for galls. Roots were rated according to

the following scale modified from the S-76 Technical Committee

work plan (3).

0 = no gall

1 = 1 2 galls per plant

2 = 3 10 galls per plant

3 = 11 30 galls per plant

4 = 31 100 galls per plant

5 = greater than 100 galls per plant




6



Res 11ts


The results of the differential host tests are shown in

Table 2.

Galls were not observed on corn, cotton, pepper, straw-

berry, or either variety of sweetpotato when inoculated with

M. arenaria. Both M. incognita populations had the same host

range, although there were differences in degree of damage;

galls were not observed on corn, cotton, peanut, strawberry or

tobacco. Following inoculation of M. javanica, galls were not

observed on corn, cotton, peanut, pepper, strawberry, or 'Porto

Rico' sweetpotato.



Discussion


Host responses of these four root-knot nematode populations

differ from the standard reaction of Meloidogyne spp. compiled

by the Southern Regional Nematology Technical Committee (3).

The committee reported that corn and pepper were susceptible

hosts for M. arenaria, that corn was a susceptible host for

M. incognita, and that 'Porto Rico' sweet potato was a suscepti-

ble host for M. javanica. Galls were not observed in these com-

binations during this experiment. Nonetheless, the host dif-

ferential test confirmed that the populations were identified

correctly.





Table 2. Meloidogyne population test on host differentials.

Host Ratings*
Host Plants M. arenaria M. incognita (S) M. incognita (A) M. javanica

Corn
(Minnesota A 401) 0,0,0,0,0 0,0,0,0,0 0,0,0,0,0 1,2,1,2,2

Cotton 0,0,0,0,0 0,0,0,0,0 0,0,0,0,0 0,0,0,0,0
(Deltapine 16)

Peanut 4,5,4,4,3 0,0,0,0,0 0,0,0,0,0 0,0,0,0,0
(Florunner)

Pepper 0,0,0,0,0 2,2,2,2,2 4,4,4,3,3 0,0,0,0,0
(California Wonder)

Strawberry 0,0,0,0,0 0,0,0,0,0 0,0,0,0,0 0,0,0,0,0
(Albritton)

SweetDotato 0,0,0,0,0 3,4,4,3,4 5,3,4,4,5 4,3,3,3,3
(allgold)

Sweetpotato 0,0,0,0,0 3,3,4,4,4 5,4,4,5,4 0,0,0,0,0
(Porto Rico)

Tobacco 2,2,2,2,2 0,0,0,0,0 0,0,0,0,0 5,5,5,5,5
(NC-95)

Watermelon 4,5,4,5,4 5,5,5,5,5 5,4,5,5,5 5,5,5,5,5
(Charleston Grey)

Tomato 4,5,4,5,4 5,5,5,5,5 5,4,5,5,5 5 5,5,5,5
(Rutgers)
*Host ratings 2 = 3-10 galls per plant
0 = no gall 3 = 11-30 galls per plant
1 = 1-2 galls per plant 4 = 31-100 galls per plant
5 = greater than 100 galls per plant













CHAPTER II

DISC ELECTROPHORESIS

Introduction


Disc electrophoresis is a very sensitive analytical

method used to separate charged protein molecules. It has

proved to be an excellent analytical method for protein char-

acterization of animal or plant tissue. Not only can the gel

pore sizes be selected for optimal resolution (59), but also

one can optimize 'charge separation' (60). By operating at any

pH between 3 and 11, the maximal difference among the net charge

of molecules can be obtained (60). Disc electrophoresis can be

carried out at 0C as well as at higher temperatures and is,

therefore, applicable to enzymes (29, 56) and other thermolabile

molecules (55). Proteins at or below microgram quantities can

be detected by polyacrylamide gel electrophoresis (68).

Therefore, the technique is ideal for separating protein from

micro-organisms.

Electrophoretic separation of water soluble proteins may

have applicability in the future in the taxonomy of a variety

of micro-organisms, such as bacteria (31, 34, 37, 70), fungi

(10, 12, 24, 53, 80), protozoa (45), and nematodes (5, 21, 22,

26, 28, 41, 42, 43, 77).



8




9


Literature Review


The enzyme acetylcholinesterase, which hydrolyzes the

synaptic nerve impulse transmitter, acetylcholine, was reported

in Trichodorus christiei, Pratylenchus penetrans, Xiphinema

americanum, Dorylaimus sp., and Helicotylenchus nannus by

Rohde in 1960 (61). Although the enzyme was detected histo-

chemically in his study, it was the first study in which a

single protein was detected in plant parasitic nematodes.

Esterases were later detected and demonstrated histochemically

in free-living nematodes by Lee (46) and in Meloidogyne spp.

by Bird (7).

During 1966, specific and distinct phosphatases, esterases,

and protein patterns were demonstrated by disc electrophoresis

in Ditylenchus triformis and Panagrellus redivivus (4).

Protein compositions of Panagrellus silusiae, Aphelenchoides

fragariae and three rhabditid species were investigated by means

of agar electrophoresis by Gysels (35). Two glycoproteins and

one lipoprotein were detected in P. silusiae. Gysels also

emphasized the importance of the biochemical information in

addition to the traditional systematic methods for species

identification.

Chow and Pasternak (11) carried out their research on

protein changes during maturation of Panagrellus silusiae.

Although differences in the intensity of certain bands at

various larval stages and the adult were reported, no differences

in the number of protein pattern bands in acrylamide gel




10


eletrophoresis were observed. Specific enzyme activities were

different during different i ival stages; however, the authors

suggested that during maturation a precise regulator program

operates to control the sequential appearance of specific enzy-

matic proteins.

Erkisson and Granbery (26) reported a noticeable variation

in staining intensity of protein bands and several bands which

differ with regard to occurrence and position within four races

of Ditylenchus dipsaci. Dickson et al. (21) worked more exten-

sively on four species of Meloidogyne, two species of Ditylenchus,

and one species each of Heterodera and Aphelenchus. They found

characteristic electrophoretic protein patterns for each genus

and differences in number of bands within genera.

Ishibashi (43) reported different patterns of esterases

and acid phosphatases for Meloidogyne spp. isolated from dif-

ferent host plants or host plants growing under different con-

ditions. Evans (28) reported differences in esterase, amylase,

and acid phosphatase profiles among seven isolates of Aphelen-

chus avenae. Nevertheless, Dickson et al. (21, 22) found that

the enzyme patterns were stable in populations cultured on

several different hosts.

Trudgill and Carpenter (77) used disc electrophoresis to

identify protein patterns of six Heterodera spp. which corre-

lated with the shape of the cyst either rounded or lemon

shaped. The authors separated electrophoretically two groups

of 1. rostochiensis pathotypes and suggested that they comprised

two species (77). Later, Stone (72) described a second species,




11

H. pallida, from what had been considered H. rostochiensis.

Distinct electrophoret i differences in enzymes and water

soluble proteins of Ditylenchus dipsaci and D. triformis were

demonstrated by Hussey and Krusberg (41). Hussey et al. (42)

later reported differences of o -glycerophosphate dehydrogenase

patterns detected when Meloidogyne spp. were propagated on bean

rather than on tobacco or tomato.

Berge and Dalmasso (5) reported considerable intraspeci-

fic and interspecific heterogeneity among various populations

of Meloidogyne spp.; 19 hydrolytic enzymes were studied in his

work.



Materials and Methods


Large quantities of nematodes were obtained by rearing

on 'Rutgers' tomato afterinoculating seedlings approximately

15 cm tall with 15-20 egg masses. The seedlings were grown

in 15-cm diameter clay pots filled with Arredondo fine sand

previously fumigated with methyl bromide at the rate of 3 kg/m3

Plants were maintained for 45 days in a greenhouse at 25-300C.

All plants used in the experiments were fertilized weekly

with 250 ml of 2 percent Nutri-Sol solution (12-10-20) (Nutri-

Sol Chemical Company, Inc., Tampa, Florida 33614).

Adult females were recovered from infected roots by a

modification of the methods reported by Dropkin et al. (23),

Dickson et al. (21), and Hussey (38). Roots from 40 heavily

infected tomato plants were washed free of soil and chopped

into 2-3 cm sections. Approximately 150 grams of chopped roots




12

were softened in a 1000 ml Erlenmeyer flask by 250 ml 1:1

(v:v) water: pectinol 59-L <'.im and Haas Co., Philadelphia,

Pennsylvania) during 8 hours of agitation at 280 oscillations

per minute on a Burrell wrist-action shaker at room tempera-

ture. Then the softened roots were placed on nested 30-and

60-mesh sieves and adult females and egg masses of Meloidogyne

were dislodged from the roots by spraying with a high pressure

stream of water. Debris on the 30-mesh sieve was discarded.

Females, egg masses, and root tissue debris collected on the

60-mesh sieve were poured into beakers.

A 60-mesh sieve was placed in a pan filled with water and

50 ml of the above mixture poured into the sieve. The sieve

was raised to the surface of the water to allow all material

to float. Then the sieve was submerged slowly and most of the

females, egg masses, and root tissue debris floated. By blow-

ing gently on the water surface, most of the egg masses and

root tissue debris fell to the bottom while the females re-

mained floating. The females and other floating matter were

skimmed from the surface and were collected and emptied into

beakers. Repetition of this process cleaned the females from

other debris. Using this technique, 10 g of adult females

from 40 heavily infected plants could be obtained within 2 hours

after the roots were softened.

Relatively large quantities of nematode eggs were obtained

by the method of McClure et al. (51). Egg masses were treated

with one percent sodium hypochlorite, and eggs were collected

on a 450-mesh sieve. In most cases, approximately 6-8 g of




13

eggs could be obtained from 40 heavily infected tomato plants.

Eggs and females of Mci idogyne spp. were washed three

times in deionized water and suspended (1:4, V/V for eggs;

1:1.5, v/v for females) in iced 0.05 M potassium phosphate

(pH 7.4) buffer containing 0.15 M sodium chloride and 0.001 M

magnesium chloride (42). Eggs and females were homogenized

using a Sonifier cell disruptor Model W185 (Branson Sonic

Power Company, Danbury, Connecticut) for one minute, set at

maximum speed and equipped with a standard tip. Homogenized

tissues were extracted at 40C for 8 hours and then centrifuged

at 60,000 x 9 (rotor type 40, at 30k rpm, Beckman Model L-2)

at 40C for one hour. The clear supernatants were retained.

Supernate protein concentrations were determined fluorometri-

cally with 4-phenylspiro (furan-2(31H), l'-phthalan)-3,3' -dione

(fluorescamine) with bovine serum albumin as a standard (8).

Buffer solution was added to the protein samples to adjust the

concentration to 2 mg/ml.

Aliquots (100 mg) of adjusted samples were electrophoresed

immediately. Buffer soluble protein extracts that were used as

immunogens were stored at -75 C for future use.

Disc electrophoresis and selected staining techniques

were used to determine the various protein classes of females

and eggs of various Meloidogyne spp. Standard 7 percent poly-

acrylamide gel was used to analyze the electrophoretic profiles

of total protein, glycoprotein, and mucoprotein (19). The

total concentration (T%) of amide and the proportion of bis-

acrylamide (C%) were T% = 7.2 and C% = 2.4. Gels were prepared




14

in 5.5 mm i.d. glass tubes, with 6 cm separating gel and

1.5 cm of stacking gel. Fi' to one hundred 1l (100-200pg)

of prepared sample mixed with 40 percent sucrose solution

(3:1, v/v) were overlaid onto the top of the gel. Electro-

phoresis was conducted at 40C and a constant current of 3 ma/

tube applied until the tracking dye (bromophenol blue) migrated

to within one cm of the anodic end of the gel. The pH of the

stacking gel was 6.6-6.8, separating gel 8.8-9.0, Tris-glycine

electrode buffer 8.2-8.4, and the running pH was 9.5. For the

demonstration of the total protein profile, a minimum of ten

gels were removed from the glass tubes, fixed and stained in

0.2 percent coomassie blue, R-250, in a mixture of methyl

alcohol: deionized water: acetic acid (5:5:1) for 2 hours and

then destained by constant washing in the dye solvent.

Glycoprotein was stained by alcian blue (79), and mucoprotein

was stained by toluidine blue (58).

A minimum of five buffer soluble protein extracts from

Meloidogyne spp. were prestained by Sudan Black B in ethyl

acetate-propylene glycol and electrophoresed in 5.5 percent

gel (T% = 5.7, C% = 3.2) without stacking gel to obtain the

lipoprotein patterns (52). Photographs of lipoprotein patterns

were taken immediately after separation because the bands will

fade on standing.

All stained gels were scanned on Beckman spectrophoto-

meter ACTA Cll, Beckman scanner 2 at 280 nm and recorded on a

Beckman 10 inch recorder.




15

The relative mobility (Ef) values of each protein band

was calculated as the distance of band migration divided by

the distance of dye migration obtained by measurements on the

densitometer tracings of stained gels.

A protein band was considered similar among the various

populations if their Ef values were within a T 0.008 range

and showed similarity in staining intensity. To compare total

number of similar protein bands among the various populations,

the percent similarity was calculated as shown below (81).

% similarity no. of pairs of similar bands x
no. of different bands + no. of pairs of
similar bands

In this study, the term 'distinctive band' is used to

refer only to darkly stained well defined protein bands having

optic densities above 0.2. Those having an optical density

below 0.2 were not employed in calculating the percent simi-

larity. This does not imply that weakly stained protein bands

are not important in taxonomic relationships. The reasons

weakly stained protein bands were not employed for percent

similarity calculations were the inconsistent migration, dif-

fuse appearance and weak staining of these bands among the

populations.




16



Res ui I t s


Total protein

Adult females

All four populations studied had similar protein patterns

(Figure 1).

Among the four populations of Meloidogyne nematodes, 15-

21 protein bands were demonstrated by disc electrophoresis,

in which M. arenaria had 15, M. incognita (S) 21, M. incognita

(A) 19, and M. javanica 17. There were 8 distinctive bands in

M. arenaria and M. javanica and 9 distinctive bands in both

M. incognita populations. The Ef values (distance of protein

migration/distance of dye migration) of all the bands in adult

females are shown in Table 3.

Bands with Ef values of 0.481-0.490, 0.662-0.668, 0.681-

0.683, and 0.717-0.725 were common in all populations and were

considered to be distinctive bands. Bands with Ef value of

0.470-0.473 and 0.644-0.647 were common to both populations of

M. incognita. The percentage similarity of adult females

among species was calculated as described previously.

The percentage similarity of adult females among species

was a follows:

1. between M. arenaria and M. incognita (S)
5
(% similarity x 100 = 41.7%)


2. between M. arenaria and M. incognita (A)

(% similarity ----- x 100 = 41.7?;)
7 + 5




17





0009 1118

















L 1 1












Figure 1. Total protein patterns of coomassie blue stained
gels and corresponding densitometer tracings of stained gels
of adult females of Meloidogyne spp.







Table 3. Disc electrophoretic analyses of buffer soluble protein extracts of adult
females of Meloidogyne spp.

Ef Values*
Protein M. arenaria M. incognita (S) M. incognita (A) M. javancia

1 ------ 0.072 0.073 0.075
2 0.080 0.082 0.086 0.081
3 ------ 0.105 0.107 0.108
4 ------ 0.134** 0.136*-" 0.136**

5 0.148** ------ ------ 0.151**
6 ------ 0.167 ------ ------
7 ------ 0. 199" 0.202** 0.214**
8 0.270** 0.260 ------ ------

9 0.285** ------ 0.288** 0.285**
10 0.299* 0.305** ---------
11 ------ 0.347 0.346 --
12 0.379 0.380 0.377 0.378

13 0.407 ------ ------ 0.393
14 0.434 0.425 0.425 0.431
15 0.459 0.452 0.445 ------
16 ------ 0.473' 0.470** ------

17 0.490** 0.490** 0.487** 0.481**
18 ------ 0.523 0.517 0.522
19 0.555 0.555 0.555 0.559
20 0.601 0.604 0.605 0.607

21 ------ 0.647** 0.644** ------
22 0.668** 0.663** 0.662** 0.663**
23 0.683** 0.681** 0.681** 0.681**
24 0.725** 0.719** 0.717*"* 0.720**
Total Bands 15 21 19 17
Total Distinctive
Bands 8 9 9 8
* Average of 2 replicates
** Distinctive Bands




19

3. between M. arenaria and M. javanica


(% similarity = x 100 = 60')
4+6
4 + 6

4. between M. incognita (S) and M-. javanica


(% similarity = x 100 = 45.5%)
5 + 6

5. between M. incognita (A) and M. javanica


(% similarity = 3 x 100 = 30%)
37

The percentage similarity of adult females within M.

incognita was:


% similarity = x 100 = 80o
2+8





The protein profiles of eggs obtained from each nematode

population were more similar than those from the adult females

(Figure 2). Among the four populations of Meloidogyne nema-

todes, 17 or 18 protein bands were demonstrated by disc elec-

trophoresis, in which M. javanica had 17, and other populations

had 18. M. incognita populations had 10 distinctive hands,

M. arenaria and M. javanica had 9. The Ef values of all bands

in eggs are shown in Table 4.

The distinctive bands with the Ef values of 0.110-0.121,

0.220-0.228, 0.238-0.246, 0.369-0.375, 0.383-0.388, 0.490-0.495,

0.658-0.666, 0.686-0.691, and 0.731-0.742 were common in all





20





000E SIE















122E 129E





















Figure 2. Total protein patterns of coomassie blue
stained gels and corresponding densitometer tracings of
stained gels of eggs of Meloidogyne spp.





Table 4. Disc electrophoretic analyses of buffer soluble protein extracts of eggs of
Meloidogyne spp.


Ef Values*
Protein
Bands M. arenaria M. incognita (S) M. incognita (A) M. javancia


1 0.121** 0.119** 0.113** 0.110**
2 0.159 ---- -- -----
3 0.228** 0.220** 0.225** 0.222**
4 0.246** 0.243** 0.238** 0.242**

5 0.292 0.294 0.289 0.288
6 0.346 0.343 0.342 0.338
7 0.374** 0.369** 0.369** 0.375**
8 0.386** 0.386** 0.383** 0.388**

9 0.416 0.417 0.414 0.419
10 0.495** 0.492** 0.490** 9.491**
11 0.530 0.529 0.529 0.533
12 0.564 0.567 0.558 0.564

13 0.601 0.597 0.597 0.597
14 ----- 0.651** 0.646** -----
15 0.664** 0.666** 0.662** 0.658**
16 0.691** 0.689** 0.685** 0.686*w

17 0.738** 0.742** 0.731** 0.734**
18 0.779 0.771 0.772 0.778
19 0.852 0.850 0.845 0.846


Total Bands 18 18 18 17

Total Distinc-
tive Bands 9 10 10 9

* Average of 2 replicates
** Distinctive Bands




22



four populations; the band 0. 4(6-0.651 was the only different

distinctive band between the :i. incognita populations and other

species.

The percentage similarity within M. incognita populations

and between M. arenaria and M. javanica was:
9
% similarity x 100 = 100%
0+ 9

This signified no differences.

The percentage similarity between M. arenaria and M.

incognita (S), M. arenaria and M. incognita (A), M. incognita

populations and M. javanica was:

% similarity = 9 x 1
x 100 = 90%
1 + 9
1+9



Adult females and eggs

The four common bands in adult females were also common

in eggs. The band 0.644-0.647 in M. incognita females was

also demonstrated in eggs (0.646-0.651). The percentage

similarity between adult females and their eggs are shown

below:

M. arenaria

% similarity = 4 3
x 100 = 33.3%*
8 + 4




*Since the distinctive band 0.285-0.288 in adult females over-
lapped the indistinctive band 0.288-0.294 in the eggs, this
band was ignored in calculation.





23



M. incognita (S)

% similarity = 5
x 100 = 35.1%
9+5


M. incognita (A)

% similarity = 5 100 3
x 100 : 38.5%
8 + 5
8+5


M. javanica

% similarity = 4
x 100 = 33.3%
8 + 4
8+4


Glycoprotein

One glycoprotein band was observed in extracts of adult

females and eggs (Figure 3). In extracts of adult females

the Ef value of the band was 0.083-0.086 and of eggs 0.854-

0.860.


Mucoprotein

There was no detectable mucoprotein bands in adult fe-

male extracts, but there were two mucoprotein bands in the

egg extracts (Figure 4), with E values of 0.325-0.356 and

0.961-0.969.


Lipoprotein

Extracts of adult females and of eggs had one lipopro-

tein band, a widely diffuse band located at a similar posi-

tion in all populations studied (Figure 5).

The Ef values of glycoprotein, mucoprotein, and lipo-

protein are shown in Table 5.



































Figure 3. Diagrammatic representation of glycoprotein
patterns of Meloidogyne spp. adult females (above) and
eggs (below) stained with alcian blue. (a = M. arenaria,
iS = M. incognita (S), iA = M. incognita (A), and
j = javanica.





25













-0

.1

.2.

.3,

A

S.5
I.U

.6.

.7.

.8

.9

+ 1.0
a iS iA







-0



.2

.3

.4

u. .5

.6

.7

.8

.9

+1.0
a iS iA





26



















0-

.1.

.2

.3.

.4.



.6

.7.

.8.

.9

+1.0 -
a iS iA j









Figure 4. Diagrammatic representation of mucoprotein
patterns of eggs of Meloidogyne spp. stained with toluidine
blue. (a = M. arenaria, iS = M. incognita (S), iA = M.
incognita (A), j = M. javanica).

































Figure 5. Diagrammatic representation of lipoprotein
patterns of adult females (above) and eggs (below) of
Meloidogyne spp. stained with Sudan Black B.
(a = M. arenaria, iS = M. incognita (S), iA = M. incognita
(A), j = M. javanica).






28












.1

.2.

.3

.4




.6

.7

.8

.9


+1.0
a iS iA j











.1

.2

.3.

.4




.6.

.7

.8.

.9.



a iS iA







Table 5. Disc electrophoretic analyses of glycoprotein, mucoprotein and lipoprotein
from buffer soluble protein extracts of Meloidogyne spp.



Ef Values*

M. arenaria M. incognita (S) M. incognita (A) M. javanica


Glycoprotein

adul females 0.085 0.083 0.084 0.036

eggs 0.854 0.859 0.854 0.860


Muconrotein

adult females no bands no bands no bands no bands
observed observed observed observed

eggs 0.325 0.336 0.355 0.356
0.963 0.961 0.968 0.969


Lipcrrotein

adult females 0.446 0.527 0.509 0.450

eggs 0.442 0.469 0.470 0.455



*Average of 2 replicates


NJ
11




30



Discu;:- i on


Differences among the protein patterns of adult females

of the four populations studied show the potential of pro-

tein analyses as a complement to other methods presently

used to identify species of Meloidogyne. There were protein

bands which existed in only one population, bands that existed

only within species, and bands common in all populations stu-

died. As pointed out by Gysels (35), Trudgill and Carpenter

(77), Dickson et al. (21, 22), and Hussey (41, 42) the dif-

ferences in protein patterns may give useful information on

nematode systematics.

The protein patterns of eggs did not show as many dif-

ferences as those of the adult females, but some characteris-

tic distinctive bands were demonstrated in both adult females

and eggs.

One specific band was present only in M. incognita. It's

Ef value was 0.644-0.647 for adult females and 0.646-0.651 for

eggs. This band may prove to be unique to M. incognita.

Unlike Chow and Pasternak's (11) report of no differences

in number of bands in Panagrellus silusiae during maturation,

there were differences between adult females and eggs of

Meloidogyne spp. The protein patterns obtained from adult

females were specific for the species, whereas eggs of the

species did not show consistent differences.

Depending upon the host plants on which they have fed,

nematodes have different total protein patterns (21, 22, 27,




31



41, 42). Since a stable prolcin profile of a certain popula-

tion is important for reliabi use in identification, more

work on the differences of protein patterns due to the host

plants is necessary.

The glycoprotein and lipoprotein patterns show little pro-

mise for purposes of identification. Although different Ef

valuatof lipoproteins were obtained the difference may have

been due to experimental error since lipoprotein patterns

were represented by a widespread band in the middle of the gel,

and the Ef values were taken at the points with the highest

density.

The percentage similarity served as an index for comparing

the different protein patterns. In this study the reasons

for counting only the distinctive band were to improve the

reproducibility of protein patterns.

Due to the low concentration of certain proteins or vari-

ability of protein preparation, some bands were not demonstra-

ted in all replicates. Therefore, only those bands which were

darkly stained and consistently present were used in order to

give reproducible patterns of every population.

It is possible that some lightly stained bands may play

an important role in the protein profile characteristics or

even be related to the host plant. However, before an accep-

table and workable system of classification based on protein

patterns is established, researchers should focus on the

accessiable problems; important information on these lightly

stained bands may add to the system later.














CHAPTER III


SEROLOGY

Introduction


Serological techniques have been used not only in diag-

nostic clinics but also in identifying bacteria, fungi, viru-

ses, protozoa, and other microorganisms. Earlier serological

work on nematodes was concentrated on animal parasitic nematodes

(44, 48, 73, 74).

Molecular mimicries of metazoan parasites to their warm

blooded hosts have been known and studied for many years. In

the comprehensive review by Damian (18) the phenomenon of hosts

and parasites sharing common antigens was discussed. Studies

of common antigens between bacterial and fungal parasites and

their plant hosts have confirmed the relationship of patho-

types and serotypes (20, 66, 82). However, the serological

similarity in plant-animal relationships is still unknown.



Literature Review

Bird (6) in 1964 injected living larvae of M. javanica

into rabbits and demonstrated that antibodies were induced and

reacted with antigenic materials exuded from the nematode's

excretory pore and buccal cavity. The gelatinous matrix and

stylet tips of adult females were also antigenic. It must be


32




33



noted that all precipitation reactions took place on the sur-

face of the nematodes; the sources of the antigenic materials

within the nematodes were not studied. Lee (47) attempted to

use immunodiffusion for species identification of Meloidogyne

spp. by injecting freshly ground females into rabbits. This

was the first incidence of using homogenized tissues to in-

duce antibody formation to plant parasitic nematode antigens.

Immunodiffusion was used in this study to demonstrate the

antigen-antibody precipitation patterns.

Gibbins (30) reported that both qualitative and quan-

titative differences were found when antigens of various races

of Ditylenchus dipsaci were used against antisera of another

race. Cross-reaction was also obtained against Aphelenchoides

ritzemabosi. Therefore, the authors suggested that serology

may eventually enable biological races of D. dipsaci to be

distinguished and may lend itself to intergeneric studies.

Taxonomically related nematodes Panagrellus redivivus

and Diplogaster spp. were found serologically common and did

not have any specificity. No similar immunoprecipitin bands

were formed by either of the two against an unrelated nematode

Aphelenchus avenae (25). Three species of Ditylenchus with

five races of D. dipsaci and six species of Heterodera with

two pathotypes of H. rostochiensis were studied serologically

by Webster and Hooper (80). They reported that the precipita-

tion response divided the Heterodera spp. tested into two groups,




34



one containing H. schachtii, 1H. trifolii and H. rostochiensis

and the other group containiinj I. cruciferae, HI. carotae and

H. goettingiana. Apparently no antigens are common to both

groups. The three species of Ditvlenchi1s tested were sero-

logically distinct. No significant intraspecific differences

were found among races or pathotypes of Ditylenchus and

Heterodera.

Scott and Riggs (67) reported that two races of the soy-

bean cyst nematode were serologically identical and were

unrelated to the birch cyst nematode. The authors also re-

ported a greater number of precipitin bands separated by

immunoelectrophoresis than by immunodiffusion. Hussey (39,

40) studied two populations each of M. incognita and M.

arenaria. Based on position and coalescence, he found most

of the precipitin bands common to both species.

The two populations of M. incognita were serologically

identical. The two populations of M. arenaria differed from

each other with respect to one weak band. Later, Hussey et

al (42) reported thirteen immunoprecipitates developed with

M. incognita antiserum and twelve formed with M. arenaria anti-

serum in an immunodisc electrophoresis test. Most of the

immunoprecipitates were common.

Misaghi and McClure (54) studied the serological rela-

tionships of the egqs and larvae of M. incognita, M. javanica,

and M. arenaria. Close serological relationships among those

three species were found. However, some species-specific

antigens were also demonstrated and confirmed by cross-




35



absorption tests. The authors suggested that fluorescently

labeled antisera may provide a rapid identification of a

single specimen of a female root-knot nematode.

Only one report was found dealing with the serology of

plant-nematode relationships. McClure et al. (50) reported

cross reactions between M. incognita and two selected hosts,

cotton and soybeans. No evidence of the existence of a

common antigen was found.



Materials and Methods


Preparation of antisera

Prepared samples (0.5 ml, containing one mg protein)

were emulsified with an equal volume of Freud's complete

adjuvant. The immunogen was injected intramuscularly into

female New Zealand white rabbits (2-3 kg) at weekly intervals

for 4-5 weeks. Blood samples were obtained by cardiac punc-

ture after 7 weeks. Antibody titer was measured by serial

dilution of antigen. If antibody titer was low, one ml of the

prepared sample was injected directly into the rabbit's muscle

until the titer of the antisera was high, which usually took

five days. High antibody titer was determined when a precipi-

tin band was formed by diluting the antigen 1 to 2 with phos-

phate buffer saline (0.05 M, piH 7.4). Antisera were stored

in small quantities at -750C until needed.



Preparation of root extracts

The host plants used in the differential host test were




36



planted in 15-cm clay pots unlor a temperature range of 25-

300C. After 40 days, plant roots were taken down, washed

clean and rinsed in ice-cold buffer. The buffer contained

0.05 M potassium phosphate, 0.05 M sodium ascorbate, 0.001 M

magnesium chloride, pH 7.2. Roots were blended in ice-cold

buffer (1 g/3 ml) for two minutes at maximum speed and extrac-

ted for 12 hours at 4 C. The extracted root homogenate was

filtered through No. 1 Whatman filter paper. Filtrates con-

taining extracted soluble root proteins were stored at -750C

until needed.



Immunodiffusion and immunoelectrophoresis

Immunodiffusion was carried out in 0.5 percent agar gel

in 0.02 M sodium phosphate buffered saline, pH 7.2 (49). The

plates were developed at room temperature for 48 hours,

washed in one percent NaC1, dried and stained with Amidoblue

Black B and destained in a solution of acetic acid:water:methyl

alcohol (1:5:5) (49).

A one percent agarose gel in 0.02 M sodium barbital, HCI

buffer, pH 8.6, was used for immunoelectrophoresis (49). A

constant current of 3 ma/slide frame was applied for 80 minutes.

The incubation, staining and destaining were the same as for

immunodiffusion.



Results

Immunodiffusion

Adult females

Based on the position and coalescence, some of the pre-




37



cipitin bands were common in ;all populations studied. There

were differences in precipitin bands both between and within

species (Figure 6). The antisera of each population demon-

strated about the same precipitin patterns against antigens

of other populations. In Figure 6A, a specific band of

identity was demonstrated between M. arenaria antigen and M.

arenaria antiserum. No cross reactions of this antigen was

observed with any other population antisera. This is an anti-

body unique to M. arenaria only, and is immunologically able

to separate M. arenaria from M. incognita and M. javanica in

this study. Differences between species may also be observed

by comparing the precipitin patterns of Figures 6B, C, and D.

The differences within species are easily observed by compar-

ing the bands of identity in Figures 6B and C. M. incognita

(A) has one more distinctive band whereas no precipitin bands

occur for M. incognita (S). In Figure 6C, as pointed out by

the arrow, M. incognita (A) has one more precipitin band than

did M. incognita (S).



Eggs

The buffer soluble protein extracts of eggs did not in-

duce as many antibodies as did that of adult females (Figure 7).

However, there were immunoprecipitate bands occurring in the

egg precipitin pattern which were not exhibited in adult females.

When comparing the identity between M. incognita (A) and

M. incognita (S) in Figure 7B and between M. incognita (A)





38

















































Figure 6. Immunodiffusion patterns of different popula-
tions of adult females of Meloidogyne spp. Arrows in
figure 6A: differences between species of Meloidogyne
spp. in 6B,C differences within species of M. incognita.





39













































Figure 7. Immunodiffusion patterns of different popula-
tions of eggs of Meloidogyne spp. Arrows in figure 7A,
upper 7B, 7C, 7D: Differences between species of Meloidogyne
spp., in lower 7B: differences within species of M. incognita.





40



Adult females and eggs

There were significant serological differences between

adult female antigens and egg antigens of each population

against the antisera of adult females and eggs (Figures 8 and

9). The non-identity serological differences between adult

females and eggs within populations are also demonstrated in

Figures -8 and 9.



Immunological relationships of Meloidogyne species to

host plants

The root extracted protein antigens of ten host plants

against the antisera of all nematode populations showed no

serological relationship except for 'Porto Rico' sweetpotato.

This variety formed two immunoprecipitin bands (Figure 10).

The precipitin between 'Porto Rico' sweetpotato and

antisera occurred with adult females and with eggs of all

populations studied. It shows no relationship to the host

range of nematode populations, since only the two populations

of M. incognita reproduced on this host.



Immunoelectrophoresis

Adult females

Significant differences in immunoelectrophoresis patterns

existed both between and within species (Figure 11). In

Figure 11A five distinctive precipitin bands were separated

on M. arenaria compared with four on M. incognita (S);





41


















































Figure 8. Immunodiffusion patterns of antigens of adult
female of Meloidogyne spp. against their own antisera
and against antisera of their own eggs.





42


















































Figure 9. Immunodiffusion patterns of antigens of eggs
of Meloidogyne spp. against their own antisera and against
antisera of their own adult female.





43












































Figure 10. Immunodiffusion pattern of antiserum
of adult female M. arenaria against plant root
extracts.





































Figure 11. Diagrammatic representation of immunoeloectrophoretic
patterns of different populations of adult females of Meloidogyne
spp.




45






07 rT ... i < i7l 1



+ ,,-flW--- i----

TTt_ ; --...






| .. .. ..TI -- I i .


F,," t 0 'v1l ...I.' "


T i -: j 17 _ _0 \" -~ -I.




7 11Ti1-k I I I I













[^nj^J.J n^^'ni T "' --**** --







______________i.-L \'.::__ (___ __|

I 0 In I 1






-;; .........




46



similar results are shown in i'iqures 11B, C, E, and F. In

Figure 11D, a positive charged antibody induced by M.

incognita (S) was separated by the electric field and moved

toward the cathode of the microslide, thereby separating

the two populations of this species.





There were too few significant precipitin patterns in

eggs to separate species or populations within species (Figure

12). In this study only M. arenaria and M. incognita popula-

tions showed slight differences (Figures 12A and B).



Adult females and eggs

Significant immunoelectrophoretic differences between

the adult females and eggs are shown in Figure 13. In this

study, 4-5 precipitin bands were separated in adult females

whereas only 2-3 precipitin bands were separated in eggs.



Host relation

An obscure immunoprecipitin band was observed when the

root extract antigen of 'Porto Rico' sweetpotato was applied

against antisera of all adult females and eggs studied (Fig-

ure 14). This suggests that 'Porto Rico' sweetpotato has

structural proteins similar to those of the antigens of

nematodes.






























Figure 12. Diagrammatic representation of the immunoelectrophoretic
patterns of different populations of eggs of Meloidogyne spp.




48






F 0 O. n LI i I F
O ii. 'l'M i _ iii t -
. ....---'--'- '-"-"-'- ---~Y-O ,) ,,, i







A .--- 1 '1--.--



















BI I i
,I i it.I





-7W I i1. I71 I-rm I1

C II... -, I ..-. -T T---- .. -- --









----- o-- M i "'*'"'"' f *' '

_lL-Mil'' _'___________ ___ 1_1 "J
iiiz- 1- *i.





[ /~~,- ^ O ^'-in.ii.(



r -Z 7i 0" 15 1 6 i 'i4 i




49








r"r*r i' rrT~' '' '------------------- --i


M. arenaria


i i -n --- -. -- i





M. incoqnita(S)



i., 1 ,-____--- 3------
0'"'



M. incognita(A)






. .,.. i 1.. . .. .


M.iavanica




Figure 13. Diagrammatic representation of the immunoelec-
trophoretic patterns of adult females compared to eggs of
Meloidogyne spp.




5U




















4-I. 1< tlD t *.. -














Figure 14. Diagrammatic representation of the immuno-
electrophoretic patterns of antisera of adult females
of Meloidogyne spp. against root extracts of 'Porto
Rico' sweetpotato.




51



Discilr!; ion


Serological tests showed that all the populations are

closely related. However, there were more identity bands

within species than between species both in immunodiffusion

and immunoelectrophoresis. Serologically, the adult females

demonstrated more non-identity precipitin bands than did the

eggs; by means of these patterns all populations studied can

be distinguished. The intraspecific differences of two M.

incognita populations are distinct as are the interspecific

differences among M. arenaria, M. incognita, and M. javanica.

There is no serological evidence on the relationship of nema-

tode populations to their host range. The precipitin bands

on 'Porto Rico' sweetpotato give no significant information

in this study.

As pointed out before, identification of Meloidogyne spp.

by morphology and host range is still confusing and time con-

suming, and serological techniques may facilitate this identi-

fication by providing the necessary information. Modern tech-

niques such as fluorescently labeling antisera, purification

of antibodies, and antibodies induced by purified species-

specific antigens will make the serological information easier

to obtain and more likely to play an important role in the

identificatior of Meloidogyne spp.














CHAPTER IV


AFFINITY CHROMATOGRAPHY

Introduction


Research on practical methods and procedures for the

isolation and purification of antibodies from antisera have

been attempted for twenty years (1, 9, 13, 32, 36, 57, 69,

71, 82, 83). The approach was to produce an insoluble pro-

tein antigen matrix which would combine specifically with

antibodies to give a complex that could be dissociated into

soluble antibodies and insoluble antigens (9).

Different methods for insolubilization of biologically

active proteins have been reported. However, covalent bind-

ing of the protein to a suitable water insoluble carrier

exhibits the greatest flexibility (13, 17, 71). Agarose or

sepharose immunoadsorbents activated by cyanogen bromide have

been used successfully to purify antibodies (32, 36, 57, 83).

Antibodies have also been coupled to agarose or sepharose to

purify antigens (1, 13). The use of affinity chromatography

for purification of antigens and antibodies shows promise as

a useful isolation procedure (14, 15, 16).



52




53



Materials l' Methods


Affi-gel 10, a N-hydroxysuccinimide ester of succinylated

aminoalkyl agarose, (Bio Rad Laboratories, 32nd and Griffin

Avenue, Richmond, California 94804) was used in this study

for the purpose of covalent binding of the antigen.

Antigens were diluted in 0.1 M sodium bicarbonate buffer,

pH 7.4 (2 mg/10 ml) and coupled with 0.33 g affi-gel 10 for

12 hours at 4 C with slight agitation. The affi-gel 10-sodium

bicarbonate suspensions then were packed in columns (8mm x

10 cm). Columns were washed with 0.1 M potassium phosphate

buffer (pH 6.8) until no detectable proteins were observed

passing from the columns.

The protein contents of the antigens were determined

flurorometrically before coupling. All the sodium bicarbonate

buffer after coupling and potassium phosphate buffer washes

were retained for protein determination in order to calculate

the percentage of proteins coupled with the affi-gel 10.

Antisera were diluted with one percent NaCl solution

(1:1, v/v) and applied into the column coupled with their own

antigens with a flow rate of 0.2ml/min. The columns then were

washed with 0.1 M potassium phosphate buffer pH = 6.8, and

eluted with 1 M acetic acid or 3 M sodium thiocyanates (NaCNS)

in 0.1 M potassium phosphate buffer p1l 6.8. Those solutions

eluted with acetic acid were brought back to pH 7.0 by 0.1 M

NaOH before freeze drying and those eluted with 3 M sodium




54



thiocyanates were dialyzed inst the potassium phosphate

buffer. Both non-absorbed antisera and the desorbed elutions

were freeze dried and then dissolved in 0.1 M potassium phos-

phate buffer pH 6.8 to increase the concentration of the

antibodies. These then were tested in immunodiffusion plates.



Results


The coupling of the antigens to the affi-gel 10 was

high. The coupling ratios of the proteins in the samples to

the affi-gel 10 were 85.8-88.5 percent.

The results of the immunodiffusion showed that the anti-

bodies did not bind to their own antigens. All the precipitin

bands formed in the antisera passed through the column

directly. Neither acetic acid nor sodium thiocyanate elutions

formed any precipitin bands.



Discussion


The purpose for using affinity chromatography was to

obtain purified population-specific antibodies. For unknown

reasons, it did not work. However, this technique can be

refined and when larger amounts of antigens and antisera are

available, purified population-seecific or species-specific

antibodies may be obtained by further investigations.















CHAPTER V



CONCLUSIONS


The total protein patterns analyzed by disc electrophoresis

in this study show promise as biochemical techniques to pro-

vide information for identification of nematodes.

The total protein patterns of adult females of the four

populations proved sufficiently distinctive for identification;

those of eggs proved to be much less distinctive.

The protein patterns of glycoprotein, mucoprotein, and

lipoprotein did not provide information for identification pur-

poses.

The results of these studies showed that the antibodies

induced by the buffer soluble protein extracts could be used

to separate all the populations studied. Population-specific

antibodies were shown. Antibodies induced by buffer soluble

proteins of eggs could not be used to separate populations,

although some differences were demonstrated.

Within the four populations of Meloidogyne spp., total

protein patterns obtained by disc electrophoresis and sero-

logical reactions showed differences between the buffer solu-

ble protein extracts of adult females and eggs.

There were two precipitin bands in immunodiffusion be-

tween 'Porto Rico' sweetpotato antigens and the antisera of


55




56



the buffer soluble protein extr-acts of all populations. This

does not indicate a serological relationship between the

Meloidogyne spp. populations and their host range, since it

does not correlate to the host response to the nematode popu-

lations.

Techniques for obtaining quantities of living Meloidogyne

females were further refined in this study. Approximately

10gm of clean Meloidogyne adult females could be obtained

within 2 hours after the infected roots were soften by Pecti-

nol 59-L.















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BIOGRAPHICAL SKETCH


Franklin Hon-ching Chow was born January 20, 1945, at

Chen-do, China, mainland. He moved to Taiwan in 1949 and

attended public school and senior high school in Chia-I.

After graduation he attended National Taiwan University from

1964 to 1968 and received a Bachelor of Science Degree with

a major in Entomology.

After one year of military service, he worked as a

research assistant in the Department of Plant Pathology and

Entomology, National Taiwan University for one year.

From 1970 to 1972, he obtained his Master's Degree in

Nematology in the Department of Entomology and Nematology,

University of Florida, Gainesville, Florida. Since then he

has been countinuing his study in the Department of Entomology

and Nematology, University of FLorida, for his Ph.D. degree.
















64









I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly pre-
sentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.

/


Vernon G. Perry, Chairman
Professor, Entomology-Nematology



I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly pre-
sentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.




-ephaa G. Zam, q^oiairman
Assocm te Professor, Microbiology
and Cell Science



I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly pre-
sentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.




Donald W. Dickson, Associate
Professor, Entomology-Nematology



I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly pre-
sentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.




Grover C. Smart, Jr. Profesb'r
Entomology-Nematology












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.


June, 1977


De College of Ag ulture






Dean, Graduate School




Full Text

PAGE 1

COMPARATIVE DISC ELECTROPHORETIC PROTEIN ANALYSES AND SEROLOGICAL RELATIONSHIPS OF SELECTED SPECIES OF Meloidogyne AND SOME HOST PLANTS By FRANKLIN HON-CHING CHOW A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REOUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF. FLORIDA 1977

PAGE 2

ACKNOWLEDGEMENTS The author expresses his sincere appreciation to the chairman of his supervisory committee. Dr. Vernon G. Perry, for his generous assistance and enthusiastic encouragement throughout this study, and to the cochairman of his supervisory committee. Dr. S. G. Zam, for his advice during the periods of research. The author also expresses his gratitude to Dr. D. W. Dickson and Dr. G. C. Smart, Jr. for serving as members of the supervisory committee and for their valuable help. The technical assistance of Mr. R. W. Patrick is also gratefully acknowledged, and the typing of the dissertation by Ms. Inez Butler and Mrs. Grace Beal is especially appreciated. Finally, heartfelt thanks to my wife, Marjorie, for her patience and assistance during the long period of this study

PAGE 3

TABLE OF CONTENTS „ Page ACKNOWLEDGEMENTS ii LIST OF TABLES V LIST OF FIGURES vi ABSTRACT viii INTRODUCTION 1 CHAPTER I DIFFERENTIAL HOST TEST 4 Materials and Methods 5 Results 6 Discussion 6 CHAPTER II DISC ELECTROPHORESIS Introduction 8 Literature Review 9 Materials and Methods 11 Results 16 Discussion 30 CHAPTER III SEROLOGY Introduction 32 Literature Review 32 Materials and Methods 35 Results 36 Discussion 51 iii

PAGE 4

Table of Contents Cont'd. Page CHAPTER IV AFFINITY CHROMATOGRAPHY 52 Introduction 52 Materials and Methods 53 Results 54 Discussion 54 CHAPTER V CONCLUSIONS 55 LITERATURE CITED 57 BIOGRAPHICAL SKETCH 64 i V

PAGE 5

LIST OF TABLES Table Page 1. Species and populations of Meloidogyne 3 2. Meloidogyne population test on host differentials 7 3. Disc elect rophoretic analyses of buffer soluble protein extracts of adult females of Meloidogyne spp 18 4. Disc electrophoretic analyses of buffer soluble protein extracts of eggs of Meloidogyne spp 21 5. Disc electrophoretic analyses of glycoprotein, mucoprotein, and lipoprotein from buffer soluble protein extracts of Meloidogyne spp 29 V

PAGE 6

LIST OF FIGURES Figure 1. Total protein patterns of coomassie blue stained gels and corresponding densitometer tracings of stained gels of adult females of Meloidogyne spp 2. Total protein patterns of coomassie blue stained gels and corresponding densitometer tracings of stained gels of eggs of Meloidogyne spp 20 3. Diagrammatic representation of glycoprotein patterns of Meloidogyne spp. adult females and eggs stained with alcian blue 25 4. Diagrammatic representation of mucoprotein patterns of eggs of Meloidogyne spp. stained with toluidine blue 26 5. Diagrammatic representation of lipoprotein patterns of adult females and eggs of Meloidogyne spp. stained with Sudan Black B 28 6. Immunodiffusion patterns of different populations of adult females of Meloidogyne spp 38 7. Immunodiffusion patterns of different populations of eggs of Meloidogyne spp 39 8. Immunodiffusion patterns of antigens of adult females of fteloidogYI]^ spp. against their own antisera and against antisera of their own eggs 41 9. Immunodiffusion patterns of antigens of eggs of Mel oid ogyne spp. against their own antisera and against antisera of their own adult females 42 10. Immunodiffusion pattern of antiserum of adult female M. arenaria against plant root extracts .... 43 11. Diagrammatic representation of the immunoelectrophoretic patterns of different populations of adult females of Meloidogyne spp 45 Page 17 vi

PAGE 7

Figure Page 12. Diagrammatic representation of the immuneolectrophoretic patterns of different populations of eggs of Meloidogyne spp 43 13Diagrammatic representation of the inimunoelectrophoretic patterns of adult females compared to eggs of Meloidogyne spp 49 14. Diagrammatic representation of the immunoelectrophoretic patterns of antisera of adult females of Meloidogyne spp. against root extracts of 'Porto Rico' sweetpotato 50 vii

PAGE 8

Abstract of Dissertation Pro^v nted to the Graduate Council of the University of Florida > Partial Fulfillment of the Requirements for the Degroo of Doctor of Philosophy COMPARATIVE DISC ELECTROPHORETI C PROTEIN ANALYSES AND SEROLOGICAL RELATIONSHIPS OF SELECTED SPECIES OF Meloidogyne AND SOME HOST PLANTS By Franklin Hon-Ching Chow June, 1977 Chairman: Vernon G. Perry Cochairman: Stephen G. Zam Major Department: Entomology and Nematology Disc electrophoresis and serological techniques v/ere used to distinguish between Meloidogyne arenaria M. j avanica and two populations of M. incognit a. The buffer soluble protein extracts obtained from adult females and eggs of each of the populations were used in this study. Root extracts of selected host plants were serologically tested with the antibo dies of the nematodes to determine their relationships to the host range of the nematodes. Total protein, glycoprotein, mucoprotein, and lipoprotein contents of nematode populations were analyzed by disc electro phoresis. Total protein electrophoresed in 7 percent polyacrylamide gel stained by coomassie blue gave the best protein separation both between and v/ithin species. The glycoprotein, mucoprotein, and lipoprotein patterns where observed were identical in all populations studied. One glycoprotein band and one lipoprotein band were observed in extracts of both adult females and eggs; however, no mucoprotein band was vi i i

PAGE 9

demonstrated in adult female extracts, but two were shown in egg extracts. The four populations of Meloidogyne could be separated both by immunodiffusion and Immunoelectrophoresis. Several attempts to obtain purified adult female and egg populationspecific antibodies by affinity chromatography were unsuccess ful. Although there were immunoprecipitin bands of root extracts on 'Porto Rico' sweetpotato when applied against nematode antisera, no evidence of the relationship between the nematodes and their host range was shown by serological techniques ix

PAGE 10

INTRODUCTION The root-knot nematodes of the genus Meloidogyne are damaging parasites of most agricultural crops grown in the world.. Some 35 species were recognized by Esser et al (27). Certain of these species are similar in morphology and host responses so that identification has proven to be difficult even when attempted by experienced neroatologists The characters used for species differentiation are variable even from a population produced on an individual plant. Yet identification is of the utmost importance if integrated pest management procedures are to be successfully employed. At the present time, identification of the root-knot nematodes is based partly on the morphology of adult males, females, and second stage larvae, and some nematologists use host responses to parasitism as an aid to identification. Many researchers, however, have found that these criteria are oft en confusing and hence not satisfactory. Modern technology in cytology (75, 76), enzyme and protein analyses (21, 22, 42), and serology (39, 40, 42, 50, 54) has proved very helpful in identifying certain nematodes. Some of the above authors reported that techniques such as disc electrophoresis, immunodiffusion, and Immunoelectrophoresis offer substantial promise for identification of root-knot nematodes I

PAGE 11

2 The objectives of the research reported herein were to further determine if disc o ictrophoresis and serological techniques could be used as aids in identifying populations of Meloidogyne ; to determine if purification of species-specific antibodies by affinity chromotograph can be utilized and; to determine if relationships could be demonstrated between host ranges and serological reactions. In order to accomplish these objectives, one population each of Meloidogyne arenar ia (Neal) and M. javanica (Treub) and two populations of M. incognita (Kofoid and White) were selected for investigation (Table 1) All populations were derived from single egg mass isolates and were maintained on tomato plants, Lycopersicon esculentum Mill 'Rutgers' in a greenhouse.

PAGE 12

o o i-i CD G 4-) B c\3 o O CO Ph g CO 0 cfl c <-> o O 0 a -H 13 •r-\ m ^ c U 5-1 0 -u a ba O X CO 0) CO CO •rl Q •H u -a •H 0 >-i QJ >.23 r-l 0 1 1 X 0 a QJ Q) •n 4J r-l QJ CO r-l •rl iJ r-l CO 4-1 CJ a, •U c CI. 0 d QJ CJ 0 CO 0 ct3 22 CO 0 r-l 1 0) P-l X 0) QJ S-i 0 d (3 QJ <-) a QJ CO 0 cO 0) CO CO CO u r-l d 0 CO CO < CO C 0 CO • --1 0 0 00 CM CT d 22 0 r-l CM CM 0 r-l r-l r-l 0 CO < CO CO CO CO u 4-) CO QJ •rl rl H CJ •rl C C •rl CJ CO w W c QJ c 0 0 CO (X QJ 0 a > in Vj c CO CO •H •H •r-l :2i s

PAGE 13

CMPTER I DIFFERENTIAL HOST TEST Allen (2) in 1952 demonstrated the intraspecif ic variations of four populations of M. incognita acrita on 12 hosts. Sasser (62, 63) described a method to identify root-knot nematodes by host reaction and identified an unknown population by his methods. He emphasized, however, that some populations may not be identifiable by this method. During a two-year tobacco rotation research program Sasser and Nusbaum (65) reported the existence of strains or races within M. incognita acrita that differ in their ability to attack crops. Van der Linde (78) identified a root-knot nematode as M. incognita acrita by host responses despite morphological evidence which showed the nematode to be more similar to M. incognita Biotypes of root-knot nematodes were further demonstrated by Goplen et al, (33) within M. incognita acrita M. hapla and M. javanica in tests with alfalfa varieties. In my investigations each of the four root-knot nematode populations was identified by examination of 10 female perineal patterns and by morphometrical measurements of 15 larvae. Host differential tests were conducted in an attempt to confirm the identifications and also to seek possible relationships between host ranges and serological reactions. 4

PAGE 14

5 Materials and Methods Nine differential host plants were used to observe the host responses to different populatioiis of root-knot nematodes. They were corn, Zea mays L. 'Minnesota A 401' ; cotton, Gossypium hirsutum L. 'Deltapine 16' ; peanut, Arachis hypogaea L. 'Florunner' ; pepper, Capsicum f ruitescens L. 'California Wonder' ; strawberry, Fragar ia ananas sa Duch. 'Albritton' ; sweetpotato, Ipomoea batatas (L ) Lam., 'Allgold' and 'Porto Rico'; tobacco, Nicotiana tabacum L. 'NC-95'; and watermelon, Citrulus vulgaris Schard., 'Charleston Grey.' 'Rutgers' tomato was used as an indicator of inoculum potential. All plants were rooted before inoculation. During transplantation into a 15-cm clay pot, each plant was inoculated with 10,000 eggs and then maintained in a greenhouse with a temperature range of 25-30C. There were 5 replicates per host plant. After 60 days, the plants were removed, the roots washed and examined for galls. Roots were rated according to the following scale modified from the S-76 Technical Committee work plan (3) 0 = no gall 1 = 1 2 galls per plant 2 = 3-10 galls per plant 3 = 11-30 galls per plant 4 = 31 100 galls per plant 5 = greater than 100 galls per plant

PAGE 15

6 Res 111 ts The results of the differential host tests are shown in Table 2. Galls were not observed on corn, cotton, pepper, strawberry, or either variety of sweetpotato when inoculated with M. arenaria Both M. incognita populations had the same host range, although there were differences in degree of damage; galls were not observed on corn, cotton, peanut, strawberry or tobacco. Following inoculation of M. j avanica galls were not observed on corn, cotton, peanut, pepper, strawberry, or 'Porto Rico' sweetpotato. Discussion Host responses of these four root-knot nematode populations differ from the standard reaction of Meloidogyne spp. compiled by the Southern Regional Hematology Technical Committee (3) The committee reported that corn and pepper were susceptible hosts for M. arenaria that corn was a susceptible host for M. incognita and that 'Porto Rico' sweet potato was a susceptible host for M. javanica Galls were not observed in these combinations during this experiment. Nonetheless, the host differential test confirmed that the populations were identified correctly.

PAGE 16

CO b£ •r\ 4-J CTi Pi CO O O C > c\3 SI i-i o o W rA C o o SI o o o 1 — \ CO u 1 CN o o o o CO o r^ o o o o o LO UO LTl o o o o CO o in m in rH o o o o <)o m in in o o o CO o in H O cC !-i 4.J 4J q:; cu -U o o JD -H Dc o 4-1 &' 4J 4-1 cfl (U I— 1 cu u U r-t CU I— 1 cu o in ^ cu o c o O iJ r-l CO CO O ^-s cu cu U a in O 0) C M 4J w: cc3 1 cu cfl nj 4-1 4-J X. 6 d O S3 c^3 U O Pd H --^ |3 4-J 4-1 C C ct) C^ rH rH u cu a. CO CO rH rH rH rH Cd CTJ bO o O CO rH I I rH CO iH 4J C c^ rH a V4 CU p. 4J CO C rH Cl3 rH rH C\) !h O CU O CXrH CO C rH rH X bO >H O CU O 4-J rH CT3 I cu rH SH CO bO II II II II CM CO
PAGE 17

CHAPTER II DISC ELECTROPHORESIS Introduction Disc electrophoresis is a very sensitive analytical method used to separate charged protein molecules. It has proved to be an excellent analytical method for protein characterization of animal or plant tissue. Not only can the gel pore sizes be selected for optimal resolution (59), but also one can optimize 'charge separation' (60). By operating at any pH between 3 and 11, the maximal difference among the net charge of molecules can be obtained (60). Disc electrophoresis can be carried out at 0C as well as at higher temperatures and is, therefore, applicable to enzymes (29, 56) and other thermolabile molecules (55) Proteins at or below microgram quantities can be detected by polyacrylamide gel electrophoresis (68) Therefore, the technique is ideal for separating protein from micro-organisms Electrophoretic separation of water soluble proteins may have applicability in the future in the taxonomy of a variety of micro-organisms, such as bacteria (31, 34, 37, 70), fungi (10, 12, 24, 53, 80), protozoa (45), and nematodes (5, 21, 22, 26, 28, 41, 42. 43, 77). 8

PAGE 18

9 Literatur e Review The enzyme acetylcholinesterase, which hydrolyzes the synaptic nerve impulse transmitter, acetylcholine, was reported in Trichodorus christiei Pratylenchus penetrans Xiphinema americanum Dorylaimus sp. and Helicotylenchus nannus by Rohde in 1960 (61) Although the enzyme was detected histochemically in his study, it was the first study in which a single protein was detected in plant parasitic nematodes. Esterases were later detected and demonstrated histochemically in free-living nematodes by Lee (46) and in Meloidogyne spp by Bird (7) During 1966, specific and distinct phosphatases, esterases, and protein patterns were demonstrated by disc electrophoresis in Ditylenchus triformis and Panagrellus redivivus (4) Protein compositions of Panagrellus silusiae, Aphelenchoides fragariae and three rhabditid species were investigated by means of agar electrophoresis by Gysels (35). Two glycoproteins and one lipoprotein were detected in P. silusiae Gysels also emphasized the importance of the biochemical information in addition to the traditional systematic methods for species identification Chow and Pasternak (11) carried out their research on protein changes during maturation of Pa nagrellus s ilusiae Although differences in the intensity of certain bands at various larval stages and the adult were reported, no differences in the number of protein pattern bands in acrylamide gel

PAGE 19

10 eletrophoresis were observed. Specific enzyme activities were different during different I rval stages; however, the authors suggested that during maturation a precise regulator program operates to control the sequential appearance of specific enzymatic proteins. Erkisson and Granbery (26) reported a noticeable variation in staining intensity of protein bands and several bands which differ with regard to occurrence and position within four races of Ditylenchus dipsaci Dickson et al. (21) worked more extensively on four species of Meloidogyne two species of Ditylenchus and one species each of Heterodera and Aphelenchus They found characteristic electrophoretic protein patterns for each genus and differences in number of bands within genera. Ishibashi (43) reported different patterns of esterases and acid phosphatases for Meloidogyne spp. isolated from different host plants or host plants growing under different conditions. Evans (28) reported differences in esterase, amylase, and acid phosphatase profiles among seven isolates of Aphelen chus avenae Nevertheless, Dickson et al. (21, 22) found that the enzyme patterns were stable in populations cultured on several different hosts. Trudgill and Carpenter (77) used disc electrophoresis to identify protein patterns of six Heterodera spp. which correlated with the shape of the cyst either rounded or lemon shaped. The authors separated electrophoretically two groups of H. rostochiensis pathotypes and sugp.ested that they comprised two species (77). Later, Stone (72) described a second species,

PAGE 20

11 H. pallida from what had been considered H. rostochiensis Distinct electrophoret i. iifferences in enzymes and water soluble proteins of Ditylenchus dipsaci and D. trif ormis were demonstrated by Hussey and Krusberg (41). Hussey et_ al. (42) later reported differences of c< -glycerophosphate dehydrogenase patterns detected when Meloidogyne spp were propagated on bean rather than on tobacco or tomato. Berge and Dalmasso (5) reported considerable intraspecific and interspecific heterogeneity among various populations of Meloidogyne spp.; 19 hydrolytic enzjnnes were studied in his work Materials and Methods Large quantities of nematodes were obtained by rearing on 'Rutgers' tomato after inoculating seedlings approximately 15 cm tall with 15-20 egg masses. The seedlings were grown in 15-cm diameter clay pots filled with Arredondo fine sand previously fumigated with methyl bromide at the rate of 3 kg/m^ Plants were maintained for 45 days in a greenhouse at 25-30C. All plants used in the experiments were fertilized weekly with 250 ml of 2 percent Nutri-Sol solution (12-10-20) (NutriSol Chemical Company, Inc., Tampa, Florida 33614). Adult fem.ales were recovered from infected roots by a modification of the methods reported by Dropkin et_ al. (23), Dickson et al. (21), and Hussey (38). Roots from 40 heavily infected tomato plants were washed free of soil and chopped into 2-3 cm sections. Approximately 150 grams of chopped roots

PAGE 21

12 were softened in a 1000 ml Erlenmeyer flask by 250 ml 1:1 (v:v) water: pectinol 59-L H ,!im and Haas Co., Philadelphia, Pennsylvania) during 8 hours of agitation at 280 oscillations per minute on a Burrell wrist-action shaker at room temperature. Then the softened roots were placed on nested 30-and 60-mesh sieves and adult females and egg masses of Meloidogyne were dislodged from the roots by spraying with a high pressure stream of water. Debris on the 30-mesh sieve was discarded. Females, egg masses, and root tissue debris collected on the 60-mesh sieve were poured into beakers. A 60-mesh sieve was placed in a pan filled with water and 50 ml of the above mixture poured into the sieve. The sieve was raised to the surface of the water to allow all material to float. Then the sieve was submerged slowly and most of the females, egg masses, and root tissue debris floated. By blowing gently on the water surface, most of the egg masses and root tissue debris fell to the bottom while the females remained floating. The females and other floating matter were skimmed from the surface and were collected and emptied into beakers. Repetition of this process cleaned the females from other debris. Using this technique, 10 g of adult females from 40 heavily infected plants could be obtained within 2 hours after the roots were softened. Relatively large quantities of nematode eggs were obtained by the method of McClure et al. (51). Egg masses were treated with one percent sodium hypochlorite, and eggs were collected on a 450-mesh sieve. In most cases, approximately 6-8 g of

PAGE 22

13 eggs could be obtained from 40 heavily infected tomato plants. Eggs and females of Mc 1 1 i dogyne spp. were washed three times in deionized water and suspended (1:4, V/V for eggs; 1:1.5, v/v for females) in iced 0.05 M potassium phosphate (pH 7.4) buffer containing 0.15 M sodium chloride and 0.001 M magnesium chloride (42) Eggs and females were homogenized using a Sonifier cell disruptor Model W185 (Branson Sonic Power Company, Danbury, Connecticut) for one minute, set at maximum speed and equipped with a standard tip. Homogenized tissues were extracted at 4C for 8 hours and then centrifuged at 60,000 X 2 (rotor type 40, at 30k rpm, Beckman Model L-2) at 4 C for one hour. The clear supernatants were retained. Supernate protein concentrations were determined fluorometrically with 4-phenylspiro uran2 (3H) 1 -phthalan) -3 3 -dione (f luorescaraine) with bovine serum albumin as a standard (8) Buffer solution was added to the protein samples to adjust the concentration to 2 mg/ml. Aliquots (100 mg) of adjusted samples were electrophoresed immediately. Buffer soluble protein extracts that were used as iramunogens were stored at -75C for future use. Disc electrophoresis and selected staining techniques were used to determine the various protein classes of females and eggs of various Meloidogyne spp. Standard 7 percent polyacrylamide gel was used to analyze the electrophoretic profiles of total protein, glycoprotein, and mucoprotein (19). The total concentration (T%) of amide and the proportion of bisacrylamide (C7o) were T7c =7.2 and C7c = 2.4. Gels were prepared

PAGE 23

14 in 5.5 ram i.d. glass tubes, with 6 cm separating gel and 1.5 cm of stacking gel. Fi' to one hundred jil (100-200 pg) of prepared sample mixed with 40 percent sucrose solution (3:1, v/v) were overlaid onto the top of the gel. Electrophoresis was conducted at 4C and a constant current of 3 ma/ tube applied until the tracking dye (bromophenol blue) migrated to within one cm of the anodic end of the gel. The pH of the stacking gel was 6.6-6.8, separating gel 8.8-9.0, Tris-glycine electrode buffer 8.2-8.4, and the running pH was 9.5. For the demonstration of the total protein profile, a minimum of ten gels were removed from the glass tubes, fixed and stained in 0.2 percent coomassie blue, R-250, in a mixture of methyl alcohol: deionized water: acetic acid (5:5:1) for 2 hours and then destained by constant washing in the dye solvent. Glycoprotein was stained by alcian blue (79) and mucoprotein was stained by toluidine blue (58). A minimum of five buffer soluble protein extracts from Meloidogyne spp. were prestained by Sudan Black B in ethyl acetate-propylene glycol and electrophoresed in 5 5 percent gel (T7c, = 5.7, C7o = 3.2) without stacking gel to obtain the lipoprotein patterns (52). Photographs of lipoprotein patterns were taken immediately after separation because the bands will fade on standing. All stained gels were scanned on Beckman spectrophotometer ACTA Cll, Beclcman scanner 2 at 280 nm and recorded on a Beckman 10 inch recorder.

PAGE 24

15 The relative mobility (E^) values of each protein band was calculated as the distam o of band migration divided by the distance of dye migration obtained by measurements on the densitometer tracings of stained gels. A protein band was considered similar among the various populations if their values were within a 't 0.008 range and showed similarity in staining intensity. To compare total number of similar protein bands among the various populations, the percent similarity was calculated as shown below (81) 7 ^-^-T^^^-,-,, no. of pairs of similar bands ^ i /o similarity = ^ — t-^-j-z ^ i t ; s tt x i no. of different bands + no of pairs of similar bands In this study, the term 'distinctive band' is used to refer only to darkly stained well defined protein bands having optic densities above 0.2. Those having an optical density below 0.2 were not employed in calculating the percent similarity. This does not imply that weakly stained protein bands are not important in taxonomic relationships. The reasons weakly stained protein bands were not employed for percent similarity calculations were the inconsistent migration, diffuse appearance and weak staining of these bands among the populations

PAGE 25

16 Res 11 1 ts^ Total protein Adult females All four populations studied had similar protein patterns (Figure 1) Among the four populations of Meloidogyne nematodes, 1521 protein bands were demonstrated by disc electrophoresis, in which M. arenaria had 15, M. incognita (S) 21, M. incognita (A) 19, and M. javanica 17. There were 8 distinctive bands in M. arenaria and M. j avanica and 9 distinctive bands in both M. incognita populations. The values (distance of protein migration/distance of dye migration) of all the bands in adult females are shown in Table 3. Bands with values of 0.481-0.490, 0.662-0.668, 0.6810.683, and 0.717-0.725 were common in all populations and were considered to be distinctive bands. Bands with E^ value of 0.470-0.473 and 0.644-0.647 were common to both populations of M. incognita The percentage similarity of adult females among species was calculated as described previously. The percentage similarity of adult females among species was a follows: 1. between M. arenaria and M. incognita (S) (% similarity = ^ x 100 = 41.7%) 2. between M. arenaria and M. incognita (A) (% similarity = — ^ — x 100 = 41.7%) 7+5

PAGE 26

17 Figure 1. gels and of adult Total protein patterns of coomassie blue stained corresponding densitometer tracings of stained gels females of Meloidogyne spp.

PAGE 27

18 03 0) r-l CO > •H O C > •H C o o c •r4 CO CO •H O u SI CO •H CO c 0) CO c •H 0) 4J O u PL. in t— I cxD vo r-CO o ro O O n-l r-l o o o o -X -|C 1 1— 1 1 1 in 1 1 tO cn !— 1 in 1 rH 1 OO 1 CTi CO r-t 1 rsj 1 Ol \ 1 m o 1 O 1 1 O 1 1 1 o o o CO ^ 00 o ro O O rH t-M o o o o CM cNi in r-l r\i I o o o o 00 o o r-l c^j a> CO rsi in o <} in in o o o o ro r-l O vD 00 CNl \£) 00 I o o o J1-. "J" V J> _v j> 1 -Ic J' J' J' <* eg 1 CO 1 in in o in in o ro m r-^ CO c> rH i—l .— I CM r-on r— I CTi -d" CO r-l vD ^ <,o o o o o 1^ J' J' 1 >.(. -;< -J; o in >— 1 1 00 n in cr. in o 1 00 CM PQ •rH CM U 0) a LM > C O -H U) -rH 4-) 0) O C CO W) c CO -rl CO -H PQ Q M iJ cn -rA CO CO < a -U 4-1 CO O O C3Q I"* H H J>

PAGE 28

19 3. between M. arenar ia and M. j avanica (7o similarity = — — x 100 = 60%) 4 + 6 4. between M. incognita (S) and M. j avanica (7o similarity = _^ x 100 = 45.57o) 5 + 6 5. between M. incognita (A) and M. j avanica (% similarity = ^ — x 100 = 307o) 3 + 7 The percentage similarity of adult females within M. incognita was : 7o similarity = x 100 = 807o 2 + 8 Eggs The protein profiles of eggs obtained from each nematode population were more similar than those from the adult females (Figure 2) Among the four populations of Meloidogyne nematodes, 17 or 18 protein bands were demonstrated by disc electrophoresis, in which M. j avanica had 17, and other populations had 18. M. incognita populations had 10 distinctive bands, M. arenaria and M. j avanica had 9. The values of all bands in eggs are shown in Table 4. The distinctive bands with the values of 0.110-0.121, 0.220-0.228, 0.238-0.246, 0.369-0.375, 0.383-0.388, 0.490-0.495, 0.658-0.666, 0.686-0.691, and 0.731-0.742 were common in all

PAGE 29

20 Figure 2. Total protein patterns of coomassie blue stained gels and corresponding densitometer tracings of stained gels of eggs of Meloidogyne spp.

PAGE 30

21 > •H U C > •1—1 +j c Cn O U C (13 4J •H C tr o u c •H SI c •^^ Q) cn O C IX PQ -X K K X X K -X K k K •X +: •X •X X O og CO 00 in 00 rH 00 1.D 00 rH CM 00 00 n cri in 00 m rCN rsj 00 n m ri LO in in ya VD in o rH ^' rH CM m CM cn CO rH n IX' o VD cn rH .H CM CM CM n n in in >X) VD O o O O o c o o o o o o o O o X •X 00 cn CM m in r-r00 o o o rHCMrO'^ inVDr~0O (TiCrHCM m ^ in vc rH rH rH rH OC CTv rH rH rH cn 0) -p CO u •H rH cn CO CTi 0) c rH m CM 1 OJ o MH > c 0 -H Ui •H -P -p cn 0) o d tD C •H c n3 -H P3 Q fC3 ^ -P Q) VI rH rH > -H (C (T3 < Q -U 4J > o 0 H •X -x E-< X

PAGE 31

22 four populations; the band 0.' 46-0.651 was the only different distinctive band between the H. incognita populations and other species The percentage similarity within M. incognita populations and between M. arenaria and M. j avanica was : % similarity = q-|— g — ^ 100 = 100% This signified no differences. The percentage similarity between M. arenaria and M. incognita (S) M. arenaria and M. incognita (A) M. incognita populations and M. j avanica was : Adult females and eggs The four common bands in adult females were also common in eggs. The band 0.644-0.647 in M. incognita females was also demonstrated in eggs (0.646-0.651). The percentage similarity between adult females and their eggs are shown below : M. arenaria % similarity = 9 X 100 = 90% 1 + 9 % similarity = 4 X 100 = 33.3%* 8+4 *Since the distinctive band 0.285-0.288 in adult females overlapped the indistinctive band 0.288-0.294 in the eggs, this band was ignored in calculation.

PAGE 32

23 M. incognita (S) % similarity M. incognita (A) % similarity M. javanica % similarity Glycoprotein One glycoprotein band was observed in extracts of adult females and eggs (Figure 3) In extracts of adult females the value of the band was 0.083-0.086 and of eggs 0.8540.860. Mucoprotein There was no detectable mucoprotein bands in adult female extracts, but there were two mucoprotein bands in the egg extracts (Figure 4), with values of 0.325-0.356 and 0.961-0.969. Lipoprotein Extracts of adult females and of eggs had one lipoprotein band, a widely diffuse band located at a similar position in all populations studied (Figure 5) The Ej values of glycoprotein, mucoprotein, and lipoprotein are shown in Table 5. — X 100 = 35.1% 9 + 5 — — X 100 = 38.5% 8+5 — — X 100 = 33. 3% 8+4

PAGE 33

Figure 3. Diagrammatic representation of glycoprotein patterns of M eloidogyne spp adult females (above) and eggs (below) stained with alcian blue. (a = M. arenaria is = M. incognita (S) iA = M. incognita (A) an3 j = M. j avanica

PAGE 34

UJ .1 A. .5. .6. .7. .8. .9. 1.0. a 0 .1 .2 .3 .4 UJ -S. .6. .7. .8. .9 + 1-0.

PAGE 35

26 UJ 0.1. .2. .3. .4. .5. .6. .7. .8. .9. 1.0. iS iA Figure 4. Diagrammatic representation of mucoprotein patterns of eggs of Meloidoqyne spp stained with toluidine blue. (a = M. arenaria iS = M. incognita (S) iA = M. incognita (A) j = M. javanica )

PAGE 36

Figure 5. Diagrammatic representation of lipoprotein patterns of adult females (above) and eggs (below) of Meloidogyne spp stained with Sudan Black B. (a = M. arenaria iS = M. incognita (S) iA = M. incognita (A) j = M. javanica )

PAGE 37

0 .1 .2J .3. .4 **III 'J.6 .7. .9 + 1.0. iS iA

PAGE 38

K rfl W 4J 0) •H C rH CP 0 > u c H w SI (0 •H M (t G 0) 'M (C SI SI 03 P •H c o u c CO O O VD 00 03 ID O CO CO in o 00 tn T) -0 0) c > W O XI C 0 XI o d 0) > a) Xi o T! 0) > X! O X! C O in 00 1/1 C > o 00 S-i X! ai o o U3 0 XJ C 0 tn 0) (U r-l H 03 c e e H c QJ 0.' •H U-i 4-) 0) c 4J 4J •P u m 0 rH a. J D' ^-l :j 0 c 'a u (U 0 03 >i u i—i u s ID 1X> o o in 00 in on cr\ o o o o in n o o o in m in CTi O in o in ^ U3 (N c •rH O P O o o a n (U rH 03 g iw -U rH T) 03 CO CP

PAGE 39

30 Discu .sr, ion Differences among the protein patterns of adult females of the four populations studied show the potential of protein analyses as a complement to other methods presently used to identify species of Meloidogyne There were protein bands which existed in only one population, bands that existed only within species, and bands common in all populations studied. As pointed out by Gysels (35), Trudgill and Carpenter (77), Dickson et al. (21, 22), and Hussey (41, 42) the differences in protein patterns may give useful information on nematode systematics The protein patterns of eggs did not show as many differences as those of the adult females, but some characteristic distinctive bands were demonstrated in both adult females and eggs. One specific band was present only in M. incognita. It's value was 0.644-0.(^47 for adult females and 0.646-0.651 for eggs. This band may prove to be unique to M. incognita. Unlike Chow and Pasternak's (11) report of no differences in number of bands in Panagrellus silusiae during maturation, there were differences between adult females and eggs of Meloidogyne spp. The protein patterns obtained from adult females were specific for the species, whereas eggs of the species did not show consistent differences. Depending upon the host plants on which they have fed, nematodes have different total protein patterns (21, 22, 27,

PAGE 40

31 41, 42). Since a stable prolan n profile of a certain population is important for reliabl use in identification, more work on the differences of protein patterns due to the host plants is necessary. The glycoprotein and lipoprotein patterns show little promise for purposes of identification. Although different valupj^of lipoproteins were obtained the difference may have been due to experimental error since lipoprotein patterns were represented by a widespread band in the middle of the gel, and the values were taken at the points with the highest density. The percentage similarity served as an index for comparing the different protein patterns. In this study the reasons for counting only the distinctive band were to improve the reproducibility of protein patterns. Due to the low concentration of certain proteins or variability of protein preparation, some bands were not demonstrated in all replicates. Therefore, only those bands which were darkly stained and consistently present were used in order to give reproducible patterns of every population. It is possible that some lightly stained bands may play an important role in the protein profile characteristics or even be related to the host plant. However, before an acceptable and worlcable system of classification based on protein patterns is established, researchers should focus on the accessiable problems; important information on these lightly stained bands may add to the system later.

PAGE 41

CHAPTER III SEROLOGY Introduction Serological techniques have been used not only in diagnostic clinics but also in identifying bacteria, fungi, viruses, protozoa, and other microorganisms. Earlier serological work on nematodes was concentrated on animal parasitic nematodes (44, 48, 73, 74) Molecular mimicries of metazoan parasites to their warm blooded hosts have been known and studied for many years. In the comprehensive review by Damian (18) the phenomenon of hosts and parasites sharing common antigens was discussed. Studies of common antigens between bacterial and fungal parasites and their plant hosts have confirmed the relationship of pathotypes and serotypes (20, 66, 82). However, the serological similarity in plant-animal relationships is still unknown. Literature Review Bird (6) in 1964 injected living larvae of M. j avanica into rabbits and demonstrated that antibodies were induced and reacted with antigenic materials exuded from the nematode's excretory pore and buccal cavity. The gelatinous matrix and stylet tips of adult females were also antigenic. It must be 32

PAGE 42

33 noted that all precipitation r<>actions took place on the surface of the nematodes; the s(.)urces of the antigenic materials within the nematodes were not studied. Lee (47) attempted to use immunodiffusion for species identification of Meloidogyne spp. by injecting freshly ground females into rabbits. This was the first incidence of using homogenized tissues to induce antibody formation to plant parasitic nematode antigens. Immunodiffusion was used in this study to demonstrate the antigenantibody precipitation patterns. Gibbins (30) reported that both qualitative and quantitative differences were found when antigens of various races of Pity lenchus dipsaci were used against antisera of another race. Cross-reaction was also obtained against Aphelenchoides ritzemabosi Therefore, the authors suggested that serology may eventually enable biological races of D. dipsaci to be distinguished and may lend itself to intergeneric studies. Taxonomically related nematodes Pan agrellus redivivus and Diplogaster spp. were found serologically common and did not have any specificity. No similar immunoprecipitin bands were formed by either of the two against an unrelated nematode Aphelenchus avenae (25). Three species of Ditylenchus with five races of D. dipsaci and six species of Heterodera with two pathotypes of H. ros tochiensis were studied serologically by Webster and Hooper (80) They reported that the precipitation response divided the Hete rodera spp. tested into two groups,

PAGE 43

34 one containing H. schachtii H. tri foli i and H. ros tochiensis and the other group containinj H. cruciferae H. carotae and H. goettingiana Apparently no antigens are common to both groups. The three species of Ditvlenchu s tested were serologically distinct. No significant intraspecif ic differences were found among races or pathotypes of Pity lenchus and Heterodera Scott and Riggs (67) reported that two races of the soybean cyst nematode were serologically identical and were unrelated to the birch cyst nematode. The authors also reported a greater number of precipitin bands separated by Immunoelectrophoresis than by immunodiffusion. Hussey (39, 40) studied two populations each of M. incognita and M. arenaria Based on position and coalescence, he found most of the precipitin bands common to both species. The two populations of M. incognita were serologically identical. The two populations of M. arenar ia differed from each other with respect to one weak band. Later, Hussey et al_(42) reported thirteen immunoprecipitates developed with M. incognita antiserum and twelve formed with M. arenaria antiserum in an immunodisc electrophoresis test. Most of the immunoprecipitates were common. Misaghi and McClure (54) studied the serological relationships of the eggs and larvae of M. incognita M. j avanica and M. arenaria Close serological relationships among those three species were found. However, some species-specific antigens were also demonstrated and confirmed by cross-

PAGE 44

35 absorption tests. The authors suggested that f luorescently labeled antisera may provide a rapid identification of a single specimen of a female root-knot nematode. Only one report was found dealing with the serology of plant-nematode relationships. McClure et. al^. (50) reported cross reactions between M. incognita and two selected hosts, cotton and soybeans. No evidence of the existence of a common antigen was found. Materials and Methods Preparation of antisera Prepared samples (0.5 ml, containing one mg protein) were emulsified with an equal volume of Freud's complete adjuvant. The immunogen was injected intramuscularly into female New Zealand white rabbits (2-3 kg) at weekly intervals for 4-5 weeks. Blood samples were obtained by cardiac puncture after 7 weeks. Antibody titer was measured by serial dilution of antigen. If antibody titer was low, one ml of the prepared sample was injected directly into the rabbit's muscle until the titer of the antisera was high, which usually took five days. High antibody titer was determined when a precipitin band was formed by diluting the antigen 1 to 2 with phosphate buffer saline (0.05 M, pH 7.4). Antisera were stored in small quantities at -75C until, needed. Preparation of root extracts The host plants used in the differential host test were

PAGE 45

36 planted in 15-cni clay pots unrlor a temperature range of 2530C. After 40 days, plant roots were taken down, washed clean and rinsed in ice-cold buffer. The buffer contained 0.05 M potassium phosphate, 0.05 M sodium ascorbate, 0.001 M magnesium chloride, pH 7.2. Roots were blended in ice-cold buffer (1 g/3 ml) for two minutes at maximum speed and extracted for 12 hours at 4C. The extracted root homogenate was filtered through No. 1 Whatman filter paper. Filtrates containing extracted soluble root proteins were stored at -75C until needed. Immunodiffusion and Immunoelectrophoresis Immunodiffusion was carried out in 0.5 percent agar gel in 0.02 M sodium phosphate buffered saline, pU 7.2 (49). The plates were developed at room temperature for 4 8 hours, washed in one percent NaCl, dried and stained with Amidoblue Black B and destained in a solution of acetic acid :water :methyl alcohol (1:5:5) (49). A one percent agarose gel in 0.02 M sodium barbital, HCl buffer, pH 8.6, was used for Immunoelectrophoresis (49). A constant current of 3 ma/slide frame was applied for 80 minutes. The incubation, staining and destaining were the same as for imravinodif fusion Results Immunodiffusio n Adult females Based on the position and coalescence, some of the pre-

PAGE 46

37 cipitin bands were common in -HI populations studied. There were differences in precipitin bands both between and within species (Figure 6) The antisera of each population demonstrated about the same precipitin patterns against antigens of other populations. In Figure 6A, a specific band of identity was demonstrated between M. arenari a antigen and M. arenaria antiserum. No cross reactions of this antigen was observed with any other population antisera. This is an antibody unique to M. arenaria only, and is immunologically able to separate M. arenaria from M. incognita and M. j avanica in this study. Differences between species may also be observed by comparing the precipitin patterns of Figures 6B, C, and D. The differences within species are easily observed by comparing the bands of identity in Figures 6B and C. M. incognita (A) has one more distinctive band whereas no precipitin bands occur for M. incognita (S) In Figure 6C, as pointed out by the arrow, M. incognita (A) has one more precipitin band than did M. incognita (S) Eggs The buffer soluble protein extracts of eggs did not induce as many antibodies as did that of adult females (Figure 7) However, there were immunoprecipitate bands occurring in the egg precipitin pattern which were not exhibited in adult females. When comparing the identity between M. incognita (A) and M. incognita (S) in Figure 7B and between M. incognita (A)

PAGE 47

Figure 6. Immunodiffusion patterns of different populations of adult females of Meloidogyne spp. Arrows in figure 6A: differences between species of Meloidogyne spp. in 6B,C differences within species of M. incognita.

PAGE 48

39 \ Antl-M. arenaria Antl-M. J^nica itiiti-H. arenaria W M. arenaria Antl-M. incognita (A) Antl-H. Incognita (S) ^ Antl-M. arenaria Antl-H. Incognita(S) Antl-M. java|^a Antl-M. arenaria ^ ^M. incognita (S) Aati-N. incognita (A) Anti-M. incognita (S) Anti^H. incognita(S) Anti-N. incognita (A) Antl-H. javapfca A itl-M. arenaria M. inco^lta(A) Antl-M. Incoiinita(A) Anti-M. Incognita(S) Antl-N. Incognita (A) ^P||J||||||||P ...... / Antl-M. Uv&^t^ Anti-M. arenaria "-""i^M. Javanica Anti-M Incoqnita(A) Anti-H. incognita(S) Antl-M. iavanica v \ Figure 7. Immunodiffusion patterns of different populations of eggs of Meloidogyne spp. Arrows in figure 7A, upper IB, 7C, 7D: Differences between species of Meloidogyne spp., in lower 7B : differences within species of M. incognita

PAGE 49

40 Adult females and eggs There were significant serological differences between adult female antigens and egg antigens of each population against the antisera of adult females and eggs (Figures 8 and 9) The non-identity serological differences between adult females and eggs within populations are also demonstrated in Figures 8 and 9. Immunological relationships of Meloidogyne species to host plants The root extracted protein antigens of ten host plants against the antisera of all nematode populations showed no serological relationship except for 'Porto Rico' sweetpotato. This variety formed two immunoprecipi tin bands (Figure 10) The precipitin between 'Porto Rico' sweetpotato and antisera occurred with adult females and with eggs of all populations studied. It shows no relationship to the host range of nematode populations, since only the two populations of M. incognita reproduced on this host. Immunoelectrophoresis Adult females Significant differences in Immunoelectrophoresis patterns existed both between and within species (Figure 11) In Figure llA five distinctive precipitin bands were separated on M. arenaria compared with four on M. incognita (S) ;

PAGE 50

41 Figure 8. Immunodiffusion patterns of antigens of adult female of Meloidogyne spp. against their own antisera and against antisera of their own eggs.

PAGE 51

42 Figure 9, Immunodiffusion patterns of antigens of eggs of Meloidogyne spp. against their own antisera and against antisera of their own adult female.

PAGE 52

Cornj S.P.P.R. Cotton Anti a sCrAXJ. Peanut Figure 10. Immunodiffusion pattern of antiseriira of adult female M. arenaria against plant root extracts

PAGE 53

Figure 11. Diagrammatic representation of immunoeloect rophoretic patterns of different populations of adult females of Meloidogyne spp.

PAGE 54

45 + [ ~TVf i r V; — nrr-nrri-' A 1 1 1 i f i 1 I T r i-f-^ft-fH^' I Apt, i -M i iii;' jnTl .1 TaI B [ Ant, i -'T TTi' < 1 111 i r, i' (D AtiL i -ri Ill' •(i')ii i t .1 In] D -O r<^__yr I 1 II i t .1 ( (~) iTl'TlS' ( )'iM i I .1 ( 1 i I .1 r a n I i M — i iicoMn i In (. [ Ant i lL i.ivnn i yn E O M i n Ill i I n ( A)

PAGE 55

46 similar results are shown in I'iqures IIB, C, E, and F. In Figure llD, a positive charged antibody induced by M. incognita (S) was separated by the electric field and moved toward the cathode of the microslide, thereby separating the two populations of this species. Eggs There were too few significant precipitin patterns in eggs to separate species or populations within species (Figure 12) In this study only M. arenaria and M. incognita populations showed slight differences (Figures 12A and B) Adult females and eggs Significant immunoelectrophoretic differences between the adult females and eggs are shown in Figure 13. In this study, 4-5 precipitin bands were separated in adult females whereas only 2-3 precipitin bands were separated in eggs. Host relation An obscure immunoprecipitin band was observed when the root extract antigen of 'Porto Rico' sweetpotato was applied against antisera of all adult females and eggs studied (Figure 14). This suggests that 'Porto Rico' sweetpotato has structural proteins similar to those of the antigens of nematodes.

PAGE 56

Figure 12. Diagrammatic representation of the immunoelectrophoretic patterns of different populations of eggs of Meloidogyne spp.

PAGE 57

48

PAGE 58

VI. arena ria .Vr incognita (S) I'll! f.-in.ir yi incQgnita (A) All! I --,1 hiTt I < ill, i |T 1 ,.;|,, VI. iavanica Figure 13. Diagrammatic representation of the immunoelectrophoretic patterns of adult females compared to eggs of Meloidogyne spp.

PAGE 59

50 I AiiLi-n 1 1 Aiit, i -W i ncnMn i 1 a r7 1 Figure 14. Diagrammatic representation of the imraunoelectrophoretic patterns of antisera of adult females of Meloidoqyne spp. against root extracts of 'Porto Rico' sweetpotato.

PAGE 60

51 Discu py; ion Serological tests showed that all the populations are closely related. However, there were more identity bands within species than between species both in immunodiffusion and Immunoelectrophoresis. Serologically, the adult females demonstrated more non-identity precipitin bands than did the eggs; by means of these patterns all populations studied can be distinguished. The intraspeci f ic differences of two M. incognita populations are distinct as are the interspecific differences among M. arenaria M. incognita and M. javanica There is no serological evidence on the relationship of nematode populations to their host range. The precipitin bands on 'Porto Rico' sweetpotato give no significant information in this study. As pointed out before, identification of Meloidogyne spp. by morphology and host range is still confusing and time consuming, and serological techniques may facilitate this identification by providing the necessary information. Modern techniques such as f luorescently labeling antisera, purification of antibodies, and antibodies induced by purified speciesspecific antigens will make the serological information easier to obtain and m.ore likely to play an important role in the identification of Meloidogy ne spp.

PAGE 61

CHAPTER IV AFFINITY CHROMATOGRAPHY Introduction Research on practical methods and procedures for the isolation and purification of antibodies from antisera have been attempted for twenty years (1, 9, 13, 32, 36, 57, 69, 71, 82, 83). The approach was to produce an insoluble protein antigen matrix which would combine specifically with antibodies to give a complex that could be dissociated into soluble antibodies and insoluble antigens (9). Different methods for insolubilization of biologically active proteins have been reported. However, covalent binding of the protein to a suitable water insoluble carrier exhibits the greatest flexibility (13, 17, 71). Agarose or sepharose immunoadsorbents activated by cyanogen bromide have been used successfully to purify antibodies (32, 36, 57, 83). Antibodies have also been coupled to agarose or sepharose to purify antigens (1, 13) The use of affinity chromatography for purification of antigens and antibodies shows promise as a useful isolation procedure (14, 15, 16). 52

PAGE 62

53 Materials i Methods Affi-gel 10, a Nhydroxy succinimide ester of succinylated aitiinoalkyl agarose, (Bio Rad Laboratories, 32nd and Griffin Avenue, Richmond, California 94804) was used in this study for the purpose of covalent binding of the antigen. Antigens were diluted in 0.1 M sodium bicarbonate buffer, pH 7.4 (2 mg/10 ml) and coupled with 0.33 g affi-gel 10 for 12 hours at 4C with slight agitation. The affi-gel 10-sodium bicarbonate suspensions then were packed in columns (8ram x 10 cm). Columns were washed with 0.1 M potassium phosphate buffer (pH 6.8) until no detectable proteins were observed passing from the columns. The protein contents of the antigens were determined f lurorometrically before coupling. All the sodium bicarbonate buffer after coupling and potassium phosphate buffer washes were retained for protein determination in order to calculate the percentage of proteins coupled with the affi-gel 10. Antisera were diluted with one percent NaCl solution (1:1, v/v) and applied into the column coupled with their own antigens with a flow rate of 0.2ml/min. The columns then were washed with 0.1 M potassium phosphate buffer pH = 6.8, and eluted with 1 M acetic acid or 3 M sodium thiocyanates (NaCNS) in 0.1 M potassium phosphate buffer pll 6.8. Those solutions eluted with acetic acid were brought back to pH 7.0 by 0.1 M NaOII before freeze drying and those eluted with 3 M sodium

PAGE 63

54 thiocyanates were dialyzed : jinst the potassium phosphate buffer. Both non-absorbed antisera and the desorbed elutions were freeze dried and then dissolved in 0.1 M potassium phosphate buffer pH 6.8 to increase the concentration of the antibodies. These then were tested in immunodiffusion plates. Results The coupling of the antigens to the affi-gel 10 was high. The coupling ratios of the proteins in the samples to the affi-gel 10 were 85.8-88.5 percent. The results of the immunodiffusion showed that the antibodies did not bind to their own antigens. All the precipitin bands formed in the antisera passed through the column directly. Neither acetic acid nor sodium thiocyanate elutions formed any precipitin bands. Discussion The purpose for using affinity chromatography was to obtain purified population-specific antibodies. For unknown reasons, it did not work. However, this technique can be refined and when larger amounts of antigens and antisera are available, purified population-specific or species-specific antibodies may be obtained by further investigations.

PAGE 64

CHAPTER V CONCLUSIONS The total protein patterns analyzed by disc electrophoresis in this study show promise as biochemical techniques to provide information for identification of nematodes. The total protein patterns of adult females of the four populations proved sufficiently distinctive for identification; those of eggs proved to be much less distinctive. The protein patterns of glycoprotein, mucoprotein, and lipoprotein did not provide information for identification purposes. The results of these studies showed that the antibodies induced by the buffer soluble protein extracts could be used to separate all the populations studied. Population-specific antibodies were shown. Antibodies induced by buffer soluble proteins of eggs could not be used to separate populations, although some differences were demonstrated. Within the four populations of Meloidogyne spp., total protein patterns obtained by disc electrophoresis and serological reactions showed differences between the buffer soluble protein extracts of adult females and eggs. There were two precipitin bands in immunodiffusion between 'Porto Rico' sweetpotato antigens and the antisera of 55

PAGE 65

56 the buffer soluble protein ex' racts of all populations. This does not indicate a serological relationship between the Meloidoqyne spp. populations and their host range, since it does not correlate to the host response to the nematode populations Techniques for obtaining quantities of living Meloidogyne females were further refined in this study. Approximately lOgm of clean Meloidogyne adult females could be obtained within 2 hours after the infected roots were soften by Pectinol 59-L.

PAGE 66

LITERATURE CITED 1. Akanuraa, Y., T. Kuzuya, M. Hayashi T. Ide, and N. Kuzuya. 1970. Immunological reactivity of insulin to sepharose coupled with insulin-antibody--! ts use for the extraction of insulin from serum. Biochem. Biophys. Res. Commun. 38:947-953. 2. Allen, M.W. 1952, Observations of the genus Meloidogyne Goeldi. Proc. Helminthol. Soc. Wash. 19:44-51. 3. Anonymous. 1975. S-76 Work Plan for 1975. Southern Regional Nematology Technical Committee, Mimeographed report. 4. Benton, A.W. and R.F. Myers. 1966. Esterases, phosphatases, and protein patterns of Pit y le nchus trif ormis and Panagrellus redivivus Nematologica 12 : 495-500. 5. Berge, J.B., and A. Dalmasso. 1975. Caracteris tiques biochimiques de quelques populations de Meloidogyne hapla et Meloidogyne spp. Cah. ORSTOM, ser. Biol. 10:263-271. 6. Bird, A.F. 1964. Serological studies on the plant parasitic nematodes, Meloidogyne javanica Exp. Parasitol. 15: 350-360. 7. Bird, A.F. 1966. Esterases in the genus Meloidogyne Nematologica 12:359-361. 8. Bohlen, P., S. Stein, W. Dairman, and S. Udenfriend. 1973. Fluorometric assay of proteins in the Nanogram Range. Arch. Biochem. Biophys. 155:213-220. 9. Campbell, D.H., E. Luescher, and L.S. Lerman. 1951. Immunologic adsorbent. I. Isolation of antibody by means of a cellulose-protein antigen. Proc. Nat. Acad. Sci. USA. 37:575-578. 10. Chang, L.O., A.M. Srb, and F.C. Steward. 1962. Electrophoretic separations of the .soluble proteins of Neurospora. Nature 193:756-759. 57

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58 11. Chow, H.H. and J. Pasternak. 1969. Protein changes during maturation of the free-living nematode, Panagrellus silusia J. Exp. Zool. 170:77-84. 12. Clare, B.C. 1963. Starch-gel electrophoresis of proteins as an aid in identifving fungi. Nature 200:803-804. 13. Cuatrecasas, P. 1970. Protein purification by affinity chromatography. J. Biol. Chem. 245:3059-3065. 14. Cuatrecasas, P. 1971. Affinity chromatography. Ann. Rev. Biochem. 40:259-278. 15. Cuatrecasas, P. 1972. Affinity chromatography of macromolecules. Advan. Enzymol. 36:29-89. 16. Cuatrecasas, P. and C.B. Anfinsen. 1971. Affinity chromatography. Methods. Enzymol. 22:345-378. 17. Cuatrecasas, P. and I. Parikh. 1972. Adsorbents for affinity chromatography. Use of N-Hydroxysuccinimide esters of agarose. Biochem. 11:2291-2299. 18. Damian, R.T. 1964. Molecular mimicry; antigen sharing by parasite and host and its consequences. Amer. Natur. 98:129-149. 19. Davis, B.J. 1964. Disc electrophoresis. II. Method and application to human proteins. Ny.Y. Acad. Sci. 121:404-427. 20. Devay, J.E., R. Charudattan, and D.L.S. Wimalajeewa. 1972. Common antigenic determinants as a possible regulator of host-pathogen compatibilitv. Amer. Natur 106:185-194. 21. Dickson, D.W., J.N. Sasser, and D. Huisingh. 1970. Comparative disc-electrophoretic protein analyses of selected Meloidogyn e, Ditylenchus Heterodera and Aphelenchus spp. J. Nematol. 2:286-293. 22. Dickson, D.W., D. Huisingh, and J.N. Sasser. 1971. Dehydrogenases, acids and alkaline phosphatases and esterases for chemotaxonomy of selected Meloidogyne Ditylenchu s Heterodera and Aphelenchus, spp. J. Nematol. 3 : 1-16. 23. Dropkin, V.H., W.L. Smith, Jr., and R.F. Myers. 1960. Recovery of nematodes from infected roots by maceration Nematologica 5:285-288.

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59 24. Durbin, R.D. 1966. Comparative gel-electrophoretic investigation of tin !>rotein patterns of Septoria species. Nature 2 1 1 : 1186-1187 25. El-Sherif, M. and W.F. Mai. 1968. The use of immunodiffusion in nematode identification. Nematologica 14:593-595. 26. Eriksson, K.B. and J. Granbery. 1969. Studies of Dity lenchus dipsaci races using electrophoresis in acrylamide gel Nematologica 15:530-534. 27. Esser, R.P., V.G. Perry, andA.L. Taylor. 1976. A diagnostic compendium of the genus Meloidogyne (Nematoda: Heteroderidae) Proc. Helminthol. Soc. Wash. 43:138-150. 28. Evans, A.A.F. 1971. Taxonomic value of gel electrophoresis of protein from mycophagus and plant-parasitic nematodes. Int. J. Biochem. 2:72-79. 29. Gabriel, L. and S.F. Wang. 1969. Determination of enzymatic activity in polyacrylamide gels. I. Enzymes catalyzing the conversion of nonreducing substrates to reducing products. Anal. Biochem. 27:545-554. 30. Gibbins, L.N. 1968. An assessment of serological procedures for the differentiation of biological races of Ditylenchus dipsaci Nematologica 14:184-188. 31. Gill, H.S., M.N. Khare, and D. Powell. 1968. The use of polyacrylamide gel electrophoresis in the identification of Xanthomonas f ragari ae Kennedy and King. Trans. 111. State AcadT Sci. 6TT177-181. 32. Goetzl. E.J., and H. Metzger. 1970. Affinity labeling of a mouse myeloma protein which binds nitrophenyl ligands. Kinetics of labeling and isolation of a labeled peptide. Biochem. 9:1267-1278. 33. Goplen, B.P., E.H. Standord, and M.W. Allen. 1959. Demonstration of physiological races within three root-knot nem.atode species attacking alfalfa. Phytopathology 49:653-565. 34. Gottlieb, D. and P.M. Hepden. 1966. The electrophoretic movement of proteins from various Streptomyces species as a taxonomic criterion. J. Gen. Microbiol 44:95-104.

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60 35. Gysels, H. 1968. Electrophoretic observations on the protein composition free-living and plant-parasitic nematodes, with a sp'cial reference to some components showing a digestive activity. Nematologica 14:489-496. 36. Howe, C.W., R. Morisset, and W.W. Spink. 1970. Isolation of specific canine antibodies by affinity chromatography. Fed. Proc. 29:830 (Abstr.). 37. Huisingh, D. and R.D. Durbin, 1967. Physical and physiological methods for differentiation among Agrobacterium rhizogenes A. tumef aciens and A. radiobacter Phytopathology 57:922-923, 38. Hussey, R.S. 1971. A technique for obtaining quantities of living Meloidogyne females. J. Nematol. 3:99-100. 39. Hussey, R.S. 1971. Serological relationships in rootknot ( Meloidogyne spp.) nematodes. J. Nematol. 3:341. 40. Hussey, R.S. 1972. Serological relationships of Meloidogyne incognita and M. arenaria. J. Nematol 4:101-104: ~ 41. Hussey, R.S. and L.R. Krusberg. 1971. Disc-electrophoretic patterns of enzymes and soluble proteins of Ditylenchus dipsaci and D. triformis. J. Namatol 3:79-84. 42. Hussey, R.S., J.N. Sasser, and D. Hisingh. 1972. Discelectrophoretic studies of soluble proteins and enzymes of Meloidogyne_ incognita snd M. arenaria. J. Nematol 4: 183-189, 43. Ishibashi, N. 1970. Variations of the electrophoretic protein patterns of Heteroderidae (Nematoda : Tylenchida) depending on the developmental stages of the nematode and on the growing conditions of the host plants Appl. Entomol. Zool. 5:23-32. 44. Jackson, G.J. 1959. Fluorescent antibody studies of T richinella spiralis infections. J. Infectious Dis 105:97-117. 45. Kates, J.R. and L, Goldstein. 1964. A comparison of the protein composition of three species of amoebae J. Protozool. 11:30-35. 46. Lee, D.L. 1964. Esterase enzvmes in two free-living nematodes. Proc. Helminthol" Soc Wash. 31:285-288. 47. Lee, S.H. 1965. Attempts to use immunodif fusing for species identification of Meloidogyne Nematologica 11:41 (Abst.). ^

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61 48. Lee, C.L. and R.M. Lewcrt. 1960. The distribution of various reactants iti human antiSchis tosoma mansoni serums fractionated starch electrophoresis. J. Infectious Dis. 10*' 69-76. 49. Maclnnis A.J. and II. Voge. 1970. Experiments and techniques in parasitology. W.H. Freeman and Company, pp. 232. 50. McClure, H.A., I. Misaghi, and E.L. Nigh. 1973. Shared antigens of parasitic nematodes and host plants. Nature 244:306-307, 51. McClure, M.A., T.H. Kurk and I. Misaghi. 1973. A method for obtaining quantities of clean Meloidogyne eggs. J. Nematol. 5:230, 52. McDonald, H.J. and J.Q. Kissane. 1960. Sudan black B in ethyl acetate-propylene glycol for prestaining lipoproteins. Anal. Biochem. 2:178-179. 53. Meyer, J. A. and J.L. Renard. 1969. Protein and esterase patterns of two formae speciales of Fusarium oxy sporum. Phytopathology 59:1409-1411. 54. Misaghi, I. and M.A. McClure. 1974. Antigenic and relationship of Meloidogyne incognita M. j avanica and M. arenaria PhytopathologyT" 64 : 698-701 55. Nikolai, T.F. and U.S. Seal. 1966. X-chromosome linked familial decrease in thyroxine-binding globulin activity. J. Clin. Endocrinol. Metab. 26:835-841. 56. Nishihara, M. A. Chrambach, and H.V. Aposhian. 1967. The deoxycy tidy late deaminase found in Bacillus subtilis infected with phage SP8 BiochenT 5T1877-1886. 57. Parker, D.C. and C. Briles. 1970. Fractionation of antibodies from group A antisera by affinity chromatography. Fed. Proc, 29:438 (Abstr.). 58. Rennert, O.M. 1967. Disc electrophoresis of acid mucopolysaccharides. Nature 213:1133. 59. Rodbard, D. and A. Chrambach. 1970. Unified theory for gelelectrophoresis and gel filtration. Proc. Nat Acad. Sci. U.S. 65:970-977. 60. Rodbard, D. and A. Chrambach. 1971. Estimation of molecular radius, free mobility, and valence using polyacrylamide gel electrophoresis. Anal. Biochem. 40:95-134.

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62 61. Rohde, R.A. 1960. Aceiylcholinesterase in plantparasitic nematodes -ul an anticholinesterase from asparagus. Proc. 11: ; i.nthol Soc Wash, 27:121-123. 62. Sasser, J.N. 1952, Identification of root-knot nematodes ( Meloidogyne spp.) by host reaction. Plant Dis. Rep. 36:84-86. ^ 63. Sasser, H.N. 1954. Identification and host-parasite relationships of certain root-knot nematodes ( Meloidogyn e spp.). Univ. Md Agr. Exp. Sta. Bull. A-77, 30 p. 64. Sasser, J.N. 1966. Behavior of Meloidogyne spp. from various geographical locations on ten host differentials Nematologica 12:97-98, 65. Sasser, J.N. and C.J. Nusbaum. 1955. Seasonal fluctuations and host specificity of root-knot nematode populations in two-year tobacco rotation plots Phytopathology 45:540-545. 66. Schnathorst. U.C. and J.E. DeVay. 1963. Common antigens in Xanthumoua s malvacearum and G ossypium hirsutum and their possible relationship to host specificity and disease resistance. Phytopathology 53:1142 (Abstr.). 67. Scott, H.A. and R.D. Riggs. 1971. Immunoelectrophoretic comparisons of three plant-parasitic nematodes Phytopathology 61:751-752. 68. Shapire, A.L. and J.V. Maizel, Jr. 1969. Molecular weight estimation of polypeptides by SDS-polyacrylamide gel electrophoresis. Anal. Biochem. 29:505-514. 69. Silman, I.H. and E. Katchalski. 1966. Water-insoluble derivatives of enzymes, antigens and antibodies. Ann Rev. Biochem. 35:873-908. 70. Smith, J.H. and D. Powell. 1968. A disc electrophoretic comparison of protein patterns of Erwinia amyloyora with other bacteria, including associated yellow fo'rms Phytopathology 58:972-975. 71. Steers, E. Jr., P. Cuatrecasas andH.B. Pollard 1971 The purification of o^-galactosldase from Escherichia' ^^in> nii^""^^^ chromatography. J. Biol-'ChiitT 246 : 196-200 72. Stone, A R. 1972. H£ter?djera pallida n sp (Nematoda: Heteroderidae) a second speciel-^ potato cyst nematode. Nematologica 18:591-606. f j

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63 73. Taffs, L.F. 1^61. Tbr in vitro iicf:ion of immune pig serum on ^3ccoud and Miird stage Ascaris suura larvae. Parasitology 51:32 74. Taffs, L.F. and A. Voller. 1962. Fluorescent antibody studies in vitro on Ascaris suum Goeze, 1782. J. Helminthol. 36:339-3567 75. Triantaphyllou A.C. 1973. Gametogenesis and reproduction of Meluidugyae graroinis and M. ottersoni (Nematoda: Heteroderidae). J. Nematol. 5:84-87. 76. Triantaphyllou, A.C. and J.M. Fisher. 1976. Gametogenesis • in Amphimictic and parthenogenetic populations of Aphelenchus avenae J. Nematol. 8:168-177. 77. Trudgill, D.L. and J.M. Carpenter. 1971. Disk electrophoresis of proteins of Heterodera species and pathotypes of Heteroder a rostochiensis Ann. Appl. Biol. 69:35-41. 78. Van der Linde, W.J. 1956. The M eloidogyne problem in Africa. Nematologica 1:177-1^3"^ 79. Wardi. A.H. and G.A. Michos. 1972. Alcian blue straining of glycoproteins in acrylamide disc electrophoresis. Anal. Biochem. 49:607-609. 80. Webster, J.M. and D.J. Hooper. 1968. Serological and morphological studies on the interand intraspecif ic differences of the plant-parasitic nematodes Heterodera and Ditylenchus Parasitology. 58:879-891. 81. Whitney, P.J., J.G. Vaughan and J.B. Heale. 1968. A disc electrophoretic study of the proteins of Verticilli um albo-atrum V erticillium dahliae and Fusarium oxyspo rum with respect to their taxonomy J. Exp. Bot 19:415-426. 82. Wimalajeewa, D.L.S. and J.E. DeVay. 1971. The occurrence and characterization of a common antigen relationship between Ustilago maydis and Zea mays. Physiol Plant Pathol. 1:523-5357^^ 83. Wofsy, L. and B. Burr. 1969. The use of affinity chromatography for the specific purification of antibodies and antigens. J. Immunol. 103:380-382.

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BIOGRAPHICAL SKETCH Franklin Hon-ching Chow was born January 20, 1945, at Chen-do, China, mainland. He moved to Taiwan in 1949 and attended public school and senior high school in Chia-I After graduation he attended National Taiwan University from 1964 to 1968 and received a Bachelor of Science Degree with a major in Entomology. After one year of military service, he worked as a research assistant in the Department of Plant Pathology and Entomology, National Taiwan University for one year. From 1970 to 1972, he obtained his Master's Degree in Nematology in the Department of Entomology and Neraatology, University of Florida, Gainesville, Florida, Since then he has been countinuing his study in the Department of Entomology and Nematology, University of FLorida, for his Ph.D. degree. 64

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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. ;'ernon G. Perry, Chai:^an Professor, Entomology-Nematology 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. -.ephdd G. Zam, qD/nairman AssocMte Professor, Microbiology and Cell Science 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. Donald W. Dickson Associate Professor, Entomology-Nematology 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. Grover C. Smart, 'Jr. P Entomology-Nematology or

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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. June, 1977 Dean, Graduate School