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Parasitism of Pratylenchus spp. to Lovell, Nemaguard, and Okinawa peach

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Title:
Parasitism of Pratylenchus spp. to Lovell, Nemaguard, and Okinawa peach
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Stokes, Donald Eugene, 1931-
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63 leaves : ill. ; 28 cm.

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Cells ( jstor )
Inoculum ( jstor )
Nematology ( jstor )
Peaches ( jstor )
Photomicrographs ( jstor )
Plant roots ( jstor )
Rootstocks ( jstor )
Roundworms ( jstor )
Soils ( jstor )
Species ( jstor )
Dissertations, Academic -- Entomology and Nematology -- UF
Entomology and Nematology thesis Ph. D
Peach ( lcsh )
Pratylenchus ( lcsh )
City of Gainesville ( local )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1972.
Bibliography:
Includes bibliographical references (leaves 59-62).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Donald Eugene Stokes.

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Parasitism of Pratylenchus spp. to
'Lovell,' 'Nemaguard' and 'Okinawa' Peach














By

DONALD EUGENE STOKES


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 1972













ACKNOWLEDGMENTS

The author wishes to express his sincerest appreciation to his supervisory committee for guidance and criticism; Dr. Vernon G. Perry, Chairman; Dr. Grover C. Smart, Jr.; and Dr. Daniel A. Roberts. Special recognition to Dr. Perry for untold assistance is hereby extended.

The author expresses his gratitude to Mr. Halwin L. Jones,

Director, Division of Plant Industry, Florida Department of Agriculture and Consumer Services, and the late Dr. William G. Cowperthwaite, former Division of Plant Industry Director, for their assistance and encouragement concerning graduate studies. Kind appreciation is extended to the Honorable Doyle Conner, Florida Commissioner of Agriculture and Consumer Services.

Acknowledgment is also extended to Dr. William G. Eden, Chairman of the Department of Entomology and Nematology, for his assistance and counsel.

The author wishes to thank Dr. Robert H. Biggs, Professor, Fruit Crops Department, University of Florida, for providing plant materials.

Special thanks to Mr. Jack 0. Holmes and other members of the Hillsborough County Florida Nurseryman and Grower's Association for providing timely financial aid.

Thanks also go to Mr. E. M. Collins, Jr., for photographic assistance.

To Norma Donovan, typist and friend, the author is indebted for prompt and efficient handling of this manuscript.

Affectionate gratitude is extended to his wife, Grace, and their children for sacrifice and patience.
















TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS .............................................. ii

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

LIST OF FIGURES.................................................. vii

ABSTRACT ..................................................... ix

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

LITERATURE REVIEW............................................... 4

MATERIALS AND METHODS............................................ 13

Soil and Inoculum Sources..................................... 13

General Methods................................................ 13

Identification of Nematode Species............................ 14

Experiment I ............................................. 15

Experiment II................................................ 17

Experiment III............................................... 18

Histopathology of Peach Roots Infected With Pratylenchus
brachyurus ................................................. 19

RESULTS AND DISCUSSION........................................... 20

Experiment I ............................................. 20

Experiment II................................................ 22

Experiment III............................................... 22

Histopathology of Peach Roots Infected With Pratylenchus
brachyurus ................................................. 34





iii











Page

Roots Stained With Safranin-Fast Green....................... 43

Study of Pratylenchus brachyurus From Experimental Plants.... 45

Study of Pratylenchus penetrans and Pratylenchus coffeae
From Experiment I............................................ 47

Fungus Associated With Nematode Colony....................... 47

Stylet Knob Variation in a Colony of Pratylenchus
brachyurns ................................................... 48

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

SUMMARY........................................................ 57

LITERATURE CITED............................................... 59

BIOGRAPHICAL SKETCH............................................ 63














LIST OF TABLES


Table Page

1. A host list of Pratylenchus brachyurus in Florida

compiled from the unpublished files of the Nematology

Bureau, Florida Department of Agriculture and Consumer Services, Gainesville, Florida....,............. 5

2. A host list of Pratylenchus coffeae in Florida compiled from the unpublished files of the Nematology

Bureau, Florida Department of Agriculture and

Consumer Services, Gainesville, Florida.............. 9

3. A host list of Pratylenchus penetrans in Florida

compiled from the unpublished files of the Nematology

Bureau, Florida Department of Agriculture and Consumer Services, Gainesville, Florida.................. 10

4. Numbers of Pratylenchus brachyurus, P. penetrans,

and P. coffeae recovered from the roots of three

peach cultivars used as rootstocks inoculated with

50 nematodes per six-inch pot......................... 21

5. Dry weights of 'Lovell,' 'Okinawa,' and 'Nemaguard' peach seedlings and number of nematodes recovered

when uninoculated and inoculated with 100 Pratylenchus brachyurus and grown for 17 weeks in methyl

bromide-treated soil.................................. 24










Table Page

6. Analysis of variance................................... 27

7. Dry weights of 'Lovell' and 'Okinawa' peach seedlings

and numbers of nematodes recovered when uninoculated

and inoculated with Pratylenchus brachyurus and

grown for 17 weeks in methyl bromide-treated soil.... 32 8. Analysis of variance................................... 40















LIST OF FIGURES


Figure Page

1. Photograph of young 'Nemaguard' seedlings growing in

vermiculite........................................... 16

2. Photograph of 'Okinawa' peaches: plant at right

inoculated with 1,000 as compared with uninoculated

plant................................................. 23

3. Photomicrograph of a transverse section of 'Okinawa' peach root from uninoculated plant.................... 35

4. A-B. Photomicrograph of a transverse section of 'Nemaguard' peach root infected by Pratylenchus

brachyurus.......................................... . 37

5. A-B. Photomicrograph of a transverse section of 'Okinawa'

peach root infected with Pratylenchus brachyurus...... 39

6. Photomicrograph of a longitudinal section of 'Lovell' peach

root infected with Pratylenchus brachyurus............ 44

7. Photomicrograph of the male tail of Pratylenchus brachyurus. No bursal rib is present................. 46

8. A-B. Photomicrographs of Arthrobotrys anchonia showing

constricting rings.................................... 50

9. Normal Pratylenchus brachurus stylet showing spheroid

knobs................................ ................. 51











Figure Page 10. A-F, a-f. Lateral views (A-F) of variations in stylet knob abnormalities of Pratylenchus brachyurus

correspond to sub-ventral views of malformities (a-f).. 53


viii















Abstract of Dissertation Presented to the
Graduate Council of the University of Florida in Partial Fulfillment
of the Requirements for the Degree of Doctor of Philosophy
PARASITISM OF PRATYLENCHUS SPP. TO
'LOVELL,' 'NEMAGUARD' AND 'OKINAWA' PEACH By

Donald Eugene Stokes

June, 1972

Chairman: V. G. Perry
Major Department: Entomology and Nematology

Three species of Pratylenchus were demonstrated to parasitize

and reproduce in roots of 'Lovell,' 'Nemaguard' and''Okinawa' peaches,

Prunus persica Batsch. Populations of P. brachyurus (Godfrey, 1929) Filipjev and Schuurmans Stekhoven, 1941, P. coffeae (Zimmerman, 1898) Goodey, 1951, and P. penetrans (Cobb, 1917) Chitwood and Oteifa, 1952,

increased when inoculated onto these peaches.

Two tests for pathogenicity of P. brachyurus were conducted

using inoculum levels of 100 and 1,000 specimens per plant. 'Okinawa,' 'Lovell' and 'Nemaguard' peach were included in the low inoculum test,

but 'Nemaguard' was not tested at the high inoculum level. Substantial nematode population increases resulted on each peach variety of each test. Statistical comparisons of plant dry weights (top,

root, and total) disclosed insignificant differences between inoculated

and uninoculated plants of all the rootstock varieties used in the 100 (low) inoculum level test. In the 1,000 (high) inoculum level











test, however, statistical significance at the 1 per cent level occurred between dry weights of tops, roots, and total plants of 'Okinawa.' Statistical significance was also obtained at the 5 per cent level on root dry weights of 'Lovell' in this test.

Roots of inoculated plants in three tests exhibited necrotic lesions. No differences in types of lesions were noted among the various peach rootstocks.

Microscopic examination of sections of each peach rootstock revealed damage to cortical parenchyma by P. brachyurus. The tissue damage varied from a slight effect on several cells to the formation of cavities due to destruction of many cells. All developmental stages of P. brachytirus were found associated with various degrees of tissue damage.

Results of the tests indicated that large numbers of Pratylenchus brachyurus may have little effect on the growth of 'Lovell' and 'Okinawa' peaches.

A nematophagous fungus, identified as Arthrobotrys anchonia Drechsler, was found associated with Pratylenchus brachyurus in greenhouse-maintained colonies.

Pratylenchus brachyurus males reared on peach did not have a small rib formed by phasmids extending into the bursa. This conforms to the original description but not to the description of P. brachyurus males recovered from citrus in Florida.

Projections extending anteriorly from the stylet knobs were

observed in some populations of P. brachyurus when reared on 'Dixie 18'











field corn but not on 'Lovell' peach or sweet orange, Citrus sinensis Osbeck. Progeny of the nematodes with this characteristic did not possess the projections when reared on peaches or citrus.














INTRODUCTION


Several factors have until recently prevented the development of a major peach-growing industry in Florida. Peach, Prunus persica Batsch, is susceptible to the root-knot nematode species Meloidogyne incognita (Kofoid and White, 1919) Chitwood, 1949 and M. javanica (Treub, 1885) Chitwood, 1949, the dominant root-knot nematodes in Central and North Florida. 'Lovell' peach, highly susceptible to both root-knot nematode species, was a common rootstock in these areas (26). Resistance in peach rootstocks to both of these species was made available with the introduction of the 'Okinawa' and 'Nemaguard' rootstock varieties. Scion varieties used for fruit production normally are propagated onto rootstocks by bud grafts. These rootstocks, plus other research developments, have recently made possible economic production of peaches in Florida. Malo (20) found that the nature of resistance of 'Nemaguard' and 'Okinawa' peach rootstocks to M. javanica lies in the ability of the host plant to inhibit nematode growth. This is accomplished by walling off the nematodes and suppressing development of giant cells (or nurse cells) on which the nematode feeds. Little or no attention has been devoted to the reaction of Pratylenchus spp. on the root-knot nematode resistant 'Okinawa' and 'Nemaguard' peach rootstocks.

The nematode genus Pratylenchus Filipjev contains certain

obligately plant parasitic species which rank among the more important organisms responsible for plant root destruction (29).











Steiner (28) ranked Pratylenchus spp. as tissue parasites

"par excellence" and major economic pests. A recent key (5) lists 35 species of Pratylenchus.

Principal factors influencing distribution of Pratylenchus

species are climate and soil type (17). Pratylenchus spp. were recovered from over 95 per cent of 215 samples of varied host and representative soil types collected from Gainesville to Homestead, Florida (8).

Steiner (28) stated that under certain conditions Pratylenchus may multiply to enormous numbers, particularly in orchards that remain undisturbed for years, but also on some annual crops such as peanuts.

Species of Pratylenchus found in Florida associated with

economic crop plants include P. brachyurus (Godfrey, 1929) Filipjev and Schuurmans Stekhoven, 1941, P. coffeae (Zimmerman, 1898) Goodey 1951, and P. penetrans (Cobb, 1917) Chitwood and Oteifa, 1952. P. brachyurus is the most commonly observed species of the genus in Florida (8) and is often recovered from soil and root samples taken from peach trees.1 P. penetrans has been identified as an agent in the peach replant problem in Canada (22) and causes appreciable injury to roses grafted on Rosa fortuneana Lindley stock in Florida

(19). P. coffeae stunted young apple trees and caused root injury to peach trees and other fruit crops when planted in infested soil (3). This nematode is associated with several economic plants in Florida.1


1Unpublished records of Nematology Bureau, Division of Plant Industry, Florida Department of Agriculture and Consumer Services, Gainesville.











Since P. brachyurus, P. coffeae, and P. penetrans are prominent parasitic nematodes, the possibility exists that they may cause damage to peach roots. Since 'Okinawa' and 'Nemaguard' rootstocks show resistance to M. incognita and M. javanica, it is important to determine the reaction of other plant parasitic nematodes to these rootstocks.

The primary objective of this study was to determine if P.

brachyurus, P. coffeae, and P. penetrans parasitized, reproduced upon, and caused damage to 'Lovell,' 'Okinawa' and 'Nemaguard' peach rootstocks. Secondary objectives were to study the general morphology, life cycle stages,and feeding habits of P. brachyurus.















LITERATURE REVIEW


Since 1880 when the first species of Pratylenchus was described as Tylenchus pratensis by de Man (21) a total of 34 species has been described. Filipjev (9) erected Pratylenchus in 1936. A monograph of the genus was published by Sher and Allen (27) in 1953. They listed the following synonyms for P. coffeae:

Tylenchus coffeae Zimmerman, 1898

Tylenchus musicola Cobb, 1919

Pratylenchus musicola (Cobb, 1919) Filipjev, 1936

Tylenchus mahogani Cobb, 1920

Anguillulina mahogani (Cobb, 1920) Goodey, 1932

Pratylenchus mahogani (Cobb, 1920) Filipjev, 1936

Anguillulina pratensis Goffart, 1929

P. coffeae attacks a wide range of economic plants including camellia, vetch, and strawberry in the United States (29). Table 2 gives a partial list of host plants of P. coffeae in Florida. Colbran

(4) reported it to be associated with Prunus persica Batsch in Australia.

Pratylenchus penetrans (Cobb, 1917) Chitwood and Oteifa, 1952, had been named Tylenchus penetrans earlier by Cobb (17).

Records indicate that P. penetrans has one of the widest

known host ranges of any species in the genus (29). In Canada, P. penetrans has been defined as a serious problem in peach replant (22).
















Table 1. A host list of Pratylenchus brachyurus in Florida compiled
from the unpublished files of the Nematology Bureau, Florida Department of Agriculture and Consumer Services, Gainesville,
Florida


Scientific Name


Acer saccharinum L. Aleurites Fordii Hemsl. Aloe sp.

Annona glabra L. Arecastrum Romanzoffianum Becc. Asclepias tuberosa L. Asimina parviflora (Michx.) Dural. Begonia sp. Carya illinoensis Koch Cassia sp.

Chamaecrista sp. Moench. Chamaecyparis obtusa Sieb. & Zucc. Chamaedorea elegans Mart. Chrysanthemum sp. Citrus sp.

Citrus aurantium L. Citrus limon Burm. Citrus paradisi Macf. Citrus reticulata Blanco


Common Name


white maple tung oil tree



pond apple green palm butterfly weed papaw



pecan





Hinoki cypress parlor palm





sour orange lemon

grapefruit tangerine















Table 1. (continued)


Scientific Name


Citrus sinensis Osbeck Cocos nucifera L. Codiaeum sp. Juss. Cynodon dactylon Pers. Digitaria sanguinalis (L.) Scop. Diospyros virginiana L. Eremochloa ophiuroides Hack. Eupatorium capillifolium (Lam.) Small. Euphorbia sp. Euphorbia pulcherrima Willd. Fragaria sp. Gladiolus sp. Glottidium vesicarium (Jacq.) Harper Hemerocallis fulva L. Hibiscus sp. Hibiscus rosa-sinensis L. Indigofera endecaphylla Jacq. Juglans nigra L. Jussiaea peruviana L. Lantana sp.


Common Name


sweet orange coconut palm



Bermuda grass crabgrass common persimmon centipede grass dog fennel



poinsettia





coffee weed common orange daylily



Chinese hibiscus indigo

black walnut primrose willow















Table 1. (continued)


Scientific Name


Ligustrum sp. Ligustrum lucidum Ait. Lonicera sp. Lycopersicon esculentum Mill. Magnolia grandiflora L. Meibomia sp. Menthe spicata L. Myrica cerifera L. Paspalum notatum Flugge. Paspalum urvillei Steud. Pennisetum glaucum R. Br. Persea americana Mill. Philodendron sodiroi Hort. Phoenix Roebelenii O'Brien Phytolacca americana L. Pinus clausa (Engelm.) Vassey. Pinus elliottii Engelm. Pinus palustris Mill. Polystichum adiantiforme J. Smith


Common Name


glossy privet



tomato bull bay



spearmint wax myrtle Bahia grass



pearl millet avocado philodendron dwarf date-palm pokeberry sand pine slash pine longleaf pine leather leaf fern















Table 1. (continued)


Scientific Name


Prunus persica Batsch Psidium guajava L. Pteridium aquilinum (L.) Kuhn Pteris latiuscula Desv. Pyrus sp.

Quercus laevis Walt: Saccharum officinarum L. Saintpaulia ionantha Wendl. Schinus terebinthifolius Redd. Serenoa repens (Barth.) Smilax sp.

Stenotaphrum secundatum Kuntze Stillingia angustifolia (Torr.) S.Wats. Vigna sinensis 'Iron' (L.) Endl. Zoisia japonica Steud. Zoisia japonica 'Emerald' Steud.


Common Name


peach

guava







turkey oak sugar cane African violet Brazilian pepper tree saw-palmetto



St. Augustine grass queen's-root cowpea

Japanese lawn grass














Table 2. A host list of Pratylenchus coffeae in Florida compiled from
the unpublished files of the Nematology Bureau, Florida Department of Agriculture and Consumer Services, Gainesville, Florida


Scientific Name


Aglaonema simplex Blume Ananas comosus Merr. Chamaedorea seifrizii Burret Citrus sp.

Hedera canariensis Willd. Hibiscus sp. Ilex glabra Gray Monstera deliciosa Liebm. (Ceriman) Pinus elliottii Engelm Quercus laurifolia Michx. Trifolium hybridum L. Vriesia imperialis Carriere


Common Name


pineapple parlor palm Algerian ivy gallberry slash pine laurel oak alsike clover


bromeliad















Table 3. A host list of Pratylenchus penetrans in Florida compiled
from the unpublished files of the Nematology Bureau, Florida Department of Agriculture and Consumer Services, Gainesville,
Florida


Scientific Name


Aglaonema commutatum Schott. Chrysanthemum sp. Eremochloa ophiuroides Hack. Ficus pandurata Sander Ilex glabra Gray Juniperus silicicola (Small) Bailey Pinus sp.

Polystichum adiantiforme J. Smith Zoisia sp.


Common Name


centipede grass fiddleleaf fig gallberry Southern red cedar



leather leaf fern Japanese lawn grass









In Florida, P. penetrans is parasitic to a rose rootstock, Rosa fortuneana Lindley (19), and leather leaf fern, Polystichum adiantiforme J. Smith (24). Other plants attacked by P. penetrans in Florida are listed in Table 3.

According to Loof (17), synonyms for P. brachyurus include:

Tylenchus brachyurus Godfrey, 1929

Pratylenchus leiocephalus Steiner, 1949

Pratylenchus pratensis Thorne, 1940

Pratylenchus steineri Lordello, Zamith and Boock, 1954

P. brachyurus is reported to attack many economic plants including pineapple, peanut, strawberry, lespedeza, cotton, okra, corn, tobacco, and potato tubers (29). Pratylenchus spp. have been the most frequently found plant parasitic nematodes associated with citrus roots (2). P. brachyurus is the species of the genus most commonly found in Florida (8) and it is reported pathogenic to citrus (1). Additional hosts of P. brachyurus in Florida are listed in Table 1.

Fliegel (10) reported Pratylenchus vulnus, P. zeae and P.

brachyurus on peach in Georgia. P. brachyurus, the most commonly found species, was not associated with severe damage to roots and populations were relatively small. P. zeae generally did not affect plant root systems.

Godfrey (11) characterized symptoms of pineapple roots caused by P. brachyurus as irregularly elongate brown spots which are light in color when young and later become darker. Lesions are not sunken and are readily distinguishable from lesions produced by other organisms. Discoloration and cell destruction were observed from the epidermal cells through the cortex.










Brooks and Perry (1) reported that injury by P. brachyurus extended two to three cells to each side of the nematode, which suggests toxic substances secreted by the nematode.

Graham (12) reported damage to tobacco and corn by Pratylenchus brachyurus and P. zeae. These two nematode species were always found in the outer parenchyma and never in the vascular tissues.

In Pretoria, Koen and Hogewind (16) observed that Pratylenchus brachyurus caused irregularly shaped purple-brown lesions 1-5 mm in diameter on potato tubers. Infected tubers lose considerable weight when stored at room temperatures and are unsuitable for use as seed pieces. By digging potatoes at an early stage and storing at 5 C no visible lesions developed.

Pratylenchus vulnus was reported (18) to be associated with

declining peach orchards in California. D'Souza (6) reported P. vulnus as a pathogen of peach in California; however, the nematode appeared most prominent as a disease incitant or a disease aggravator and alone did not stunt peach seedlings at the population levels occurring in peach orchards.

Feeding by Pratylenchus spp. is of two obvious types. One type (15) is exhibited by P. crenatus which feeds ectoparasitically on epidermal cells, migrating from cell to cell and causing light necrotic spots on the roots. Another type (23) is exhibited by P. scribneri, which feeds endoparasitically on amaryllis, forming large necrotic areas in the cortex with "nests" of eggs and larvae.














MATERIALS AND METHODS


Soil and Inoculum Sources

Soil for all experiments was obtained from the "Agronomy Farm," Institute of Food and Agricultural Sciences, University of Florida, Gainesville, and was of the Lakeland fine sand series. The soil used in each experiment was fumigated with methyl bromide (2 pounds per cubic yard) and aerated at least 3 weeks before use.

The three species of Pratylenchus under investigation were

obtained from different sources. Specimens of Pratylenchus brachyurus were obtained from peach growing in a nursery near Belleview, Florida; P. penetrans from leather leaf fern, Crescent City, Florida; and P. coffeae from Chinese evergreen growing in a greenhouse at Gainesville, Florida.


General Methods

Populations of P. brachyurus used as inoculum were maintained and increased on 'Lovell' peach; 'Dixie 18' field corn, Zea mays L.; or sweet orange, Citrus sinensis Osbeck. P. penetrans and P. coffeae populations were maintained and increased on the hosts from which they had been collected, leather leaf fern and Chinese evergreen, respectively. Inoculated host plants used for colonizing respective nematode species were grown in wooden boxes,approximately 12 x 24 x 10 inches in dimension,containing fumigated soil. Adequate quantities of nematodes for










inoculum were thus always available when needed. All colonization and pathogenicity tests were conducted in an environmental chamber at 24 � 2 C and illuminated 2200 ft c for 16 hours per day.

Nematode specimens used for inoculum were obtained by incubating appropriate infected plant roots for about 2 days in glass pint jars containing a small amount of water. Root washings from the jars were placed onto a facial tissue in a Baermann funnel partially filled with distilled water and the nematodes were recovered in the bottom of the funnel. Nematodes recovered were subsequently hand-picked into distilled water before use. A ratio of nine females to one male was used for P. penetrans and P. coffeae where males are plentiful, and only females, of course, for P. brachyurus where males are rare.


Identification of Nematode Species

Using the following method Pratylenchus spp. were identified both at the beginning of the test and after plants were harvested.

Specimens were hand-picked into distilled water on a glass slide. The slide was moved about over a small gas flame for 5-6 seconds duration and repeated until the nematodes ceased twisting and straightened out. Glass rods of a diameter near that of the nematodes were then placed in a triangle around the specimens and a cover glass applied. Zut was used to seal the edges of the cover glasses to the slides. Other specimens so killed were fixed in 3 per cent formalin and mounted in formalin. Some of those fixed in the 3 per cent formalin were processed to anhydrous glycerine and mounted, using











Seinhorst's method (25). Specimens from each method were then studied in detail with the aid of a research microscope equipped with an oil immersion objective.


Experiment I

Peach seedlings of 'Lovell,' 'Okinawa,' and 'Nemaguard' varieties grown in vermiculite (Fig. 1) to a height of about 2 cm were obtained from the Fruit Crops Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, for experimental purposes. When seedlings were approximately 3 cm in height each was transplanted into a 6-inch clay pot partially filled with fumigated soil. Each peach variety was replicated three times for each of the three nematode species. One pot of each peach variety served as an uninoculated control for each replicate.

The plants were inoculated with nematodes 16 days after transplanting, when they were approximately 4 cm in height. Fifty handpicked specimens of the appropriate Pratylenchus species in a water suspension were withdrawn into a 10 cc capacity hypodermic syringe (without needle) and ejected about three quarters of an inch deep into the soil of a designated pot. A thin layer of moistened sterilized soil was used to cover the existing soil and distilled water was added. This procedure was followed in all inoculations. Distilled water was added to each pot as needed to maintain adequate soil moisture. A liquid fertilizer solution (OrthoR 12-6-6) at the rate of 1 tablespoon per gallon of water was applied every 2 weeks. Plants were sprayed with Kelthane as needed to control mites. The plants were visually inspected every day.

























































Fig. 1. Photograph of young 'Nemaguard' seedlings
growing in vermiculite.










Sixteen weeks after inoculation, the plant tops were removed just above the soil line and discarded. Roots were removed from the soil and two 5-gram samples (net weight) were placed into a food blender and comminuted for approximately 20 seconds. The blender contents were then washed onto a facial tissue held in a Baermann funnel partially filled with distilled water and left overnight. The nematode suspension was drawn from the Baermann funnel and poured onto a 325-mesh sieve. The sievings were washed into a Syracuse watch glass and the nematodes counted with the aid of a dissecting microscope. Soil was mixed thoroughly and two-100 ml samples were processed by a centrifugal-flotation technique (13). Nematodes recovered were placed into a Syracuse watch glass and counted. The total number of nematodes per pot was calculated.


Experiment II

Five replicates of 'Lovell,' 'Okinawa,' and 'Nemaguard' peach approximately 4 cm high,growing in 8-inch clay pots partially filled with sterilized soil, were each inoculated with 100 hand-picked Pratylenchus brachyurus. This experiment was of 17 weeks duration.

Watering, fertilizing, spraying for pest control, and inspection for Experiments II and III were as described for Experiment I.

Seventeen weeks after inoculation tops were removed from plants. Roots were individually removed from the soil and washed to remove adhering soil particles. The washings were collected in a 2-liter capacity can, were roiled, and a 500 ml sample removed and processed by the centrifugation-flotation technique (13). Root washings in Experiment I were not processed separately, but included with soil processing.










The soil recovered from each treatment was mixed thoroughly, a 100-ml sample removed and processed by the centrifugation-flotation technique.

Nematodes recovered from root washings and soil were counted

with the aid of a dissecting microscope and the total number of specimens per replicate was calculated.

Roots were incubated (30) by placing each replicate individually in a 4-pound (4 x 3 x 13 inches) polyethylene bag. After 3, 7, 14, 21, and 28 days, emerged nematodes were collected by washing the roots and the interior of the polyethylene bags with tap water by a jet nozzle attachment. Washings were collected in cans and poured onto a 325-mesh screen. Nematodes collected on the screen were washed into Syracuse watch glasses for counting. Examinations and counting of nematodes were accomplished with the aid of a dissecting microscope. Total numbers of nematodes from each root system was obtained from roots, root washings, and soil samples.

Plant tops and roots (after incubation) were maintained for

2 weeks at 92 C in a plant-drying oven to obtain dry weights. Identity of tops and roots was maintained throughout the experiments.


Experiment III

Material and methods for Experiment III were the same as for

Experiment II except 1,000 hand-picked Pratylenchus brachyurus per replicate were used and only 'Okinawa' and 'Lovell' peach were included.

All handling of plants and soil was the same as for Experiment II.










Histopathology of Peach Roots Infected With Pratylenchus brachyurus


Peach roots infected and not infected by P. brachurus were

killed and fixed in FAA (formalin, 6.5 ml; glacial acetic acid, 7.5 ml; 50 per cent ethanol, 100 ml), dehydrated to tertiary-butyl alcohol and embedded in paraffin. Sections cut 12 p thick with a rotary microtome were affixed to glass microscope slides with Haupt's adhesive, stained with safranin-fast green and mounted in Fisher's "Permount" according to Johansen's (14) procedure.















RESULTS AND DISCUSSION


Experiment I

Plant growth was virtually uniform throughout the 16-week experiment and no noticeable differences occurred on the above-ground plant parts. Each replicate in each treatment yielded greater nematode populations than had been used as inoculum, indicating parasitism and reproduction by P. brachyurus, P. penetrans, and P. coffeae. Using an average figure for all three rootstocks, P. brachyurus reproduced

3.2 times; P. penetrans, 2.7 times;and P. coffeae, 2.3 times the original inoculum levels (Table 4). And using an average figure for all three nematodes, 'Okinawa' peach supported the largest total populations, which was 3.3 times the inoculum level. 'Lovell' and 'Nemaguard' peach supported total nematode populations of 2.3 and 1.8 times, respectively, the inoculum level (Table 4).

P. brachyurus was chosen for further study because it reproduced at a greater rate than did P. penetrans or P. coffeae. P. penetrans and P. coffeae should not be discounted as potential pathogens of peach, because reproduction occurred and population increases were noted in 'Lovell,' 'Okinawa,' and 'Nemaguard' cultivars.

Roots of inoculated plants had very small brownish lesions that did not occur on roots of uninoculated plants.










Table 4.


Numbers of Pratylenchus brachyurus, P. penetrans, and P. coffeae recovered from the roots of three peach cultivars used as rootstocks inoculated with 50 nematodes per 6-inch pot


Peach P. brachyurus P. penetrans P. coffeae Rootstock totals


'Lovell' 212 79 127 418 'Lovell' 88 154 80 322 'Lovell' 149 112 45 306
Total 449 345 252 1046

Average 150 115 84 315


'Okinawa' 280 218 0 498 'Okinawa' 140 200 167 507 'Okinawa' 229 129 124 482
Total 649 547 291 1487

Average 216 182 97 496


'Nemaguard' 111 0 0 111 'Nemaguard' 124 94 218 436 'Nemaguard' 141 106 59 306

Total 376 200 277 853

Average 125 67 92 284

Total 1474 1092 820











Experiment II

Above-ground plant symptoms caused by nematode injury were not observed during this experiment making it impossible to distinguish between inoculated and uninoculated plants. But P. brachyurus, at the 100 per pot inoculum level on 'Okinawa' peach, reached a density of

3.5 times the original population. 'Lovell' and 'Nemaguard' peach at the 100 inoculum level supported P. brachyurus populations of 2.7 and

2.4 times, respectively, the original inoculum level (Table 5). An analysis of variance showed no statistical significance between root, top, or total dry weights (Table 6).

Experiment II results substantiate that P. brachyurus is capable of reproducing in 'Okinawa,' 'Nemaguard,' and 'Lovell' peach roots. 'Okinawa' again supported the largest nematode population with 'Lovell' and 'Nemaguard' following in descending numbers.

Small brownish lesions were again observed on roots of plants inoculated with P. brachyurus.


Experiment III

No differences in growth were observed between inoculated and uninoculated plants during this experiment and no symptoms of injury were detected on above-ground plant parts (Fig. 2). At the 1,000 inoculum level, P. brachyurus reproduced 2.4 and 2 times the original population on 'Okinawa' and 'Lovell' peach, respectively (Table 7). 'Nemaguard' was unavailable for use in this experiment.























































Fig. 2. Photograph of 'Okinawa' peaches: plant at
right inoculated with 1,000 as compared
with uninoculated plant.











Table 5. Dry weights of 'Lovell,' 'Okinawa,' and 'Nemaguard' peach seedlings and number of
nematodes recovered when uninoculated and inoculated with 100 Pratylenchus brachyurus
and grown for 17 weeks in methyl bromide-treated soil.




Peach and Dry Weight in Grams No. of Treatment Root Top -Plant Nematodes


'Lovell' inoculated


Total Mean


'Lovell' uninoculated


Total


1.03 1.10
1.42 .94
1.39
5.88


1.13 1.16
1.49 1.14
1.46
6.38


2.16 2.26 2.91 2.08 2.85
12.26


1.18 1.28 2.45


1.42 1.31 1.29 1.04 1.17
6.23


1.47 1.38 1.32 1.07 1.22 6.46


2.89 2.69 2.61 2.11 2.39
12.69


Mean 1.25 1.29 2.54


268
232 290 249 306
1345


269











Table 5. (continued)


Peach and Dry Weight in Grams No. of Treatment Root Top Plant Nematodes


'Okinawa' inoculated


1. 1.41 2. 1.22 3. 1.10 4. .96 5. 1.01 Total 5.70


Mean


'Okinawa' uninoculated


1.44 1.21 1.12 1.01 1.07
5.85


2.85 2.43 2.22 1.97 2.08
11.55


412 344 387 322 260
1725


1.14 1.17 2.31


1. 1.32 2. 1.17 3. 1.24 4. 1.06 5. 1.14 Total 5.93


1.37 1.22 1.26 1.10 1.21
6.16


2.69 2.39 2.50 2.16 2.35
12.09


1.18 1.23 2.42


Mean










Table 5. (continued)


Peach and Dry Weight in Grams No. of Treatment Root Top Plant Nematodes


'Nemaguard' 1. 1.21 1.24 2.45 185 inoculated 2. 1.32 1.39 2.71 139 3. 1.04 1.06 2.10 315 4. 1.47 1.51 2.98 260 5. .94 .99 1.93 306 Total 5.98 6.19 12.17 1205

Mean 1.20 1.24 2.43 241


'Nemaguard' 1. 1.47 1.52 2.99 0 uninoculated 2. 1.40 1.45 2.85 0 3. 1.13 1.14 2.27 0 4. 1.29 1.35 2.64 0 5. 1.62 1.62 3.24 0 Total 6.91 7.08 13.99

Mean 1.38 1.42 2.80











Table 6. Analysis of variance


Source of variation df Sum of Squares Mean Square F


Analysis of variance: 'Lovell' top dry weight in grams (from data of Table 1) Treatment 1 .002 .002 .07547 Error 8 .212 .0265 Total 9 .214 P. brachyurus uninoculated
6.38 6.46


Analysis of variance: 'Lovell' root dry weight in grams (from data of Table 1) Treatment 1 .011 .001 .314 Error 8 .279 .035 Total 9 .290 P. brachyurus uninoculated
5.88 6.23











Table 6. (continued)


Source of variation df Sum of Squares Mean Square F


Analysis of variance:

Treatment Error Total


'Lovell' total dry weight in grams (from data of Table 1)


1
8
9


.186
.784
.97


.186 .098


P. brachyurus
12.26


Analysis of variance:


Treatment Error Total


uninoculated
12.69


'Okinawa' root dry weights in grams (from data of Table 1)


1
8
9


.011
.169
.180


P. brachyurus
5.70


uninoculated
5.93


1.90


.011 .021125











Table 6. (continued)


Source of variation df Sum of Squares Mean Square F


Analysis of variance:


Treatment Error Total


'Okinawa' top dry weight in grdms (from data of Table 1)


.013 .137
.15


.013 .017


P. brachyurus
5.85


Analysis of variance:


Treatment Error Total


uninoculated
6.16


'Okinawa' total dry weight in grams (from data of Table 1)


1
8
9


.034 .626
.66


P. brachyurus
11.55


uninoculated
12.09


.034 .078











Table 6. (continued)


Source of variation df Sum of Squares Mean Square F


Analysis of variance:


Treatment Error Total


'Nemaguard' root dry weight in 'grams (from data of Table 1)


1
8
9


.086 .294
.380


.086
.036


P. brachyurus
5.98


Analysis of variance:


Treatment Error Total


uninoculated
6.91


'Nemaguard' top dry weights in grams (from data of Table 1)


1
8
9


.079 .329
.408


P. brachyurus
6.19


uninoculated
7.08


2.34


.079 .041


1.92











Table 6. (continued)




Source of variation df Sum of Squares Mean Square F Analysis of variance: 'Nemaguard' total dry weight in'grams (from data of Table 1) Treatment 1 .33 .331 4.96 Error 8 .53 .066 Total 9 1.86 P. brachyurus uninoculated 12.17 13.99











Table 7. Dry weights of 'Lovell' and 'Okinawa' peach seedlings and numbers of nematodes
recovered when uninoculated and inoculated with Pratylenchus brachyurus and
grown for 17 weeks in methyl bromide-treated soil



Peach and Dry Weight in Grams No. of Treatment Root Top Total Nematodes


'Lovell' inoculated


1. .92 2. .97 3. .89 4. 1.01 5. 1.10 Total 4.89


Mean


.978 1.17 2.14


'Lovell' uninoculated


1. 1.20 2. 1.37 3. 1.09 4. 1.32 5. 1.04 Total 6.07


1.21 1.27 2.48


1.09 1.21 1.10 1.17 1.26
5.83


2.01 2.18 1.99 2.18 2.36
10.72


2140 1979 2244 1715 1827 9905

1981


1.29 1.44
1.14 1.40 1.07
6.34


2.49 2.81 2.23 2.77 2.11
12.41


I


Mean










Table 7. (continued)


.Peach and Dry Weight in Grams No. of Treatment Root Top Total Nematodes


'Okinawa' 1. .96 1.02 1.98 2319 inoculated 2. .89 .97 1.86 2614 3. .94 1.01 1.95 1974 4. .76 .90 1.66 2149 5. 1.02 1.10 2.12 2730 Total 4.57 5.00 9.51 11,786

Mean .91 1.00 1.91 2357


'Okinawa' 1. 1.29 1.34 2.63 0 uninoculated 2. 1.17 1.26 2.43 0 3. 1.15 1.24 2.39 0 4. 1.06 1.12 2.18 0 5. 1.24 1.30 2.54 0 Total 5.91 6.26 12.17

Mean 1.18 1.25 2.45











Statistical analysis of data obtained from this experiment and the analysis of variance table show that top, root, and total dry weights of 'Okinawa' plants inoculated with P. brachyurus are statistically different from uninoculated plants at the 1 per cent level of probability (Table 8). This statistical method also shows that root weights of inoculated and uninoculated 'Lovell' plants were statistically different at the 5 per cent level of probability (Table 8). No statistically significant difference existed at either the 1 or 5 per cent levels of probability when top dry weights or total dry weights of 'Lovell' peach were compared.

Obvious disease symptoms such as stunting, chlorosis, and leaf

drop being absent on test plants suggests that injury to peach is very gradual and subtle.


Histopathology of Peach Roots Infected With Pratylenchus brachyurus

Microscopic examination of sections of infected roots revealed the presence of P. brachyurus within and damage to cortical regions (Fig. 4). Various developmental stages of the parasite were found within root tissues showing various stages of damage. Eggs were noted singly and in groups of three to four inside single cavities. Tissue damage varied from slight effects on several cells to the formation of small cavities due to destruction of many cells (Fig. 5). Small root lesions and ruptures were obvious in root epidermis in some instances, but injury was not extensive.

















































,:V









Fig. 3. Photomicrograph of a transverse section of
'Okinawa' peach root from uninoculated plant.






















Fig. 4. A-B.


Photomicrograph of a transverse section of 'Nemaguard' peach root infected by Pratylenchus brachyurus. A. Portions of nematode bodies (arrows) are adjacent to cavities in the cortical region.


B. Head portion of a nematode in cortical region. Note the absence of
cavity formation.






























I


e


e.14






















Fig. 5. A-B.


Photomicrograph of a transverse section of 'Okinawa' peach root infected with Pratylenchus brachyurus. A. Portion of a nematode (a) is in one of the cortical cavities (b). Note proximity of cavities to epidermis.


B. Head portion of a nematode in cortical
region near epidermis.









f r











Table 8. Analysis of variance


Source of variation df Sum of Squares Mean Square F


Analysis of variance:


'Lovell' top dry weight in grams (from data of Table 4)


Treatment Error Total


P. brachyurus inoculated
5.83


Analysis of variance:

Treatment Error Total


uninoculated
6.34


'Lovell' root dry weight in grams (from data of Table 4)


8
9


.13924 .12865
.26789


P. brachyurus inoculated
4.89


uninoculated
6.07


.0101
1.80901
1.81911


.0101 .202


.13924 .01608


8.66**


M M M M











Table 8. (continued)


Source of variation df Sum of Squares Mean Square F Analysis of variance: 'Lovell' total weight in grams (from data of Table 4)


Treatment Error Total


1
8
9


.28561
.47470
.76031


4.81


.28561
.05933


P. brachyurus inoculated
10.72


Analysis of variance:

Treatment Error Total


uninoculated
12.41


'Okinawa' top weight in grams (from data of Table 4)


.15876 .05248
.21124


P. brachyurus inoculated
5.00


uninoculated
6.26


.15876 .00656


24.20***











Table 8. (continued)


Source of variation df Sum of Squares Mean Square F


Analysis of variance: 'Okinawa' root weight in grams (from data of Table 4) Treatment 1 .179560 .179560 25.02 Error 8 .0574 .007175 Total 9 .23696 P. brachyurus inoculated uninoculated
4.57 5.91


Analysis of variance: 'Okinawa' total weight in grams (from data of Table 4) Treatment 1 .6760 .6760 25.60 Error 8 .2112 .0264 Total 9 .8872 P. brachyurus inoculated uninoculated
9.57 12.17

**Significant at 5 per cent level.

***Significant at 1 per cent level.


***


***











The nematodes observed inside peach roots were mostly oriented along the transverse root axis with heads away from root tips. Occasionally, the heads were observed at right angles to the transverse axis. The stele was not generally infected; however, in two instances single specimens of P. brachyurus were found inside the stele (Fig. 6).


Roots Stained With Safranin-Fast Green

The walls of plant cells not affected by the feeding actions of P. brachyurus appeared green as compared to a red color of the cell walls associated with feeding by the nematodes. Cytoplasm of cells affected by nematode feeding was more granular than that of normal cells. Apparent nematode injury extended sometimes two or three cells away from the nematode, which is suggestive of chemical damage by nematode secretions. Safranin typically stains lignified and cutinized cell walls a brilliant red; whereas, fast green should be prominent on cellulose cell walls (14). No evidence of hypertrophy or hyperplasia was observed.

The damage caused by P. brachyurus to each of the three peach rootstocks was quite similar. There were no apparent differences in the kind or degree of damage caused by the feeding of the different species of nematodes on the root tissue. Both mechanical and chemical factors appeared involved in total plant damage. Cavities located in the cortical parenchyma resulted from feeding and migration of the nematode. Injury to the vascular system was limited to the cells occupied by the nematode or being fed upon.

The ability of Pratylenchus brachyurus to successfully parasitize and reproduce on 'Lovell,' 'Okinawa,' and 'Nemaguard' suggests adequate nutrition furnished by this host to the nematodes. There
























































Fig. 6. Photomicrograph of a longitudinal section of
'Lovell' peach root infected with Pratylenchus
brachyurus. Head of nematode (arrow) is inside
the stele.











was no evidence in Pratylenchus brachyurus-infected peach to indicate presence of a "walling off" process (20), a mechanical type of plant resistance that develops when Meloidogyne javanica feeds on 'Okinawa' or 'Nemaguard,' or any other type of plant resistance. "Walling off" causes a breakdown of giant cells which in effect removes the nematode's food source. This type root-knot nematode resistance does not occur in 'Lovell' peach. P. brachyurus reproduced to large numbers on 'Lovell,' 'Okinawa,' and 'Nemaguard,' but caused slight root damage. Further testing of the three rootstocks is needed to determine the effects of longer exposures to large numbers of P. brachyurus.

P. brachyurus normally fed solitary in the cortical region of peach; however, nematodes in groups of two to three occasionally were observed.

As many as six P. brachyurus in solitary sites were observed

in a single transverse peach root section, but in no case was "nesting" found in peach roots, even where cortical necrotic cavities occurred.

"Nesting" often occurs in necrotic cavities resulting from the feeding of this species of Pratylenchus.


Study of Pratylenchus brachyurus From Experimental Plants

Measurements and characteristics of P. brachyurus recovered from peach roots in Experiments I, II, and III conformed to those described by Sher and Allen (27), but males differed from those described by Brooks and Perry (1) from citrus in Florida. In more than 100 males examined there was no extension of the phasmid into the bursa (Fig. 7), in agreement with Sher and Allen (27). Brooks and Perry (1) reported
























































Fig. 7. Photomicrograph of the male tail of Pratylenchus
brachyurus. No bursal rib is present.











that the phasmid on males from citrus extended into the bursa. Even when the experimental peach population was reared on citrus, males maintained their bursal characteristic forming no phasmidial rib. Thus two discrete populations of P. brachyurus exist in Florida; however, these differences do not warrant species separation at this time. Males of the peach population apparently do not inseminate females, as no sperm were observed in the uteri of the females.


Study of Pratylenchus penetrans and
Pratylenchus coffeae From Experiment I

Morphological and dimensional characteristics of P. penetrans and P. coffeae recovered from experimental plants conformed to the species descriptions by Sher and Allen. Males recovered from experimental plants comprised approximately 25 per cent and 35 per cent of the total populations of P. coffeae and P. penetrans, respectively.


Fungus Associated With Nematode Colony

Populations of Pratylenchus brachyurus were maintained on

'Lovell,' 'Nemaguard,' and 'Okinawa' peach in a greenhouse for biotype identity in 8-inch clay pots containing a soil mixture of approximately one-third peat and two-thirds sandy soil by volume. A periodic examination of soil and peach roots revealed a fungus associated with certain specimens of P. brachyurus. A fungus structure of the constricting ring type often encircled nematodes in the region of the stylet knob. Other nematodes without the constricting ring fungus were constricted in the stylet knob region as evidence of previous association with the fungus.











Three nematodes trapped in a constricting ring with fungus

mycelium attached were placed on Petri plates containing 2 per cent potato-dextrose agar. Cultures of the fungus grown on this agar were transferred to 10 x 50 mm glass test tubes containing 2 per cent corn meal agar plus dextrose. Characteristics of the fungus conformed to the description of Arthrobotrys anchonia Drechsler. Type material of this fungus was collected in Ft. Lauderdale, Florida (7).

A. anchonia (Fig. 8-A) was reported (7) to trap and parasitize Eucephalobus sp., but it has never before been reported to trap a plant parasitic nematode (Fig. 8-B).


Stylet Knob Variation in a Colony of Pratylenchus brachyurus

Colonies of P. brachyurus maintained in a greenhouse were examined periodically, particularly to determine nematode vigor, presence of contaminants, or nematode predators and diseases. One collection of P. brachyurus colonized on 'Dixie 18' field corn contained specimens exhibiting an atypical morphological characteristic of the stylet knobs of the species. The typical shape of stylet knobs of P. brachyurus is spheroid to oval (Fig. 9). Abnormal anterior projections of varying lenths (Fig. 10 A-F and a-f) were formed on one to all three stylet knobs of the atypical specimens grown on 'Dixie 18' corn. These stylet knobs appeared in lateral view to have apical projections. In no instance was the projection observed extending anteriorly more than half the stylet length. Margins of the stylet knob extensions were within the marginal bounds of the stylet extruder




















Fig. 8. A-B. Photomicrographs of Arthrobotrys anchonia
showing constricting rings. A. Constricting rings are attached to hyphal strands.































B. Fratylenchus brachyurus female caught in constricting ring.























A 4****

























* 44 9,9
























































Fig. 9. Normal Pratylenchus brachyurus stylet showing
spheroid knobs.






















































Fig. 10. A-F, a-f. Lateral views (A-F) of variations in
stylet knob abnormalities of Pratylenchus brachyurus
correspond to sub-ventral views of malformities (a-f).











A a B b lc D d E e F f






54



muscles. The abnormal stylet occurred on approximately 40 per cent of the females in certain colonies on 'Dixie 18' field corn but not in all colonies. The abnormality was not found on P. brachyurus reared on other hosts, and progeny of females possessing the abnormal stylet characteristic reared on peach or citrus did not have the atypical condition.















CONCLUSIONS


1. Plant parasitic nematodes belonging to the genus Pratylenchus

have been recovered from roots of declining peach trees.

2. Three species of this nematode genus, Pratylenchus brachyurus

(Godfrey, 1929) Filipjev and Schuurmans Stekhoven, 1941, Pratylenchus coffeae (Zimmerman, 1898) Goodey, 1951, and Pratylenchus

penetrans (Cobb, 1917) Chitwood and Oteifa, 1952, will parasitize and reproduce on peach, Prunus persica Batsch: 'Lovell,' 'Nemaguard,' and 'Okinawa' rootstocks.

3. Pratylenchus brachyurus reproduced a greater population than

either P. coffeae or P. penetrans in one inoculation test suggesting greater potential damage to hosts.

4. Damage by Pratylenchus brachyurus to peach roots is typified by

lesions on epidermal tissues and injury to cortical regions.

5. Levels of inoculum influence severity of P. brachyurus damage to

'Okinawa' rootstock. Longer exposures of the host to the nematode

could influence severity in plant injury, particularly with perennial crops such as peach in areas with little seasonal climatic variation.

It is possible that population thresholds were not reached in

17 weeks.

6. A variable stylet characteristic among Pratylenchus brachyurus

populations occurred in certain populations.





56



7. A ring-forming fungus, Arthrobotys anchonia Drechsler, is capable

of ensnaring Pratylenchus brachyurus; however, the efficiency of

this fungus to control the nematode is unknown.

8. Pratylenchus brachyurus males reared on peach lack a small rib

formed by phasmids extending into the bursa and differ from P.

brachyurus reared on citrus in this respect.















SUMMARY


P. brachyurus occurs in peach nurseries and orchards in Florida, but its possible role in peach decline was heretofore undefined. An initial test was established to ascertain parasitism of P. brachyurus to 'Lovell,' 'Okinawa,' and 'Nemaguard' peach rootstocks. P. brachyurus was more prolific than P. penetrans or P. coffeae on these hosts. Two pathogenicity tests of 17 weeks duration were conducted using 100 or 1,000 specimens of P. brachyurus per plant, each treatment being replicated five times. 'Okinawa,' 'Nemaguard,' and 'Lovell' peach were used in the first test, but 'Nemaguard' was not available for the second. The parasite reproduced at rates of 2.7, 3.5, and 2.4 times the inoculum level on 'Lovell,' 'Okinawa,' and 'Nemaguard' peach, respectively (low inoculum level test) and 2.4 and 2 times on 'Okinawa' and 'Lovell', respectively (high inoculum level test). Temperatures of 24 � 2 C and a photoperiod at 2200 ft c for 16 hrs were maintained throughout all tests.

Statistical comparisons of plant dry weights (top, root, and total) disclosed no significant difference between inoculated and uninoculated plants except in the high inoculum level test. Dry weights of top, roots, and total plant of 'Okinawa' peach were statistically significant at the 1 per cent level; with 'Lovell' the dry weight of the roots only was significantly different from the control and that was at the 5 per cent level.










Microscopic examination of sections of each peach rootstock revealed damage to cortical parenchyma by P. brachyurus. Tissue damage varied from slight effects on several cells to formation of small cavities due to cell destruction.

Results indicate that even large numbers of P. brachyurus may have little effect on early growth of 'Okinawa' and 'Lovell' peach seedlings.

Abnormal stylet knob extensions were discovered on some colonies of P. brachyurus.

Arthrobotrys anchonia ensnared P. brachyurus specimens in colonies maintained on peach growing in soil containing about onethird peat.

Pratylenchus brachyurus males reared on peach did not have a small rib formed by the phasmids extending into the bursa which is characteristic of P. brachyurus males found on citrus in Florida.















LITERATURE CITED


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control. Fla. Agr. Exp. Sta. 256 p.

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southern states. Mycologia 46:762-782.

8. Feldmesser, J., W. A. Feder, and J. A. Pinckard. 1956. The occurrence of Pratylenchus spp. in Florida soils. Phytopathology

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10. Fliegel, P. 1969. Population dynamics and pathogenicity of three

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12. Graham, T. W. 1951. Nematode root rot of tobacco and other plants.

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Tylenchorhynchus dubius, and Hemicycliophora similis. Nematologica

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Proc. Fla. State Hort. Soc. 74:388-392.










20. Malo, S. E. 1967. Nature of resistance of 'Okinawa' and 'Nemaguard' peach to the root-knot nematode Meloidogyne javanica.

Proc. Amer. Soc. Hort. Sci. 90:39-46.

21. Man, J. G. de. 1880. Die einheimischen, frei in der reinen

Erde und im sussen Wasser lebenden Nematoden. Tijdschr. Ned.

Dierk. Ver. 5:1-104.

22. Mountain, W. B., and Z. A. Patrick. 1959. The peach replant

problem in Ontario. VII. The pathogenicity of Pratylenchus

penetrans (Cobb, 1917) Filip. & Stek., 1941. Can. J. Bot.

37:459-470.

23. Nong, L., and G. F. Weber. 1965. Pathological effects of Pratylenchus scribneri and Scutellonema brachyurum on amaryllis.

Phytopathology 55:228-230.

24. Rhoades, H. L. 1968. Pathogenicity and control of Pratylenchus

penetrans on leatherleaf fern. Plant Disease Reptr. 52:383-385. 25. Seinhorst, J. W. 1959. A rapid method for the transfer of nematodes from a fixative to anhydrous glycerine. Nematologica

4:67-69.

26. Sharpe, R. H., and R. Parker. 1963. Peach production in Florida.

Fla. Agr. Exp. Sta. Circ. 264. 16 p.

27. Sher, S. A., and M. W. Allen. 1953. Revision of the genus Pratylenchus (Nematoda:Tylenchidae). Univ. Calif. Publ. Zool. 57:

441-470.

28. Steiner, G. 1949. Plant nematodes the grower should know. Soil

Sci. Soc. Fla. Proc. 46:72-117.





62



29. Thorne, G. 1961. Principles of nematology. McGraw-Hill Book Co.,

Inc., N. Y. 553 p.

30. Young, T. W. 1954. An incubation method for collecting migratory

endo-parasitic nematodes. Plant Disease Reptr. .38:794-795.














BIOGRAPHICAL SKETCH

Donald Eugene Stokes was born August 25, 1931, at Andalusia,

Alabama. After attending public schools in DeFuniak Springs, Florida, he enrolled in the University of Florida, where he received the degree of Bachelor of Science in Agriculture in January, 1956. He was employed by the State Plant Board of Florida in 1956 and served as District Inspector until he entered the Graduate School in 1962. He received a Master of Agriculture degree in April, 1963. He continued graduate work toward the Doctor of Philosophy degree while continuing employment with the Division of Plant Industry, Florida Department of Agriculture and Consumer Services (formerly the State Plant Board) until completion of requirements for the degree in June, 1972.

He is married to the former Alma Grace Pollman, and has five

children: Mary Donna, Jack, Nelle, Bertha, and Susan. He is a member of Alpha Zeta, Phi Sigma, the Society of Nematologists, the European Society of Nematologists, the Organization of Tropical American Nematologists, The Florida Nematology Forum, and the Florida State Horticulture Society.














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.



Virnon G erry, Chairman
Professor of 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.



Gr6er C. Smart, Jr.
Associate Professor of 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.



Daniel A. Roberts
Professor of Plant Pathology

This dissertation was submitted to the Dean 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, 1972 ,


e College of Ag culture


Dean, Graduate School




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Parasitism of Pratylenchus spp. to 'Lovell,' 'Nemaguard' and 'Okinawa' Peach By DONALD EUGENE STOKES 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 1972

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ACKNOWLEDGMENTS The author wishes to express his sincerest appreciation to his supervisory committee for guidance and criticism; Dr. Vernon G. Perry, Chairman; Dr. Grover C. Smart, Jr.; and Dr. Daniel A. Roberts. Special recognition to Dr. Perry for untold assistance is hereby extended. The author expresses his gratitude to Mr. Halwin L. Jones, Director, Division of Plant Industry, Florida Department of Agriculture and Consumer Services, and the late Dr. William G. Cowperthwaite, former Division of Plant Industry Director, for their assistance and encouragement concerning graduate studies. Kind appreciation is extended to the Honorable Doyle Conner, Florida Commissioner of Agriculture and Consumer Services. Acknowledgment is also extended to Dr. William G. Eden, Chairman of the Department of Entomology and Nematology, for his assistance and counsel. The author wishes to thank Dr. Robert H. Biggs, Professor, Fruit Crops Department, University of Florida, for providing plant materials. Special thanks to Mr. Jack 0. Holmes and other members of the Hillsborough County Florida Nurseryman and Grower's Association for providing timely financial aid. Thanks also go to Mr. E. M. Collins, Jr., for photographic assistance. To Norma Donovan, typist and friend, the author is Indebted for prompt and efficient handling of this manuscript. Affectionate gratitude is extended to his wife, Grace, and their children for sacrifice and patience. 11

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TABLE OF CONTENTS Page ACKNOWLEDGMENTS ii LIST OF TABLES V LIST OF FIGURES • vii ABSTRACT ix INTRODUCTION 1 LITERATURE REVIEW 4 MATERIALS AND METHODS 13 Soil and Inoculum Sources 13 General Methods 13 Identification of Nematode Species 14 Experiment 1 15 Experiment II 17 Experiment III 18 Hlstopathology of Peach Roots Infected With Pratylenchus brachyurus 19 RESULTS AND DISCUSSION 20 Experiment 1 20 Experiment II 22 Experiment III 22 Hlstopathology of Peach Roots Infected With Pratylenchus brachyurus 34 iii

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Page Roots Stained With Saf ranin-Fast Green 43 Study of Pratylenchus brachyurus From Experimental Plants . ... 45 Study of Pratylenchus penetrans and Pratylenchus coffeae From Experiment 1 47 Fungus Associated With Nematode Colony . 47 Stylet Knob Variation in a Colony of Pratylenchus brach3rurjus 48 CONCLUSIONS 55 SUMMARY 57 LITERATURE CITED 59 BIOGRAPHICAL SKETCH 63 iv

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LIST OF TABLES Table Page 1. A host list of Pratylenchus brachyurus in Florida compiled from the unpublished files of the hematology Bureau, Florida Department of Agriculture and Consumer Services, Gainesville, Florida...., 5 2. A host list of Pratylenchus coff eae in Florida compiled from the unpublished files of the Nematology Bureau, Florida Department of Agriculture and Consumer Services, Gainesville, Florida 9 3. A host list of Pratylenchus penetrans in Florida compiled from the unpublished files of the Nematology Bureau, Florida Department of Agriculture and Consumer Services, Gainesville, Florida 1^ 4. Numbers of Pratylenchus brachyurus , P^. penetrans , and P^. coff eae recovered from the roots of three peach cultivars used as rootstocks inoculated with 50 nematodes per six-inch pot 5. Dry weights of 'Lovell,' 'Okinawa,' and 'Nemaguard' peach seedlings and number of nematodes recovered when uninoculated and inoculated with 100 Pratylenchus brachyurus and grown for 17 weeks in methyl bromidetreated soil 24 V

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Table Page 6. Analysis of variance 27 7. Dry weights of 'Lovell' and 'Okinawa' peach seedlings and numbers of nematodes recovered when uninoculated • and inoculated with Pratylenchus brachyurus and grown for 17 weeks in methyl bromide-treated soil.... 32 8. Analysis of variance 40 vi

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LIST OF FIGURES Figure Page 1. Photograph of young 'Nemaguard' seedlings growing in vermiculite 2. Photograph of 'Okinawa' peaches: plant at right inoculated with 1,000 as compared with uninoculated plant 3. Photomicrograph of a transverse section of 'Okinawa' 35 peach root from uninoculated plant 4. A-B. Photomicrograph of a transverse section of 'Nemaguard' peach root infected by Pratylenchus 37 brachyurus 5. A-B. Photomicrograph of a transverse section of 'Okinawa' 39 peach root infected with Pratylenchus brachyurus 6. Photomicrograph of a longitudinal section of 'Lovell' peach root infected with Pratylenchus brachyurus 7. Photomicrograph of the male tail of Pratylenchus brach3njrus . No bursal rib is present 8. A-B. Photomicrographs of Arthrobotrys anchonia showing constricting rings 50 9. Normal Pratylenchus brachurus stylet showing spheroid knobs 5i vii

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Figure Page 10. A-F, a-f. Lateral views (A-F) of variations in stylet knob abnormalities of Pratylenchus brachyurus correspond to sub-ventral views of malformities (a-f).. viii

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Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy PARASITISM OF PRATYLENCHUS SPP. TO 'LOVELL,' 'NEMAGUARD' AND 'OKINAWA' PEACH By Donald Eugene Stokes June, 1972 Chairman: V. G. Perry Major Department: Entomology and Nematology Three species of Pratylenchus were demonstrated to parasitize and reproduce in roots of 'Lovell,' 'Nemaguard' and '' Okinawa ' peaches, Prunus persica Batsch. Populations of P^. brachyurus (Godfrey, 1929) Filipjev and Schuurmans Stekhoven, 1941, P^. cof f eae (Zimmerman, 1898) Goodey, 1951, and P. penetrans (Cobb, 1917) Chitwood and Oteifa, 1952, increased when inoculated onto these peaches. Two tests for pathogenicity of P^. brachyurus were conducted using inoculum levels of 100 and 1,000 specimens per plant. 'Okinawa,' 'Lovell' and 'Nemaguard' peach were included in the low inoculum test, but 'Nemaguard' was not tested at the high inoculum level. Substantial nematode population increases resulted on each peach variety of each test. Statistical comparisons of plant dry weights (top, root, and total) disclosed insignificant differences between inoculated and uninoculated plants of all the rootstock varieties used in the 100 (low) inoculum level test. In the 1,000 (high) inoculum level ix

PAGE 10

test, however, statistical significance at the 1 per cent level occurred between dry weights of tops, roots, and total plants of 'Okinawa.' Statistical significance was also obtained at the 5 per cent level on root dry weights of 'Lovell' in this test. Roots of inoculated plants in three tests exhibited necrotic lesions. No differences in types of lesions were noted among the various peach rootstocks. Microscopic examination of sections of each peach rootstock revealed damage to cortical parenchyma by P^. brachyurus . The tissue damage varied from a slight effect on several cells to the formation of cavities due to destruction of many cells. All developmental stages of P^. brachyurus were found associated with various degrees of tissue damage. Results of the tests indicated that large numbers of Pratylenchus brachyurus may have little effect on the growth of 'Lovell' and 'Okinawa' peaches. A nematophagous fungus, identified as Arthrobotrys anchonia Drechsler, was found associated with Fratylenchus brachyurus in greenhouse-maintained colonies . Fratylenchus brachyurus males reared on peach did not have a small rib formed by phasmids extending into the bursa. This conforms to the original description but not to the description of P^. brachyurus males recovered from citrus in Florida. Projections extending anteriorly from the stylet knobs were observed in some populations of P^. brachyurus when reared on 'Dixie 18' X

PAGE 11

field corn but not on 'Lovell' peach or sweet orange. Citrus sinensis Osbeck. Progeny of the nematodes with this characteristic did not possess the projections when reared on peaches or citrus. I

PAGE 12

INTRODUCTION Several factors have until recently prevented the development of a major peach-growing industry in Florida. Peach, Prunus persica Batsch, is susceptible to the root-knot nematode species Meloidogyne Incognita (Kofoid and White, 1919) Chitwood, 1949 and M. javanica (Treub, 1885) Chitwood, 1949, the dominant root-knot nematodes in Central and North Florida. 'Lovell' peach, highly susceptible to both root-knot nematode species, was a common rootstock in these areas (26). Resistance in peach rootstocks to both of these species was made available with the introduction of the 'Okinawa' and 'Nemaguard' rootstock varieties. Scion varieties used for fruit production normally are propagated onto rootstocks by bud grafts. These rootstocks, plus other research developments, have recently made possible economic production of peaches in Florida. Malo (20) found that the nature of resistance of 'Nemaguard' and 'Okinavja' peach rootstocks to M. javanica lies in the ability of the host plant to inhibit nematode growth. This is accomplished by walling off the nematodes and suppressing development of giant cells (or nurse cells) on which the nematode feeds. Little or no attention has been devoted to the reaction of Pratylenchus spp. on the root-knot nematode resistant 'Okinawa' and 'Nemaguard' peach rootstocks. The nematode genus Pratylenchus Filipjev contains certain obligately plant parasitic species which rank among the more important organisms responsible for plant root destruction (29). 1

PAGE 13

2 Steiner (28) ranked Pratylenchus spp. as tissue parasites "par excellence" and major economic pests. A recent key (5) lists 35 species of Pratylenchus . Principal factors influencing distribution of Pratylenchus species are climate and soil type (17). Pratylenchus spp. were recovered from over 95 per cent of 215 samples of varied host and representative soil types collected from Gainesville to Homestead, Florida (8). Steiner (28) stated that under certain conditions Pratylenchus may multiply to enormous numbers, particularly in orchards that remain undisturbed for years, but also on some annual crops such as peanuts. Species of Pratylenchus found in Florida associated with economic crop plants include P. brachyurus (Godfrey, 1929) Filipjev and Schuurmans Stekhoven, 1941, P. cof feae (Zimmerman, 1898) Goodey 1951, and P. penetrans (Cobb, 1917) Chitwood and Oteifa, 1952. P. brachyurus is the most commonly observed species of the genus in Florida (8) and is often recovered from soil and root samples taken from peach trees. P^. penetrans has been identified as an agent in the peach replant problem in Canada (22) and causes appreciable injury to roses grafted on Rosa f ortuneana Lindley stock in Florida (19). P. cof feae stunted young apple trees and caused root injury to peach trees and other fruit crops when planted in infested soil (3). This nematode is associated with several economic plants in Florida.'^ ''Unpublished records of Nematology Bureau, Division of Plant Industry, Florida Department of Agriculture and Consumer Services, Gainesville.

PAGE 14

3 Since P. brachyurus , P. coffeae, and ?_. penetrans are prominent parasitic nematodes, the possibility exists that they may cause damage to peach roots. Since 'Okinawa' and 'Nemaguard' rootstocks show resistance to M. incognita and M. javanica, it is important to determine the reaction of other plant parasitic nematodes to these rootstocks. The primary objective of this study was to determine if P^. brachyurus , P^. coffeae , and P^. penetrans parasitized, reproduced upon, and caused damage to 'Lovell,' 'Okinawa' and 'Nemaguard' peach rootstocks. Secondary objectives were to study the general morphology, life cycle stages, and feeding habits of P^. brachyurus .

PAGE 15

LITERATURE REVIEW Since 1880 when the first species of Pratylenchus was described as Tylenchus pratensis by de Man (21) a total of 34 species has been described. Filipjev (9) erected Pratylenchus in 1936. A monograph of the genus was published by Sher and Allen (27) in 1953. They listed the following synonyms for P^. coffeae: Tylenchus coffeae Zimmerman, 1898 Tylenchus musicola Cobb, 1919 Pratylenchus musicola (Cobb, 1919) Filipjev, 1936 Tylenchus mahogani Cobb, 1920 Anguillulina mahogani (Cobb, 1920) Goodey, 1932 Pratylenchus mahogani (Cobb, 1920) Filipjev, 1936 Anguillulina pratensis Goffart, 1929 P. coffeae attacks a wide range of economic plants including camellia, vetch, and strawberry in the United States (29). Table 2 gives a partial list of host plants of P^. coffeae in Florida. Colbran (4) reported it to be associated with Prunus persica Batsch in Australia. Pratylenchus penetrans (Cobb, 1917) Chitwood and Oteifa, 1952, had been named Tylenchus penetrans earlier by Cobb (17). Records indicate that P^. penetrans has one of the widest known host ranges of any species in the genus (29). In Canada, P^. penetrans has been defined as a serious problem in peach replant (22). 4

PAGE 16

5 Table 1. A host list of Pratylenchus brachyurus in Florida compiled from the unpublished files of the Nematology Bureau, Florida Department of Agriculture and Consumer Services, Gainesville, Florida Scientific Name Common Name Acer saccharinum L. Aleurites Fordii Hemsl. Aloe sp. Annona glabra L. Arecastrum Romanzof f ianum Becc. Asclepias tuberosa L. Asimina parviflora (Michx.) Dural, Begonia sp. Carya illinoensis Koch Cassia sp. Chamaecrista sp. Moench. Chamaecyparis obtusa Sieb. & Zucc. Chamaedorea elegans Mart. Chrysanthemum sp. Citrus sp. Citrus aurantium L. Citrus limon Burm. Citrus paradisi Macf . Citrus reticulata Blanco white maple tung oil tree pond apple green palm butterfly weed papaw pecan Hinoki cypress parlor palm sour orange lemon grapefruit tangerine

PAGE 17

6 Table 1. (continued) Scientific Name Common Name Citrus sinensis Osbeck Cocos nucif era L. Codiaeum sp. Juss. Cynodon dactylon Pers. Digitaria san^uinalis (L.) Scop. Diospyros virginiana L. Eremochloa ophiuroides Hack. Eupatorium capillif o.lium (Lam.) Small. Euphorbia sp. Euphorbia pulcherrima Willd. Fragaria sp. Gladiolus sp. Glottidium vesicarium (Jacq.) Harper Hemerocallis f ulva L. Hibiscus sp. Hibiscus rosa-sinensis L. Indigof era endecaphylla Jacq. Juglans nigra L. Jussiaea peruviana L. Lantana sp. sweet orange coconut palm Bermuda grass crabgrass common persimmon centipede grass dog fennel poinsettia coffee weed common orange daylily Chinese hibiscus indigo black walnut primrose willow

PAGE 18

Table 1. (continued) Scientific Name Common Name Ligustrum sp. Li gust rum lucidum Ait. Lonicera sp. Lycopersicon esculentum Mill. Magnolia grandif lora L. Meibomia sp. Menthe spicata L. Myrica cerif era L. Paspalum notatum Flugge. Paspalum urvillei Steud. Pennisetum glaucum R. Br. Persea americana Mill. Philodendron sodiroi Hort. Phoenix Roebelenii O'Brien Phytolacca americana L. Pinus clausa (Engelm.) Vassey. Pinus elliottii Engelm. Pinus palustris Mill. Polystichum adiantif orme J. Smith glossy privet tomato bull bay spearmint wax myrtle Bahia grass pearl millet avocado philodendron dwarf date-palm pokeberry sand pine slash pine longleaf pine leather leaf fern

PAGE 19

8 Table 1. (continued) Scientific Name Common Name Prunus persica Batsch Psldlum gua j ava L. Pterldlum aquilinum (L . ) Kuhn Pterls latluscula Desv. Pyrus sp. Quercus laevls Walt.Saccharum of f icinarum L. Salntpaulia ionantha Wendl. Schlnus terebinthifolius Redd. Serenoa repens (Barth.) Smllax sp. Stenotaphrum secundatum Kuntze Stillingia angustlfolia (Torr.) S.Wats. Vlgna sinensis 'Iron' (L.) Endl. Zoisia japonica Steud. Zolsia japonica 'Emerald' Steud. peach guava turkey oak sugar cane African violet Brazilian pepper tree saw-palmetto St. Augustine grass queen 's-root cowpea Japanese lavm grass

PAGE 20

9 Table 2. A host list of Pratylenchus cof f eae in Florida compiled from the unpublished files of the Nematology Bureau, Florida Department of Agriculture and Consumer Services, Gainesville, Florida Scientific Name Common Name Aglaonema simplex Blume Ananas comosus Merr. Chamaedorea self rizii Burret Citrus sp. Hedera canariensis Willd. Hibiscus sp. Ilex glabra Gray Monstera deliciosa Liebm. (Ceriman) Pinus elliottli Engelm Quercus laurif olia Michx. Trif olium hybridum L. Vriesia imperialis Carriere pineapple parlor palm Algerian ivy gallberry slash pine laurel oak alsike clover bromeliad

PAGE 21

10 Table 3. A host list of Pratylenchus penetrans in Florida compiled from the unpublished files of the Nematology Bureau, Florida Department of Agriculture and Consumer Services, Gainesville, Florida Scientific Name Common Name Aglaonema commutatum Schott. Chrysanthemum sp. Eremochloa ophluroides Hack. Ficus pandurata Sander Ilex glabra Gray Juniperus silicicola (Small) Bailey Pinus sp. Polys tichum adiantif orme J. Smith Zoisia sp. centipede grass flddleleaf fig gallberry Southern red cedar leather leaf fern Japanese lawn grass

PAGE 22

11 In Florida, P^. penetrans is parasitic to a rose rootstock, Rosa fortuneana Lindley (19), and leather leaf fern, Polystichum adiantiforme J. Smith (24). Other plants attacked by P^. penetrans in Florida are listed in Table 3. According to Loof (17), synonyms for P^. brachyurus include: Tylenchus brachyurus Godfrey, 1929 Pratylenchus leiocephalus Steiner, 1949 Pratylenchus pratensis Thorne, 1940 Pratylenchus steinerl Lordello, Zamith and Boock, 1954 P^. brachyurus is reported to attack many economic plants including pineapple, peanut, strawberry, lespedeza, cotton, okra, corn, tobacco, and potato tubers (29). Pratylenchus spp. have been the most frequently found plant parasitic nematodes associated with citrus roots (2). P^. brachyurus is the species of the genus most commonly found in Florida (8) and it is reported pathogenic to citrus (1). Additional hosts of P^. brachyurus in Florida are listed in Table 1. Fliegel (10) reported Pratylenchus vulnus , P. zeae and P^. brachyurus on peach in Georgia. P^. brachyurus , the most commonly found species, was not associated with severe damage to roots and populations were relatively small. P^. zeae generally did not affect plant root systems. Godfrey (11) characterized symptoms of pineapple roots caused by P. brachyurus as irregularly elongate brown spots which are light in color when young and later become darker. Lesions are not sunken and are readily distinguishable from lesions produced by other organisms. Discoloration and cell destruction were observed from the epidermal cells through the cortex.

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12 Brooks and Perry (1) reported that injury by P. brachyurus extended two to three cells to each side of the nematode, which suggests toxic substances secreted by the nematode. Graham (12) reported damage to tobacco and corn by Pratylenchus brachyurus and P^. zeae. These two nematode species were always found in the outer parenchyma and never in the vascular tissues. In Pretoria, Koen and Hogewind (15) observed that Pratylenchus brachyurus caused irregularly shaped purple-brown lesions 1-5 mm in diameter on potato tubers. Infected tubers lose considerable weight when stored at room temperatures and are unsuitable for use as seed pieces. By digging potatoes at an early stage and storing at 5 C no visible lesions developed . Pratylenchus vulnus was reported (18) to be associated with declining peach orchards in California. D'Souza (6) reported vulnus as a pathogen of peach in California; however, the nematode appeared most prominent as a disease incitant or a disease aggravator and alone did not stunt peach seedlings at the population levels occurring in peach orchards. Feeding by Pratylenchus spp. is of two obvious types. One type (15) is exhibited by P^. crenatus which feeds ectoparasitically on epidermal cells, migrating from cell to cell and causing light necrotic spots on the roots. Another type (23) is exhibited by scribneri , which feeds endoparasitically on amaryllis, forming large necrotic areas in the cortex with "nests" of eggs and larvae.

PAGE 24

MATERIALS AND METHODS Soil and Inoculum Sources Soil for all experiments was obtained from the "Agronomy Farm," Institute of Food and Agricultural Sciences, University of Florida, Gainesville, and was of the Lakeland fine sand series. The soil used in each experiment was fumigated with methyl bromide (2 pounds per cubic yard) and aerated at least 3 weeks before use. The three species of Pratylenchus under investigation were obtained from different sources. Specimens of Pratylenchus brachyurus were obtained from peach growing in a nursery near Belleview, Florida; penetrans from leather leaf fern. Crescent City, Florida; and £. cof f eae from Chinese evergreen growing in a greenhouse at Gainesville, Florida. General Methods Populations of P^. brachyurus used as inoculum were maintained and increased on 'Lovell' peach; 'Dixie 18' field corn, Zea mays L. ; or sweet orange. Citrus sinensis Osbeck. P^. penetrans and P. cof feae populations were maintained and increased on the hosts from which they had been collected, leather leaf fern and Chinese evergreen, respectively. Inoculated host plants used for colonizing respective nematode species were grown in wooden boxes, approximately 12 x 24 x 10 inches in dimension, containing fumigated soil. Adequate quantities of nematodes for 13

PAGE 25

14 inoculum were thus always available when needed. All colonization and pathogenicity tests were conducted in an environmental chamber at 24 ± 2 C and illuminated 2200 ft c for 16 hours per day. Nematode specimens used for inoculum were obtained by incubating appropriate infected plant roots for about 2 days in glass pint jars containing a small amount of water. Root washings from the jars were placed onto a facial tissue in a Baermann funnel partially filled with distilled water and the nematodes were recovered in the bottom of the funnel. Nematodes recovered were subsequently hand-picked into distilled water before use. A ratio of nine females to one male was used for P^. penetrans and P. cof feae where males are plentiful, and only females, of course, for P. brachyurus where males are rare. Identification of Nematode Species Using the following method Pratylenchus spp. were identified both at the beginning of the test and after plants were harvested. Specimens were hand-picked into distilled water on a glass slide. The slide was moved about over a small gas flame for 5-6 seconds duration and repeated until the nematodes ceased twisting and straightened out. Glass rods of a diameter near that of the nematodes were then placed in a triangle around the specimens and a cover glass applied. Zut was used to seal the edges of the cover glasses to the slides. Other specimens so killed were fixed in 3 per cent formalin and mounted in formalin. Some of those fixed in the 3 per cent formalin were processed to anhydrous glycerine and mounted, using

PAGE 26

15 Selnhorst's method (25). Specimens from each method were then studied in detail with the aid of a research microscope equipped with an oil Immersion objective. Experiment I Peach seedlings of 'Lovell,' 'Okinawa,' and 'Nemaguard' varieties grown in vermiculite (Fig. 1) to a height of about 2 cm were obtained from the Fruit Crops Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, for experimental purposes. When seedlings were approximately 3 cm in height each was transplanted into a 6-inch clay pot partially filled with fumigated soil. Each peach variety was replicated three times for each of the three nematode species. One pot of each peach variety served as an uninoculated control for each replicate. The plants were inoculated with nematodes 16 days after transplanting, when they were approximately 4 cm in height. Fifty handpicked specimens of the appropriate Pratylenchus species in a water suspension were withdrawn into a 10 cc capacity hypodermic syringe (without needle) and ejected about three quarters of an inch deep into the soil of a designated pot. A thin layer of moistened sterilized soil was used to cover the existing soil and distilled water was added. This procedure was followed in all inoculations. Distilled water was added to each pot as needed to maintain adequate soil moisture. A liquid fertilizer solution (Ortho^ 12-6-6) at the rate of 1 tablespoon per gallon of water was applied every 2 weeks. Plants were sprayed with Kelthane as needed to control mites. The plants were visually inspected every day.

PAGE 27

16 Fig. 1. Photograph of young 'Nemaguard' seedlings growing in vermiculite .

PAGE 28

17 Sixteen weeks after inoculation, the plant tops were removed just above the soil line and discarded. Roots were removed from the soil and two 5-gram samples (net weight) were placed into a food blender and comminuted for approximately 20 seconds. The blender contents were then washed onto a facial tissue held in a Baermann funnel partially filled with distilled water and left overnight. The nematode suspension was drawn from the Baermann funnel and poured onto a 325-mesh sieve. The sievings were washed into a Syracuse watch glass and the nematodes counted with the aid of a dissecting microscope. Soil was mixed thoroughly and two-100 ml samples were processed by a centrifugal-flotation technique (13). Nematodes recovered were placed into a Syracuse watch glass and counted. The total number of nematodes per pot was calculated. Experiment II Five replicates of 'Lovell,' 'Okinawa,' and 'Nemaguard' peach approximately 4 cm high, growing in 8-inch clay pots partially filled with sterilized soil, were each inoculated with 100 hand-picked Pratylenchus brachyurus . This experiment was of 17 weeks duration. Watering, fertilizing, spraying for pest control, and inspection for Experiments 11 and III were as described for Experiment I. Seventeen weeks after inoculation tops were removed from plants. Roots were individually removed from the soil and washed to remove adhering soil particles. The washings were collected in a 2-liter capacity can, were roiled, and a 500 ml sample removed and processed by the centrifugation-f lotation technique (13). Root washings in Experiment I were not processed separately, but included with soil processing.

PAGE 29

18 The soil recovered from each treatment was mixed thoroughly, a 100-ml sample removed and processed by the centrlfugation-f lotation technique. Nematodes recovered from root washings and soil were counted with the aid of a dissecting microscope and the total number of specimens per replicate was calculated. Roots were incubated (30) by placing each replicate individually in a 4-pound (4% x 3^5 x 13 inches) polyethylene bag. After 3, 7, 14, 21, and 28 days, emerged nematodes were collected by washing the roots and the interior of the polyethylene bags with tap water by a jet nozzle attachment. Washings were collected in cans and poured onto a 325-mesh screen. Nematodes collected on the screen were washed into Syracuse watch glasses for counting. Examinations and counting of nematodes were accomplished with the aid of a dissecting microscope. Total numbers of nematodes from each root system was obtained from roots, root washings, and soil samples. Plant tops and roots (after incubation) were maintained for 2 weeks at 92 C in a plant-drying oven to obtain dry weights. Identity of tops and roots was maintained throughout the experiments. Experiment III Material and methods for Experiment III were the same as for Experiment II except 1,000 hand-picked Pratylenchus brachyurus per repli cate were used and only 'Okinawa' and 'Lovell' peach were included. All handling of plants and soil was the same as for Experiment II

PAGE 30

19 Hlstopathology of Peach Roots Infected With Pratylenchus brachyurus Peach roots infected and not infected by ]P. brachurus were killed and fixed in FAA (formalin, 6.5 ml; glacial acetic acid, 7.5 ml; 50 per cent ethanol, 100 ml), dehydrated to tertiary-butyl alcohol and embedded in paraffin. Sections cut 12 y thick with a rotary microtome were affixed to glass microscope slides with Haupt's adhesive, stained with safranin-f ast green and mounted in Fisher's "Permount" according to Johansen's (14) procedure.

PAGE 31

RESULTS AND DISCUSSION Experiment I Plant growth was virtually uniform throughout the 16-week experiment and no noticeable differences occurred on the above-ground plant parts. Each replicate in each treatment yielded greater nematode populations than had been used as inoculum, indicating parasitism and reproduction by P^. brachyurus , P^. penetrans , and P, cof f eae. UslAg an average figure for all three rootstocks, P^. brachyurus reproduced 3.2 times; P. penetrans . 2.7 times; ard P. coffgae, 2.3 times the original inoculum levels (Table 4) . And using an average figure for all three nematodes, 'Okinawa' peach supported the largest total populations, which was 3.3 times the inoculum level. 'Lovell' and 'Nemaguard' peach supported total nematode populations of 2.3 and 1.8 times, respectively, the inoculum level (Table 4). P^. brachyurus was chosen for further study because it reproduced at a greater rate than did P^. penetrans or P. coffeae. P. penetrans and P^. coffeae should not be discounted as potential pathogens of peach, because reproduction occurred and population Increases were noted in 'Lovell,' 'Okinawa,' and 'Nemaguard' cultivars. Roots of inoculated plants had very small brownish lesions that did not occur on roots of uninoculated plants. 20

PAGE 32

21 o to o rH »*-< u to to 4-1 4J O e o U o 0) o (U r\ \j CO u 6 (U 0) > C o CJ o u J= (U u (U cd •H CO 0) 0) 14-1 X) 14-1 14-1 o f-i •H 4-1 rH 3 3 O ^ O to C u 3 0) to u iH Q) 3 P. u to (U o ^1 Q> to V4 J= 43 4-1 4J O O IH OCO O U * 0) rH a ^ to Oi 0) H PL4 00 eg vO rH fv4 O ro m in t» CSl \o rH VO vO CO O 00 00 a\ rH CO O m oo o ro > > o o o l-J I-) h-J to to to CO c c c •H -H A! ^ ^ O O O CO 3 V4 M CO to 3 3 00 bO 00 tO to tO B e ^ (U a> e

PAGE 33

22 Experiment II Above-ground plant symptoms caused by nematode Injury were not observed during this experiment making it impossible to distinguish between inoculated and uninoculated plants. But P. brachyuruis , at the 100 per pot inoculum level on 'Okinawa' peach, reached a density of 3.5 times the original population. 'Lovell' and 'Nemaguard' peach at the 100 inoculum level supported P^. brachyurus populations of 2.7 and 2.4 times, respectively, the original inoculum level (Table 5). An analysis of variance showed no statistical significance between root, top, or total dry weights (Table 6). Experiment II results substantiate that P^. brachyurus is capable of reproducing in 'Okinawa,' 'Nemaguard,' and 'Lovell' peach roots. 'Okinawa' again supported the largest nematode population with 'Lovell' and 'Nemaguard' following in descending numbers. Small brownish lesions were again observed on roots of plants inoculated with P^. brachyuru s. Experiment III No differences in growth were observed between inoculated and uninoculated plants during this experiment and no symptoms of injury were detected on above-ground plant parts (Fig. 2). At the 1,000 inoculum level, P^. brachyurus reproduced 2.4 and 2 times the original population on 'Okinawa' and 'Lovell' peach, respectively (Table 7). 'Nemaguard' was unavailable for use in this experiment.

PAGE 34

23 Fig. 2. Photograph of 'Okinawa' peaches: plant at right inoculated with 1,000 as compared with uninoculated plant.

PAGE 35

n 3 U 3 «4-l O o u CO 0) U B 3 cn c 3 x: -a o c c CO (U m 00 -u c CO •H V4 rH 1^ X) ^ CO (0 H TJ 3 3 0) 60 O 4J CO O CO 6 c 0) 0) •H )^ 2: 4J T3 1 c 0) CO 13 c •H CO e 0) o u U CO CO iH & 3 1-1 CO O c O •H c 4-1 •H o J 13 0) M 0) > o o (U CD Q) O CD •H TJ <1» o > 4J CO E T3 0) C O C CO o 60 cn U-l -C3 o o CO s 4-1 cn c E CO CO H u Pl, O C •H PO 6£ H •H OJ 4-1 O P o Pi T3 4-1 C C CO (U E 4= 4J CJ CO CO 0) (U M C30 CN O CJN vO vo cTi O O O 0) +j CO rH i-H 3 rH O 0) O > c O -H J c 3

PAGE 36

25 4J m C E tS cfl iH M PLi O C •H u &. O M •H H 0) > U o P O CNI CNJ O rH <} 00 CN O <• CO fo m CM in m CO i-l o o o o o m CN 00 in rH cri O in cyi CN 00 OCN o m vO CO m rH CO O rH rH CM O VO rH 3 to O G O •H C A! -H O C 3

PAGE 37

01 >4-l T) o o lO o vO m rH o o o O o 4-1 00 ro t-l vD o o O B CO ,—1 o\ , — 1 CO CM CM 00 r-i M PL. CS CM CN CN CM CN CM CM CM CM CO CO CM O rH rH c vO a\ oCM m CN rH 00 u O o CN 0) O •H (U u z c z c H -H 3

PAGE 38

u to cr CO c nJ d) CO 0) M n) 3 cr to of E 3 CO df c o •H to •H to > of 0) CJ 3 o CO to H O to u to •o B o u CO 6 to u 60 H 0) U 73 (X O a C to •H M to > o CO •H CO to -am o u-i O CM o o CU 3 O o c •H 3 IN CM O rH O CM U CO iH 00 CJ\ 3 )-i rH 3 H >i (U Xi 00 > u ro O to 1-1 M vO a 6 4-1 to o 0) o 01 •H M Hi > tn iH to -aCO rH in O CO o o OJ 4-1 CO i-H CO 3 CM u • o c 3 rH O rH O CM CM rH 00 CTl CO 3 l-i 00 u 00 CIt • u c CU B 4J to (U M H U rH O to H 4J U O W H

PAGE 39

o CM J3 H T3 6 O S-l <4-l 03 E 1-1 60 00 •H (U u o c nJ •H > o CO •w CO >^ iH cd vO 00 00 a^ -H O 00 CO o 4-1 CO -H CO c^ 3 l-i rH 3 rH > o CM o CO CM rH p u C » u o o u CO c O 0) u c Id •H Cd > o CO CO CM iH CN O O T3 0) 3 CJ o c •H c 3 r-l CTi iH VO O rH O 00 00 CO 3 >-i 3 ^ o CJ r-CO U in JO 4-1 c B 4J CO cu M H W H

PAGE 40

M nj 3 cr w c to CO 0) )-l CO 3 cr CO e 3 •a o CO > 0) o u 3 o J3 CO H CO 4J CO £ o CO e CO u 4-1 J= 00 H 0) & u XI A. O CO & CO C •H O a C CO •H M CO > <4-l o CO >. CO iH •-< o o (U U CO iH vO 3 <-{ U O vO C •H c 3 «H CO O .H 00 3 3 >. x: to o 00 CO m c B CO (U O CO u u u o H W H J3 CO H O CO XI e o <4-l w § 00 oo •H u o 4J CO u CO C O 0) o c nt •H 5 CO •H CO rH CO 00 fO 1^ o o X) 01 4-1 CO •H 3 O O o CN c .H c 3 m CM o vO CX3 CTi CO 3 u 3 CJ to CO u iH rH c 01 B •u Vj CO O 0) M M Vi O H W H CO

PAGE 41

CO H 4-1 n) 0 o 0) e u M •nl 0) •a o o -a u 3 tiO nj E (U Z o c to •H > 14-1 o to •H CO >. r-( to 00 ro o o 3 a o C •H c 3 O (>J o 00 cn iH 00 CO 3 1-1 3 00 O to U in 4-> 3 0) e u to 0 CO e tu z ON rH o o -a (U 3 o o c •H 3 <3\ 00 CM o O cn 00 H W H 0) u cd •H >^ to > of 4-1 3 •H eu to e 4J to O to to (U 1-1 4-1 M U o H w H CO 3 3 >^ o rH CO u vO

PAGE 42

\0 0) U (0 3 cr to C ca CO (U d cr CO o B D XI C o H 4-1 es •H >-i to > o 01 o (U to H o to a o u CO B to M 00 x: 60 •H u to o XJ to D 60 to a <4-( o CO >^ to n o XI 0) 3 o o e H c 3 m cn \D m in tn 3 VI 3 CJ rH to u fsl c; cu a to cu u H to U o >-l u o W H

PAGE 43

32 n o c CO to d) to \-* uru O >i w o to t-Q B P (0 C hu T3 u c c to le (0 >i bO u C to •H tH PM •H ^ •u u o o 0 Pi 73 4J c c to rH rH 00 ON CO rH CO CN rH <• CN rH CN tN O CN CN CN CN CM CM CM rH CM CT> O rH CN O rH rH CM CO 00 rH rH H rH rH rH to H a> -0vt o CM o o o rH 3 rH O 0) o > c O -H C 3

PAGE 44

o o O CT^ iH vD CT\ <3\0 CM CT> 00 CTs r-. o iH CM rn olo o H o o o o o iH CO CO 00
PAGE 45

34 Statistical analysis of data obtained from this experiment and the analysis of variance table show that top, root, and total dry weights of 'Okinawa' plants inoculated with ?_. brachyurus are statistically different from uninoculated plants at the 1 per cent leyel of probability (Table 8). This statistical method also shows that root weights of inoculated and uninoculated 'Loyell' plants were statistically different at the 5 per cent level of probability (Table 8). No statistically significant difference existed at either the 1 or 5 per cent levels of probability when top dry weights or total dry weights of 'Lovell' peach were compared. Obvious disease symptoms such as stunting, chlorosis, and leaf drop being absent on test plants suggests that injury to peach is very gradual and subtle. Histopathology of Peach Roots Infected With Pratylenchus brachyurus Microscopic examination of sections of infected roots revealed the presence of P^. brachyurus within and damage to cortical regions (Fig. 4). Various developmental stages of the parasite were found within root tissues showing various stages of damage. Eggs were noted singly and in groups of three to four inside single cavities. Tissue damage varied from slight effects on several cells to the formation of small cavities due to destruction of many cells (Fig. 5). Small root lesions and ruptures were obvious in root epidermis in some instances, but injury was not extensive.

PAGE 46

Fig. 3. Photomicrograph of a transverse section of 'Okinawa' peach root from uninoculated plant.

PAGE 47

fig. 4. A-B. Photomicrograph of a transverse section of 'Nemaguard' peach root infected by Pratylenchus brachyurus . A. Portions of nematode bodies (arrows) are adjacent to cavities in the cortical region. B. Head portion of a nematode in cortical region. Note the absence of cavity formation.

PAGE 48

37

PAGE 49

Fig. 5. A-B. Photomicrograph of a transverse section of 'Okinawa' peach root infected with Pratylenchus brachyurus . A. Portion of a nematode (a) is in one of the cortical cavities (b) . Note proximity ojC cavities to epidermis. B. Head portion of a nematode in cortical region near epidermis.

PAGE 51

u as < 3 cr 0) w iH ,o c H 0) S O CO CO e o IM 0) O c o o C •H CO •U •H nJ U to > > >4-l o <4-l o CO •H ^ 3 U at u .a o 01 u cd u cd > cn (0 to •K vO 00 < 00 CM O ON VO CO rH H O 0) 3 o C •H 3 o u-l 00 t-^ CO CM VO rH iH eg 0) u CO tH 3 a o c tH 00 CT> to 3 u 3 u Id .13 00 u a B U U <-! to O CO
PAGE 52

41 to 00 K * O CM (U « 3 a" w C to 0) 05 OJ (-1 cr CO 4-1 -a CO H 14-1 o 6 o M CO B nJ 60 bO •H CO 4-) o > O H-1 i-l CO m ON 00 m c-j o O H \o in <^ O 00 CSI i-i 00 oi •a (U 3 O o c •H c 3 0) 3 u o c 1-1 CO 3 u 3 X! O to to H M-l O to 4-1 CO •o B o I-l M-l cn E CO u C •H 4-1 x: bO •H 0) fx o to CO C •H O in 00 vO m O iH o 0) 4-1 CO rH VO 3 o o c •H C 3 vO 00 •oCN 00 CN rH m in rH r-i o CN i-H 00 cr> T3 cu o 3 o CO 4) a C CO •H U CO > 4-1 O 4-1 to c •H tI CO > o CO CO C 0) S 4J >.| rH CO O to lU M 4-1 V4 O H W H

PAGE 53

42 43 n) H o 6 o u <4-l (1) o o u nJ C •H O (U o c ca •H u CO > o CO to >^ cd •K K O to O IT) ON O tH o T3 0) 3 CJ o c •H c 3 in CO (U CO o cfl e vD 3 ca in 1-1 \o CO 60 in o CN d O •H 6 U 3 CO cu 3 O o c iH CX) CT> CO 3 3 u (d M JO c E cd ,13 CO H iw O Cd B o u CO S CO u 00 c 00 •H CU CO o CO 3 CO •H * * o m CSI O -if r~ CN vO O cu 4-1 CO 3 o O CM c c 3 o CM CM iH CO vO C-J 00 (U 3 o o c r^ 00 H W H 4) U C 10 •H u (O > «4-l O 01 C •H (U CO e u -H fH CO o CO CO cu 4-1 o H W H u at (U a o p. m 4J CO a CO o •H IH •H C 00 •H CO *

PAGE 54

43 The nematodes observed inside peach roots were mostly oriented along the transverse root axis with heads away from root tips. Occasionally, the heads were observed at right angles to the transverse axis. The stele was not generally infected; however, in two instances single specimens of P^. brachyurus were found inside the stele (Fig. 6). Roots Stained With Saf ranin-Fast Green The walls of plant cells not affected by the feeding actions of P^. brachyurus appeared green as compared to a red color of the cell walls associated with feeding by the nematodes. Cytoplasm of cells affected by nematode feeding was more granular than that of normal cells. Apparent nematode injury extended sometimes two or three cells away from the nematode, which is suggestive of chemical damage by nematode secretions. Safranin typically stains lignified and cutinized cell walls a brilliant red; whereas, fast green should be prominent on cellulose cell walls (14). No evidence of hypertrophy or hyperplasia was observed. The damage caused by P^. brachyurus to each of the three peach rootstocks was quite similar. There were no apparent differences in the kind or degree of damage caused by the feeding of the different species of nematodes on the root tissue. Both mechanical and chemical factors appeared involved in total plant damage. Cavities located in the cortical parenchyma resulted from feeding and migration of the nematode. Injury to the vascular system was limited to the cells occupied by the nematode or being fed upon. The ability of Pratylenchus brachyurus to successfully parasitize and reproduce on 'Lovell,' 'Okinawa,' and 'Nemaguard' suggests adequate nutrition furnished by this host to the nematodes. There

PAGE 55

44 Fig. 6. Photomicrograph of a longitudinal section of 'Lovell' peach root infected with Pratylenchus brachyurus . Head of nematode (arrow) is inside the stele.

PAGE 56

45 was no evidence in Pratylenchus brachyurusInfected peach to indicate presence of a "walling off" process (20), a mechanical type of plant resistance that develops when Meloidogyne javanica feeds on 'Okinawa' or 'Nemaguard, ' or any other type of plant resistance. "Walling off" causes a breakdown of giant cells which in effect removes the nematode's food source. This type root-knot nematode resistance does not occur in 'Lovell' peach. V_. brachyurus reproduced to large numbers on 'Lovell,' 'Okinawa,' and 'Nemaguard,' but caused slight root damage. Further testing of the three roots tocks is needed to determine the effects of longer exposures to large numbers of P^. brachyurus . P. brachyurus normally fed solitary in the cortical region of peach; however, nematodes in groups of two to three occasionally were observed. As many as six P^. brachyurus in solitary sites were observed in a single transverse peach root section, but in no case was "nesting" found in peach roots, even where cortical necrotic cavities occurred. "Nesting" often occurs in necrotic cavities resulting from the feeding of this species of Pratylenchus . Study of Pratylenchus brachyurus From Experimental Plants Measurements and characteristics of P^. brachyurus recovered from peach roots in Experiments I, II, and III conformed to those described by Sher and Allen (27), but males differed from those described by Brooks and Perry (1) from citrus in Florida. In more than 100 males examined there was no extension of the phasmid into the bursa (Fig. 7), in agreement with Sher and Allen (27). Brooks and Perry (1) reported

PAGE 57

Fig. 7. Photomicrograph of the male tail of Pratylenchus brachyurus . No bursal rib is present.

PAGE 58

47 that the phasmid on males from citrus extended into the bursa. Even when the experimental peach population was reared on citrus, males maintained their bursal characteristic forming no phasmidial rib. Thus two discrete populations of P^. brachyurus exist in Florida; however, these differences do not warrant species separation at this time. Males of the peach population apparently do not inseminate females, as no sperm were observed in the uteri of the females. Study of Pratylenchus penetrans and Pratylenchus coffeae From Experiment I Morphological and dimensional characteristics of P^. penetrans and P^. coffeae recovered from experimental plants conformed to the species descriptions by Sher and Allen. Males recovered from experimental plants comprised approximately 25 per cent and 33 per cent of the total populations of P^. coffeae and P^. penetrans , respectively. Fungus Associated With Nematode Colony Populations of Pratylenchus brachyurus were maintained on 'Lovell,' 'Nemaguard,' and 'Okinawa' peach in a greenhouse for biotype identity in 8-inch clay pots containing a soil mixture of approximately onethird peat and two-thirds sandy soil by volume. A periodic examination of soil and peach roots revealed a fungus associated with certain specimens of P. brachyurus . A fungus structure of the constricting ring type often encircled nematodes in the region of the stylet knob. Other nematodes without the constricting ring fungus were constricted in the stylet knob region as evidence of previous association with the fungus.

PAGE 59

48 Three nematodes trapped in a constricting ring with fungus mycelium attached were placed on Petri plates containing 2 per cent potato-dextrose agar. Cultures of the fungus grown on this agar were transferred to 10 x 50 mm glass test tubes containing 2 per cent corn meal agar plus dextrose. Characteristics of the fungus conformed to the description of Arthrobotrys anchonia Drechsler. Type material of this fungus was collected in Ft. Lauderdale, Florida (7). A. anchonia (Fig. 8-A) was reported (7) to trap and parasitize Eucephalobus sp., but it has never before been reported to trap a plant parasitic nematode (Fig. 8-B) . Stylet Knob Variation in a Colony of Pratylenchus brachyurus Colonies of P^. brachyurus maintained in a greenhouse were examined periodically, particularly to determine nematode vigor, presence of contaminants, or nematode predators and diseases. One collection of P^. brachyurus colonized on 'Dixie 18' field corn contained specimens exhibiting an atypical morphological characteristic of the stylet knobs of the species. The typical shape of stylet knobs of P. brachyurus is spheroid to oval (Fig. 9). Abnormal anterior projections of varying lenths (Fig. 10 A-F and a-f) were formed on one to all three stylet knobs of the atypical specimens grown on 'Dixie 18' corn. These stylet knobs appeared in lateral view to have qiical projections. In no instance was the projection observed extending anteriorly more than half the stylet length. Margins of the stylet knob extensions were within the marginal bounds of the stylet extruder

PAGE 60

Fig. 8. A-B. Photomicrographs of Arthrobotrys anchcnia showing constricting rings. A. Constricting rings are attached to hyphal strands. B. jPratylenchus brachyurus female caught in constricting ring.

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50

PAGE 62

51 Fig. 9. Normal Pratylenchus brachyurus stylet showing spheroid knobs.

PAGE 63

Fig. 10. A-F, a-f . Lateral views (A-F) of variations in stylet knob abnormalities of Pratylenchus brachyurus correspond to sub-yentral views of malforjnities (a-f).

PAGE 65

54 muscles. The abnormal stylet occurred on approximately 40 per cent of the females in certain colonies on 'Dixie 18' field corn but not in all colonies. The abnormality was not found on P^. brachyurus reared on other hosts, and progeny of females possessing the abnormal stylet characteristic reared on peach or citrus did not have the atypical condition.

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CONCLUSIONS Plant parasitic nematodes belonging to the genus Pratylenchus have been recovered from roots of declining peach trees. Three species of this nematode genus, Pratylenchus brachyurus (Godfrey, 1929) Filipjev and Schuurmans Stekhoven, 1941, Pratylenchus cof f eae (Zimmerman, 1898) Goodey, 1951, and Pratylenchus penetrans (Cobb, 1917) Chitwood and Oteifa, 1952, will parasitize and reproduce on peach, Prunus persica Batsch: 'Lovell,' 'Nemaguard,' and 'Okinawa' rootstocks. Pratylenchus brachyurus reproduced a greater population than either P^. cof f eae or P^. penetrans in one inoculation test suggesting greater potential damage to hosts. Damage by Pratylenchus brachyurus to peach roots is typified by lesions on epidermal tissues and injury to cortical regions. Levels of inoculum influence severity of P^. brachyurus damage to 'Okinawa' rootstock. Longer exposures of the host to the nematode could influence severity in plant injury, particularly with perennial crops such as peach in areas with little seasonal climatic variation. It is possible that population thresholds were not reached in 17 weeks. A variable stylet characteristic among Pratylenchus brachyurus populations occurred in certain populations. 55

PAGE 67

56 7. A ring-forming fungus, Arthrobotys anchonia Drechsler, is capable of ensnaring Pratylenchus brachyurus ; however, the efficiency of this fungus to control the nematode is unknown. 8. Pratylenchus brachyurus males reared on peach lack a small rib formed by phasmids extending into the bursa and differ from P^. brachyurus reared on citrus in this respect.

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SUMMARY P. brachyurus occurs in peach nurseries and orchards in Florida, but its possible role in peach decline was heretofore undefined. An initial test was established to ascertain parasitism of P^. brachyurus to 'Lovell,' 'Okinawa,' and 'Nemaguard' peach rootstocks. P^. brachyurus was more prolific than P^. penetrans or P^. cof feae on these hosts. Two pathogenicity tests of 17 weeks duration were conducted using 100 or 1,000 specimens of P^. brachyurus per plant, each treatment being replicated five times. 'Okinawa,' 'Nemaguard,' and 'Lovell' peach were used in the first test, but 'Nemaguard' was not available for the second. The parasite reproduced at rates of 2.7, 3.5, and 2.4 times the inoculum level on 'Lovell,' 'Okinawa,' and 'Nemaguard' peach, respectively (low inoculum level test) and 2.4 and 2 times on 'Okinawa' and 'Lovell', respectively (high inoculum level test). Temperatures of 24 ± 2 C and a photoperiod at 2200 ft c for 16 hrs were maintained throughout all tests. Statistical comparisons of plant dry weights (top, root, and total) disclosed no significant difference between inoculated and uninoculated plants except in the high inoculum level test. Dry weights of top, roots, and total plant of 'Okinawa' peach were statistically significant at the 1 per cent level; with 'Lovell' the dry weight of the roots only was significantly different from the control and that was at the 5 per cent level. 57

PAGE 69

58 Microscopic examination of sections of each peach rootstock revealed damage to cortical parenchyma by P. brachyurus . Tissue damage varied from slight effects on several cells to formation of small cavities due to cell destruction. Results indicate that even large numbers of P^. brachyurus may have little effect on early growth of 'Okinawa' and 'Lovell' peach seedlings. Abnormal stylet knob extensions were discovered on some colonies of ^. brachyurus . Arthrobotrys anchonia ensnared P^. brachyurus specimens in colonies maintained on peach growing in soil containing about onethird peat. Pratylenchus brachyurus males reared on peach did not have a small rib formed by the phasmids extending into the bursa which is characteristic of P^. brachyurus males found on citrus in Florida.

PAGE 70

LITERATURE CITED 1. Brooks, T. L. , and V. G. Perry. 1967. Pathogenicity of Pratylen chus brachyurus to citrus. Plant Disease Reptr. 51:569-573. 2. Christie, J. R. 1959. Plant nematodes, their bionomics and control. Fla. Agr. Exp. Sta. 256 p. 3. Colbran, R. C. 1953. Problems in tree replacement. I. The root lesion nematode, Pratylenchus coffeae Zimmerman, as a factor in the growth of replant trees in apple orchards. Australian J. Agr. Res. 4:384-389. 4. Colbran, R. C. 1955. A preliminary survey of plant nematodes in Queensland. J. Australian Inst. Agr. Sci. 21:167-169. 5. Corbett, D. C. M. 1969. Pratylenchus pinguicaudatus n. sp. (Pratylenchinae :Nematoda) with a key to the genus Pratylenchus . Nematologica 15:550-556. 6. D'Souza, G. I. 1964. The pathogenicity of Pratylenchus vulnus to peach. Ph.D. Thesis, Univ. Calif., Davis. 63 p. (unpublished) 7. Drechsler, C. 1954. Some hyphomycetes that capture eelworms in southern states. Mycologia 46:762-782. 8. Feldmesser, J., W. A. Feder, and J. A. Pinckard. 1956. The occurrence of Pratylenchus spp. in Florida soils. Phytopathology 46:11. (Abstr.) 9. Filipjev, I. N. 1936. On the classification of the Tylenchinae. Proc. Helminthol. Soc. Wash. 3:80-82. 59

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60 10. Fliegel, P. 1969. Population dynamics and pathogenicity of three species of Pratylenchus on peach. Phytopathology 59:120-124. 11. Godfrey, G. H. 1929. A destructive root disease of pineapples and other plants due to Tylenchus brachyurus n. sp. Phytopathology 19;611-629. 12. Graham, T. W. 1951. Nematode root rot of tobacco and other plants. I S.C. Agr. Exp. Sta. Bull. 390. 25 p. I 13. Jenkins, W. R. 1964. A rapid centrifugation-f lotation technique for separating nematodes from soil. Plant Disease Reptr. 48:692. 14. Johansen, D. A. 1940. Plant microtechnique. McGraw-Hill Book Co., Inc., N. Y. 523 p. 15. Klinkenberg, C. H. 1963. Observations on the feeding habits of Ro tylenchus unif ormis , Pratylenchus crenatus , P^. penetrans , Tylenchorhynchus dubius , and Hemicycliophora similis . Nematologica 9:502-506. 16. Koen, H. , and W. L. Hogewind. 1967. Symptoms and characteristics of Pratylenchus brachyurus infestation on stored potatoes. S. Afr. J. Agr. Sci. 10:543-549. 17. Loof , P. A. A. 1960. Taxonomic studies on the genus Pratylenchus (Nematoda). Tijdschr. PlZiekt. 66:29-90. 18. Lownsbery, B. F. , and E. F. Serr. 1963. Fruit and nut tree rootstocks as hosts for a root-lesion nematoda, Pratylenchus vulnus . Proc. Amer. Soc. Hort. Sci. 82:250-254. 19. McFadden, S. E., and A. A. DiEdwardo. 1961. Control of the rootlesion nematode, Pratylenchus penetrans on Rosa f ortuneana stock. Proc. Fla. State Hort. Soc. 74:388-392.

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61 20. Malo, S. E. 1967. Nature of resistance of 'Okinawa' and 'Nemaguard' peach to the root-knot nematode Meloidogyne javanlca . Proc. Amer. Soc. Hort. Sci. 90:39-46. 21. Man, J. G. de. 1880. Die einheimischen, frei in der reinen Erde und im siissen Wasser lebenden Nematoden. Tijdschr. Ned. Dierk. Ver. 5:1-104. 22. Mountain, W. B., and Z. A. Patrick. 1959. The peach replant problem in Ontario. VII. The pathogenicity of Pratylenchus penetrans (Cobb, 1917) Filip. & Stek. , 1941. Can. J. Bot. 37:459-470. 23. Nong, L,, and G. F. Weber. 1965. Pathological effects of Pratylenchus scribneri and Scutellonema brachyurum on amaryllis . Phytopathology 55:228-230. 24. Rhoades, H. L. 1968. Pathogenicity and control of Pratylenchus penetrans on leatherleaf fern. Plant Disease Reptr. 52:383-385. 25. Seinhorst, J. W. 1959. A rapid method for the transfer of nematodes from a fixative to anhydrous glycerine. Nematologica 4:67-69. 26. Sharpe, R. H. , and R. Parker. 1963. Peach production in Florida. Fla. Agr. Exp. Sta. Circ. 264. 16 p. 27. Sher, S. A., and M. W. Allen. 1953. Revision of the genus Pratylenchus (Nematoda:Tylenchidae) . Univ. Calif. Publ. Zool. 57: 441-470. 28. Steiner, G. 1949. Plant nematodes the grower should know. Soil Sci. Soc. Fla. Proc. 46:72-117.

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62 29. Thorne, G. 1961. Principles of nematology. McGraw-Hill Book Co., Inc., N. Y. 553 p. 30. Young, T. W. 1954. An incubation method for collecting migratory endo-parasitic nematodes. Plant Disease Reptr. 38:794-795.

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BIOGRAPHICAL SKETCH Donald Eugene Stokes was born August 25, 1931, at Andalusia, Alabama. After attending public schools in DeFuniak Springs, Florida, he enrolled in the University of Florida, where he received the degree of Bachelor of Science in Agriculture in January, 1956. He was employed by the State Plant Board of Florida in 1956 and served as District Inspector until he entered the Graduate School in 1962. He received a Master of Agriculture degree in April, 1963. He continued graduate work toward the Doctor of Philosophy degree while continuing employment with the Division of Plant Industry, Florida Department of Agriculture and Consumer Services (formerly the State Plant Board) until completion of requirements for the degree in June, 1972. He is married to the former Alma Grace Pollman, and has five children; Mary Donna, Jack, Nelle, Bertha, and Susan. He is a member of Alpha Zeta, Phi Sigma, the Society of Nematologists , the European Society of Nematologists, the Organization of Tropical American Nematologists, The Florida Nematology Forum, and the Florida State Horticulture Society. 63

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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. ;rnon G. Perry, Chairman Professor of 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. •t, Jr. Grm/er C. Smart Associate Professor of 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. Daniel A. Roberts Professor of Plant Pathology This dissertation was submitted to the Dean 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, 1972 College of Agriculture Dean, Graduate School