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An evaluation of two cultivars of Digitaria decumbens as biological control agents of nematodes with emphasis on Meloidogyne incognita and Belonolaimus longicaudatus

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Title:
An evaluation of two cultivars of Digitaria decumbens as biological control agents of nematodes with emphasis on Meloidogyne incognita and Belonolaimus longicaudatus
Added title page title:
Digitaria decumbens
Creator:
Haroon, Sanaa A., 1951-
Publication Date:
Language:
English
Physical Description:
xi, 108 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Bioassay ( jstor )
Eggs ( jstor )
Larvae ( jstor )
Mortality ( jstor )
Plant roots ( jstor )
Precipitates ( jstor )
Roundworms ( jstor )
Solvents ( jstor )
Species ( jstor )
Tomatoes ( jstor )
Belonolaimus longicaudatus ( lcsh )
Dissertations, Academic -- Entomology and Nematology -- UF
Entomology and Nematology thesis Ph. D
Meloidogyne incognita ( lcsh )
Nematode diseases of plants -- Biological control ( lcsh )
Pangolagrass ( lcsh )
City of Gainesville ( local )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1982.
Bibliography:
Bibliography: leaves 105-107.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Sanaa A. Haroon.

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AN EVALUATION OF TWO CULTIVARS OF DIGITARIA DECUMBENS AS
BIOLOGICAL CONTROL AGENTS OF NEMATODES WITH EMPHASIS ON MELOIDOGYNE INCOGNITA AND BELONOLAIMUS LONGICAUDATUS





BY


SANAA A. HAROON


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


1982





















Dedicated to


the memory of my father,

the love of my mother, my devoted husband, Samir, and my son, Ahmed,

and

My professors

Dr. G. C. Smart, Jr., and

Dr. R. A. Dunn














ACKNOWLEDGMENTS


I extend special thanks to Dr. G. C. Smart, Jr., chairman of the supervisory committee, for the supervision of this research since its beginning. Deep appreciation is extended, also, to him for his friendly attitude, patience, time-consuming assistance, encouragement, and suggestions. I express my appreciation to Dr. J. L. Nation, cochairman of the supervisory committee, for providing guidance and supervision of this research. Also, I express gratitude to Mrs. A. J. Overman,

Dr. H. L. Rhoades, and Dr. 0. C. Ruelke, members of my graduate committee, for their valuable assistance in conducting this study and for their critical reading of the manuscript.

Special appreciation goes to Dr. R. A. Dunn for encouragement,

inspiration, and friendship and for providing me with an assistantship during my graduate program. Appreciation is extended also to the American Association of University Women for providing a scholarship during 1980. Finally, to my husband, Dr. Samir El-Agamy, and to my son, Ahmed, I convey sincere appreciation for their understanding and cooperation during this period of my studies.


iii














TABLE OF CONTENTS


PAGE


ACKNOWLEDGMENTS. . . . . . . . . . . . . . . . . . . . . .

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

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


. . .iii


vi


viii


INTRODUCTION. . . . . . . . . . . . . . . . .

EFFECTS OF PANGOLA DIGITGRASS ON MELOIDOGYNE ARENARIA, M. JAVANICA, AND M. HAPLA . . . .

Introduction. . . . . . . . . . . . . . .
General Methods . . . . . . . . . . . . .
Materials and Methods . . . . . . . . . .
Results . . . . . . . . . . . . . . . . .
Discussion. . . . . . . . . . . . . . . .


ISOLATION, PURIFICATION, AND CHARACTERIZATION OF A
CHEMICAL SUBSTANCE FROM PANGOLA DIGITGRASS THAT
IS TOXIC TO MELOIDOGYNE INCOGNITA . . . . . . . .

Introduction. . . . . . . . . . . . . . . . . .
Materials and Methods . . . . . . . . . . . . .
Results and Discussion. . . . . . . . . . . . .
Conclusion. . . . . . . . . . . . . . . . . . .

THE EFFECT OF TRANSVALA DIGITGRASS ON MELOIDOGYNE
INCOGNITA AND BELONOLAIMUS LOGILADATUS .....


Introduction. . . . . . . . . . . . . . . . .
Materials and Methods . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . .
Discussion. . . . . . . . . . . . . . . . . .


EFFECTS OF ROOT LEACHATE FROM PANGOLA AND TRANSVALA DIGITGRASSES ON NELOIDOGYNE INCOGNITA AND BELONOLAIMUS LONGICAUDATUS.

Introduction. . . . . . . . . . . . . .
Materials and Methods . . . . . . . . .


iv


ABSTRACT CHAPTER 1 CHAPTER 2


x


1


4

4
4
5
6
6


CHAPTER 3


CHAPTER 4


8


8
9
16
22


45


CHAPTER 5


45 45 47 48


61


. . . . 61
. . . . 62








PAGE


CHAPTER 6


CHAPTER 7


CHAPTER 8


Results . . . . . . . . . . . . . . . . . . .
Discussion. . . . . . . . . . . . . . . . . .

THE EFFECTS OF VARIOUS COMBINATIONS OF PANGOLA
DIGITGRASS, TRANSVALA DIGITGRASS AND TOMATO ON MELOIDOGYNE INCOGNITA AND BELONOLAIMUS LONGICAUDATUS . . . . . . . . . . . . . . . . .

Introduction. . . . . . . . . . . . . . . . .
Materials and Methods . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . .
Discussion. . . . . . . . . . . . . . . . . .

EFFECT OF TWO DIGITGRASS CULTIVARS OF DIGITARIA
DECUMBENS ON EIGHT SPECIES OF NEMATODES . . . .

Introduction. . . . . . . . . . . . . . . . .
Materials and Methods . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . .
Discussion. . . . . . . . . . . . . . . . . .

DISCUSSION AND CONCLUSIONS. . . . . . . . . . . .


63 65


74


74 75 76 78


90


90 90
91 92


101


REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

BIOGRAPHICAL SKETCH. . . . . . . . . . . . . . . . . . . .. . . . 108


v














LIST OF TABLES


TABLE PAGE

2-1 Effect of Pangola digitgrass on Meloidogyne arenaria,
M. hapla, M. javanica, and M. incognita. . . . . . . . . . 7

3-1 Test of increasing solvent polarity for efficacy in
extracting nematicidal substances from Pangola digitgrass roots. In each bioassay 0.5 gram equivalent
of Pangola digitgrass root extract was tested. . . . . . . 24

3-2 Dose-response relationship between gram equivalents of
roots in an aqueous extract and egg hatch and mortality of larvae of Meloidogyne incognita. . . . . . . . . 25

3-3 Successive water extractions of Pangola digitgrass
roots. . . . . . . . . . . . . . . . . . . . . . . . . . . 26

3-4 Effect of heat, cold storage, and autoclaving upon the
active principle in aqueous extracts of Pangola digitgrass roots. . . . . . . . . . . . . . . . . . . . . . . . 27

3-5 Bioassay of silica gel thin layer chromatogram of an
aqueous extract of Pangola digitgrass roots. . . . . . . . 28

3-6 Bioassay of Whatman 3M paper chromatogram of an
aqueous extract of Pangola digitgrass roots. . . . . . . . 29

3-7 Tests of chromatograms of Pangola digitgrass root
precipitate with spray reagents for indicating
functional groups. . . . . . . . . . . . . . . . . . . . . 30

3-8 Effect of potassium hydroxide and hydrochloric acid on
the active principle in roots of Pangola digitgrass as
measured by egg hatch of Meloidogyne incognita . . . . . . 31

3-9 Dose-response relationship between concentration of
precipitate from Pangola digitgrass root extract and
mortality of Meloidogyne incognita larvae. . . . . . . . . 32

3-10 Determination of the LD50 value for mortality of
Meloidogyne incognita larvae in a 48 hr. bioassay
with an aqueous extract. . . . . . . . . . . . . . . . . . 33


vi










3-11 Determination of the LD50 value for mortality of
Meloidogyne incognita larvae in a 48 hr. bioassay of
the supernatant after removal by centrifugation of pH induced precipitate from the aqueous extract of
Table 3-10 . . . . . . . . . . . . . . . . . . . . . . . . 34

3-12 Determination of the LD50 value for mortality of
Meloidogyne incognita larvae in a 48 hr. bioassay of the dry precipitate obtained from the aqueous extract
of Table 3-10. . . . . . . . . . . . . . . . . . . . . . . 35

3-13 Confirmation of gram-equivalents of aqueous extract,
and of supernatant and precipitate obtained from the
aqueous extract required to give 50% mortality of a
test population of Meloidogyne incognita larvae in a
48 hr. bioassay. . . . . . . . . . . . . . . . . . . . . . 36

4-1 Effects of Transvala digitgrass, compared with Pangola
digitgrass, tomato and fallow, on Meloidogyne incognita. 50

4-2 Effect of Transvala digitgrass interplanted with Rutgers
tomato on Meloidogyne incognita. . . . . . . . . . . . . . 51

4-3 Effects of Transvala digitgrass, compared with Pangola
digitgrass and tomato, on Belonolaimus lonqicaudatus. . . . 52

5-1 Fresh weight of Pangola digitgrass, Transvala digitgrass,
and tomato roots after 14 weeks. . . . . . . . . . . . . . 67

5-2 Average number of hatched eggs and dead larvae of
Meloidogyne incognita when 500 eggs were exposed
to leachate from different ages of Pangola and
Transvala digitgrasses, tomato, and fallow soil. . . . . . 68

5-3 The number of live Belonolaimus longicaudatus after
exposure for 48 hours to root leachate from Pangola and
Transvala digitgrasses, tomato, and fallow soil. . . . . . 69

6-1 Influence of Pangola digitgrass, Transvala digitgrass,
tomato and combinations of the three on populations of
Meloidogyne incognita and Belonolaimus longicaudatus . . . 80

7-1 Effect of eight genera of plant parasitic nematodes on
Pangola and Transvala digitgrasses . . . . . . . . . . . . 93


vii


TABLE


PAGE














LIST OF FIGURES


FIGURE PAGE

3-1 The probits method of determining the LD50 Of
different concentrations of a precipitate (mg/ml)
from Pangola digitgrass root extract . . . . . . . . . . . 38

3-2 The probits method of determining the LD50 of
different concentrations of the original extract
(GE/ml) from Pangola digitgrass root . . . . . . . . . . . 40

3-3 The probits method of determining the LD50 Of
different combinations of supernatant (GE/ml) from
Pangola digitgrass root extract. . . . . . . . . . . . . . 42

3-4 The probits method of determining the LD50 of
different concentrations of precipitate (GE/ml) from
Pangola digitgrass root-extract. . . . . . . . . . . . . . 44

4-1 Effect of Transvala digitgrass interplanted with
tomato on relative mass of the tomato plants . . . . . . . 54

4-2 Development of Heloidoqyne incognita in roots of
Transvala digitgrass and tomato. . . . . . . . . . . . . . 56

4-3 Life stages of Meloidoqyne incognita in roots of
Transvala digitgrass (a) invading second stage larva,
(b) late second stage larva, (c) third or fourth
stage larva, (d) mature female, (e) eggs . . . . . . . . . 58

5-1 The influence of root leachates from Pangola and
Transvala digitgrass and tomato from plants 4 to 13
weeks old and from fallow soil of egg hatch of
Meloidogyne incognita. . . . . . . . . . . . . . . . . . . 71

5-2 The influence of root leachate from two digitgrass
plants from 4 to 13 weeks old on the survival of
Belonolaimus longicaudatus over a 48 hour period ..... 73

6-1 Pangola digitgrass interplanted with Transvala
digitgrass and inoculated with Meloidogyne incognita
and Belonolaimus longicaudatus . . . . . . . . . . . . . . 83

6-2 Pangola digitgrass interplanted with Transvala
digitgrass and left uninoculated . . . . . . . . . . . . . 83


viii








FIGURE PAGE

6-3 Transvala digitgrass inoculated with Meloidogyne
incognita and Belonolaimus longicaudatus . . . . . . . . . 85

6-4 Pangola digitgrass inoculated with Meloidogyne
incognita and Belonolaimus longicaudatus . . . . . . . . . 85

6-5 Rutgers tomato inoculated with Meloidogyne incognita
and Belonolaimus longicaudatus . . . . . . . . . . . . . . 87

6-6 Pangola digitgrass interplanted with Transvala
digitgrass and tomato and inoculated with Meloidogyne
incognita and Belonolaimus longicaudatus . . . . . . . . . 87

6-7 Transvala digitgrass interplanted with tomato and
inoculated with Meloidogyne incognita and
Belonolaimus lonqicaudatus . . . . . . . . . . . . . . . . 89

6-8 Pangola digitgrass interplanted with tomato and
inoculated with Meloidogyne incognita and
Belonolaimus longicaudatus . . . . . . . . . . . . . . . . 89

7-1 Effect of eight genera of plant parasitic nematodes
on Pangola and Transvala digitgrasses when each was inoculated with (a) Pratylenchus brachyurus, (b) Helicotylenchus erythrinae, (c) Trichodorus christiei, (d) Xiphinema americanum, (e) Tylenchorhynchus martini, (f) Hemicycliophora
parvana, and (g) Heterodera glycines . . . . . . . . . . . 95

7-2 The effect of Pangola and Transvala digitgrass on
populations of eight genera of plant parasitic nematode . . 100


ix













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

AN EVALUATION OF TWO CULTIVARS OF DIGITARIA DECUMBENS AS
BIOLOGICAL CONTROL AGENTS OF NEMATODES WITH EMPHASIS
ON MELOIDOGYNE INCOGNITA AND BELONOLAIMUS LONGICAUDATUS By

Sanaa A. Haroon

December 1982

Chairman: G. C. Smart, Jr.
Cochairman: J. L. Nation
Major Department: Entomology and Nematology

Pangola and Transvala digitgrasses are cultivars of Digitaria

decumbens Stent., which originated in Africa and have been released in Florida. Pangola digitgrass is a host for Belonolaimus longicaudatus but is antagonistic to Meloidogyne incognita; some larvae entered the roots but none developed beyond the late second stage. The roots of Transvala digitgrass were entered by larvae of four Meloidogyne spp., and some developed to maturity and produced eggs; root galls were small. Transvala was antagonistic to B. longicaudatus. When soil was infested with both B. longicaudatus and M. incognita and interplanted with Pangola digitgrass and Transvala digitgrass, populations of both nematodes declined.

An aqueous root extract of Pangola digitgrass (20 weeks old) killed many larvae of M. incognita prior to hatching and reduced survival of those that did hatch.


x







The active material is stable after heating for 7 hours at 90'C but not after heating for 20 minutes at 121'C. It is not soluble in nonpolar solvents. The active material was precipitated from concentrated aqueous extracts by adjusting to pH 10 with dilute NaoH. The dried precipitate at a dose of 1.0 mg precipitate/ml water produced about 50% mortality of M. incognita larvae in a laboratory bioassay.

The active material migrates as one spot in several paper chromatographic systems and is detectable with a spray of 1% diphenylcarbazone reagent.

Root leachate from Pangola digitgrass three months old or older limits egg hatch and kills larvae of M. incognita, while that from Transvala digitgrass kills B. longicaudatus. Pangola digitgrass and

Transvala digitgrass were hosts to and damaged by Trichodorus christiei, Xiphinema americanum, Hoplolaimus galeatus, and Tylenchorhynchus martini. Transvala digitgrass was a poor host for Hemicycliophora parvana, Helicotylenchus erythrinae, and Pratylenchus brachyurus, but Pangola digitgrass was an excellent host for all three. Transvala digitgrass was a good host for Heterodera glycines, but Pangola digitgrass was a poor host.


xi













CHAPTER 1

INTRODUCTION


Root-knot nematodes, Meloidogyne spp., and the sting nematode, Belonolaimus longicaudatus Rau 1958, are economically important parasites of plants. Meloidogyne spp. affect many species of plants, including most of the major crops of the world such as tobacco, peanut, cotton, rice, wheat, soybean, and vegetables. Although Belonolaimus longicaudatus severely damages many crops including corn, cotton, vegetable crops, turfgrasses, and pasture grasses, the nematode is restricted to sandy soil, mostly in the southeastern United States. Nematicides are used to control both nematodes; however, environmental

considerations and costs of nematicides dictate that other methods of control be investigated.

One alternative method is the use of antagonistic plants in rotation with or interplanted with crop plants. Antagonistic plants reduce

populations of plant parasitic nematodes to a greater extent than do nonhost plants or fallow.

Digitaria decumbens Stent cv. Pangola (P.I. 111110) and cv.

Transvala (P.I. 299601) are two forage digitgrasses that originated in Africa and have been introduced to Florida.

Winchester (1962A) and Haroon (1979) reported a rapid

reduction of established populations of Meloidogyne incognita (Kofoid and White 1919) Chitwood 1949 (reported as Meloidogyne





2


incognita acrita Chitwood and Oteifa 1952 by Winchester and Hayslip (1960) under Pangola digitgrass. Additionally, Haroon (1979) showed that although second stage larvae entered the roots of Pangola digitgrass, they failed to develop beyond the late second stage. The plants did not develop giant cells (nurse cells) on which the sedentary larvae and adult females feed. That could account for lack of development in plants, but some other mechanism is involved also, since larvae in the soil are killed and larvae die quickly in root extract from plants 11 weeks old and older. In extract from younger plants eggs hatch sooner than in extract from older plants or in water (Haroon, 1979).

Although Pangola digitgrass is antagonistic to Meloidoqyne

incognita, it is a host for Belonolaimus longicaudatus and some other nematodes (Overman 1961). Boyd and Perry (1969), while testing various grasses for their suitability in Florida, discovered that the related cultivar, Transvala digitgrass, is resistant to B. longicaudatus.

In order to learn more about the influence of Pangola and Transvala digitgrasses on nematodes, the following research was conducted to:

1. Determine whether Pangola digitgrass is antagonistic to

Meloidogyne arenaria (Neal 1839) Chitwood 1949, M. hapla

Chitwood 1949, and M. javanica (Treub 1885) Chitwood 1949;

2. To isolate, purify and characterize the chemical substance

or substances from Pangola digitgrass which is toxic to

M. incognita;

3. Determine the effects of Transvala digitgrass on M. incognita

and B. longicaudatus;






3

4. Determine the effect of root leachates from Pangola and

Transvala digitgrasses on hatch and survival of M.

incognita and survival of B. longicaudatus;

5. Determine the effects of various combinations of Transvala

and Pangola digitgrasses on M. incognita and B. longicaudatus;

6. Determine the effects of Pangola and Transvala digitgrasses

on some other nematodes.













CHAPTER 2

EFFECTS OF PANGOLA DIGITGRASS ON MELOIDOGYNE
ARENARIA, M. JAVANICA, AND M. HAPLA


Introduction


Pangola digitgrass is antagonistic to Meloidogyne incognita

(Winchester and Hayslip 1960, Haroon 1979), but its effect on other species of Meloidogyne has not been tested. The four most common and widespread species of Meloidogyne in the world are M. arenaria, M. hapla, M. incognita, and M. javanica (Taylor and Sasser 1978). All four species are present in tropical and subtropical regions of the world (M. hapla at high altitudes) which might be suitable for production of the digitgrasses. Since Pangola digitgrass is known to be antagonistic to M. incognita, experiments were conducted to determine whether it is antagonistic to the three other primary species of Meloidogyne.


General Methods


The population of Meloidogyne incognita used in these investigations was increased on tomato, Lycopersicon esculentum Mill. cv. Rutgers.

The following were common to all experiments. The soil type used was Arredondo fine sand (90.6% sand, 3.9% silt, 5.5% clay, and 1.9% organic matter). It was autoclaved for 15 min. under pressure


4




5


of 1.41 kg/cm2 (15 psi). All containers used were 15-cm diameter clay pots unless otherwise stated. All experiments were conducted in a greenhouse with temperatures at approximately 25CC 3. The plants were watered as needed and once a week each received about 100 ml of solution made up with lg/l of Nutrisol� fertilizer 12-10-12 analysis. Pangola and Transvala digitgrasses were obtained from cuttings that were rooted for five weeks before inoculation unless otherwise stated. A randomized block design was used in all experiments except those reported in Chapter 6 where a split plot design was used. All experiments were statistically analyzed by analysis of variance (ANOVA) and Duncan's Multiple Range Test (Steel and Torrie 1960).


Materials and Methods


Forty pots were filled with autoclaved soil, 20 of them planted with one unrooted cutting of Pangola digitgrass and 20 with one tomato seed to serve as a control. Treatments were M. arenaria, M. hapla, M. javanica, and M. incognita (standard) and tomato (control). Each treatment was replicated five times. Five weeks later the soil in each treatment was infested with 15 egg masses. All pots were placed in a greenhouse at about 25'C and watered as needed. The experiment was terminated after 90 days. The plants were removed from the soil, the tops removed and weighed, the roots washed, weighed, and the number of galls and egg masses determined. Then the roots were stained with acid fuchsin in lactophenol, destained in lactophenol, mounted on slides, and examined for the presence of different life stages of Meloidogyne. The population of second stage larvae in the





6


soil in each pot was determined by removing larvae from a 100 cm3 aliquot of soil by a centrifugation-flotation technique (Hussey and Barker 1973).


Results


After 90 days, the soil populations of second stage larvae of all four species of Meloidogyne in pots were low ranging from 3 to 36.

Soil populations in the tomato controls ranged from 2,412 to 12,180 (Table 2-1). In roots of Pangola digitgrass, the number of second stage larvae was low ranging from 9 to 48. Only one third or fourth stage larva was found in one replicate of M. arenaria. No other life stages were present in Pangola digitgrass roots nor were the roots galled or egg masses present (Table 2-1). In the tomato controls all life stages were present for all four species of Meloidogyne and galls and egg masses were on the roots.


Discussion


Pangola digitgrass exhibits the same antagonistic traits to M.

arenaria, rM. hapla, and M. javanica as to M. incognita as shown by the failure of any species to develop and mature in the roots and the small soil populations of second stage larvae. Thus, Pangola digitgrass should be a suitable crop to use wherever it can be grown for pasture or forage or as an antagonisitc plant to control the four major species of Meloidogyne before a desired crop is planted. It is probable that the grass is antagonistic to other species of Meloidogyne, but experiments must be conducted before one could be certain.








Table 2-1.


Effect of Panyola digitgrass on Meloidogyne arenaria, M. hapla, M. javanica, and M. incognita.


Fresh weight (g) Number Life stages in roots
Trea tent
Roots Tops Larvae2 Galls on Egg masses3 L24 L3-L Females Eggs
in soil roots on roots 4

M. arenaria
Pangola 10.9 17.8 24 0 0 48 0.2 0 0
Tomato (Control) 3.1 8.4 12,180 211 173 4,120 55 281 101

M. hapla
Pangola 19.2 18.8 12 0 0 11 0 0 0
Tomato (Control) 6.5 5.5 4,609 163 106 307 19 67 26

M. javanica
Pangola 14.6 14.1 3 0 0 9 0 0 0
Tomato (Control) 5.5 3.6 2,412 95 81 475 83 259 112

M. incognita
Pangola 9.2 17.9 36 0 0 12 0 0 0
Tomato (Control) 2.5 9.6 7,152 106 84 429 68 166 91


1Initial inoculum 2Per pot, average 3Per root system.


was 15 egg masses/pot of 1200 cm3 soil. of five replicates.


L2 = second stage larva; L3 = third stage larva; and L4 = fourth stage larva.














CHAPTER 3

ISOLATION, PURIFICATION, AND CHARACTERIZATION OF A CHEMICAL
SUBSTANCE FROM PANGOLA DIGITGRASS THAT IS TOXIC TO MELOIDOGYNE INCOGNITA


Introduction

Plant parasitic nematodes feed by piercing plant cells and ingesting the cell contents. Some nematodes feed as ectoparasites and others as endoparasites--either migratory or sedentary. Meloidoqyne incognita is a sedentary endoparasite that spends most of its life embedded in the roots of the host, and host plants respond to infection by developing specialized cells referred to as giant cells or nurse cells on which the nematode feeds. Nonhost plants usually do not develop nurse cells and thus the nematode cannot develop. Antagonistic plants not only do not produce nurse cells but also produce substances that are toxic to nematodes. Tyler (1938) and Steiner (1941) reported resistance by Tagetes spp. (Marigolds) to Meloidogyne. Suatmadji (1969) tested several species and hybrids of Tagetes for nematicidal properties and found Tagetes patula var. "Golden Harmony" to be most effective in reducing populations of certain nematodes. Uhlenbroek and Bijloo (1959) identified the nematicidal compounds terthienyl and dithienyl from Tagetes. The structure of terthienyl was confirmed by Horn and Lamberton (1963). Thus, the first naturally occurring chemical compound which had nematicidal properties against the endoparasites


8





9


Pratylenchus penetrans (Cobb, 1917) Filipjev, Schuurmans-Stekhoven, 1941, M. hapla, M. incognita, and M. javanica was reported. Asparagus, Asparagus officinalis var. "Mary Washington," effectively reduced populations of Paratrichodorus teres; an unidentified nematicidal compound was isolated from the roots (Rohde and Jenkins 1958). Spraying leaves of tomato with asparagus root extract indicated that the material acted systematically and was related to an acetylcholinesterase effect as described by Rohde (1960). Schaffer et al. (1962) found catechol as a nematicidal compound in roots of Eragrostis curvula. Taylor and Murant (1966) noted nematicidal activity of aqueous extracts of raspberry roots and canes.

Winchester (1962B) reported that an aqueous extract of mature roots of Pangola digitgrass reduced larval emergence of M. incognita acrita while extract from young roots increased emergence compared to a water control. Haroon (1979) reported that eggs of M. incognita hatch faster in root extract from Pangola digitgrass plants up to 10 weeks old than in extract from older plants.

Tests with leachates (Chapter 5) showed that a nematicidal substance (or substances) were present in Pangola digitgrass growing in pots killed IM. incognita. This investigation was initiated to further characterize and to isolate and purify the nematicidal substance in roots of Pangola digitgrass.


Materials and Methods


General Methods


All investigations reported in this chapter were conducted with roots of 20-week-old plants of digitgrass, Digitaria decumbens Stent.






10


cv. Pangola. The roots were surface sterilized by immersing them in one part Clorox� to 9 parts water for 3 minutes and rinsing them in three separate changes of sterile water. Extracts were prepared by comminuting a weighed portion of roots in a food blender for 30 seconds in sterile water, and the solution was passed through a micropore filter. Extracts were stored at -870C. The test nematode used was M. incognita. All eggs used in these tests were extracted from egg masses using the method of Hussey and Barker (1973) but modified by shaking the egg masses for 40 seconds instead of 4 minutes, and a 10% Cloroxa solution was used instead of a 20% Clorox� solution.

In order to compare the nematicidal activity of extracts prepared in different ways or subjected to various experimental treatments, a procedure was adopted that characterized an extract on the basis of the number of grams of Pangola digitgrass roots contained in the extract. Thus, 1 gram equivalent (1 GE) was defined as 1 gram of roots extracted with 10 ml of water. One milliliter of such an extract would contain 0.1 GE of roots. A method of computing the effectiveness of the nematicide in the root extract was calculated by using Abbott's formula (1925).


Survey of Solvents for Extracting Nematicidal Substances from
Pangola Digitgrass Roots


The objective of this experiment was to determine the best extraction medium for the nematicidal principle by using a series of solvents of increasing polarity, including pentane (nonpolar), acetone, methanol, and water (most-polar).

Ten grams of roots of Pangola digitgrass (20 weeks old) were

surface sterilized and comminuted for 60 seconds in 100 ml pentane in





Il


a food blender. The solution was passed through a micropore filter and designated as the pentane extract. The remaining root mass was blotted dry and found to weigh 7.32 grams. Sterile water (73.2 ml in order to keep the roots:water ratio equal to 1:10) was added to the

root fragments, and they were extracted again in the food blender for 60 seconds. This extract, designated as the "water after pentane" extract, was also passed through the micropore filter. A second sample of 10 grams of roots was extracted in a similar manner with acetone and then with water after acetone. A third sample of 10 g of roots was extracted with methanol and then water after methanol. The final solutions of pentane, acetone, and methanol were evaporated to dryness under vacuum on a rotary bath (temp. about 40'C), and then 100 ml of water was added to dissolve the residue before assay for nematicidal activity. The bioassay of extracts was run by placing 400 eggs of M. incognita in 5 ml of each treatment solution, with each replicated four times. Sterile water was used as a control. Egg hatch and larval survival were measured each day for 10 days. Dose-Repsponse Relationship with Aqueous Extract


An extract was prepared by comminuting 10 g of Pangola digitgrass

roots in 100 ml of sterile water to produce an extract with 10 GE of roots. Aliquots of the extract were diluted to give test solutions containing 1 GE, 0.5 GE, and 0.1 GE of roots in a final assay volume of 10 ml, and each assay was replicated three times. Two hundred eggs of M. incognita were placed into each treatment solution contained in

5.5-cm diameter petri dishes. The number of eggs that hatched each 24 hours was recorded daily for 10 days. The larvae that emerged





12


during each 24 hour period were removed and placed in a new dish containing extract with the same GE of roots in which they had hatched to determine their survival over a period of 10 days. Successive Extraction of Roots by Water


The purpose of this experiment was to determine whether all of

the active factor(s) in the roots of Pangola digitgrass could be removed with three successive extractions in water.

For the first extraction, 100 grams of roots were placed in a food blender with 100 ml water and comminuted for 60 seconds. The solution was filtered as described earlier. The remaining root residue weighed 75 grams. To keep the same ratio of roots to water, 75 ml water was added and the above steps repeated. The remaining root residue weighed 62 g; 62 ml water was added and the above steps repeated. The three extracts were tested for activity by diluting aliquots to give 0.1 GE of roots in a final assay volume of 5 ml in

3.5-cm diameter petri dishes containing 500 eggs of M. incognita. The number of eggs that hatched after 10 days and the number of dead and live larvae were determined. Each treatment was replicated four times.


Effect of Heat, Autoclaving, and Cold Storage on the Active Principle in Pangola Digitgrass Roots


This experiment was conducted to determine if the active principle(s) in Pangola digitgrass root extract is heat stable and if it remains stable in cold storage at 4'C. An extract containing 10 GE of roots in 100 ml of water was divided so that 70 ml (7 GE) were placed






13


in an oven at 90C for 7 hr. until all water had evaporated. The

residue was dissolved in 20 ml sterile water that was further divided in four replicate bioassay volumes of 5 ml each (1.75 GE/bioassay volume).

Portions of the original extract were also stored at 4*C for two months or were autoclaved at 1.41 kg/cm2 per square inch (15 psi) at 1210C (250*F) for 20 min. in a laboratory autoclave.

Each of the extracts subjected to the three treatments, and a

water control, were bioassayed with 600 eggs of M. incognita in each of four replicates. Number of eggs that hatched and mortality of larvae were noted after 10 days.


Chromatographic Study of Extracts


An aqueous solution of root extract was concentrated by heating at 90C until it contained 10 GE of roots/ml. A total of 50 lil was placed in each of 10 spots at the origin of a 20 x 20 cm sheet of TLC silica gel on plastic. The chromatograms were developed in chloroform:ethyl acetate:formic acid (75 ml:60 ml: 15 ml) or in isopropanol:water (10:3).

Chromatograms on Whatman 3M paper were also prepared with 50 PI

of the extract in each of 10 spots across the origin, and the chromatograms were developed in isopropanol:water (10:3). The paper was suspended above the solvent for 3-1/2 hours to saturate the paper with solvent vapor and then developed for 13 hours. Paper chromatograms

developed in isopropanol :water (10:3) were sprayed or dipped in spray reagents prepared according to Stahl (1965) in order to look for a detection reagent for the nematicidal compound in extracts.





14


Stability of Nematicidal Principle in Extract to Acid and Alkali


Two milliliters of root extract (31.4 GE) were added to 4 ml of

1 N hydrochloric acid (HCl), the mixture was heated at 90'C until dry, and 5 ml of sterile water was added to dissolve the residue. The pH of the solution was neutralized to pH 7.0 by adding 0.5 14 potassium hydroxide (KOH) dropwise. The final volume was adjusted to give

1.57 GE/ml by adding sterile water. The solution was divided into four equal portions and placed in 5.5 cm petri dishes for bioassay. Each bioassay dish contained 7.85 GE of roots in a final volume of

5 ml.

Effect of alkali was tested by adding 5 ml of 0.1 N KOH to 2 ml of extract (31.4 GE) to produce a strongly alkaline pH. The solution was neutralized with HCl. Without evaporating this solution to dryness the volume was adjusted to 20 ml and 5 ml was added to each of four petri dishes. The bioassay of both acid and alkali treated extract was determined with 300 eggs per dish. Hatch of eggs only was tested.

In the tests of stability of the nematicidal compound(s) to acid and alkali, a brown precipitate occurred when KOH was added to the water extract. The precipitate redissolved when the pH was adjusted to about 6. In other tests it was found that the precipitate could also be obtained from aqueous solutions without using KOH by adjusting

the pH to 8 or greater with buffer (for example, 0.1 1 TRIS buffer pH 8), and also by adding excess acetone to aqueous solutions. It thus appeared that the precipitated material was not very soluble at

alkaline pH's and that it could be forced out of solution also by a solvent that was miscible with water but in which the precipitated





15


material was not soluble, such as acetone. The precipitate obtained in either way was readily redissolved in weakly acidic water (i.e., final pH of solution was 6).

These observations on solubility of the precipitated substance

suggested that weak acid might be a better extracting medium for roots than water. To test this idea 284 g of roots that previously had been extracted in a blender with sterile water were reextracted with water acidified to pH 3 with hydrochloric acid. The final volume of extract was 1550 ml with a final pH of 4. The extract was concentrated at 90'C to 60 ml during 24 hours of heating. The 60 ml were divided into two 30 ml portions. To one 30 ml portion 120 ml of acetone was added and the mixture was shaken and allowed to sit in the refrigerator for about 30 minutes. The precipitate that formed was removed by centrifugation, washed by acetone, dried, and found to weigh 74.6 mg. To

the second 30 ml portion, 4 N KOH was added to give a pH of 10 or greater. A precipitate formed immediately and was removed by centrifugation, washed by acetone, and dried. It weighed 78.3 mg. Thus, essentially the same weight of precipitated material was obtained by each method for inducing precipitation. The precipitate obtained in each way was dissolved in water that was acidified to pH 6 with 1 N HCl and bioassayed. Each precipitate was active and killed the test nematodes.


Dose-response Relationship with the Precipitate from Pangola
Digitgrass Root Extracts


The purpose of this experiment was to test the precipitate and also to determine a dose-response relationship. Each treatment level





16


was replicated three times. The final volume of solution was 5 ml into which 250 larvae were placed. The treatment levels were 1 mg,

0.5 mg, 0.3 mg, and 0.1 mg precipitate per bioassay dish. The control consisted of sterile water. Determination of Completeness of Precipitation of Nematicidal Compound(s) from an Aqueous Extract of Pangola Digitgrass Roots


In order to determine if precipitation resulted in all nematicidal activity in the precipitate, four different experiments were conducted to determine the relative killing effect of an aqueous extract (412 g/1236 ml water--evaporated to 80 ml), the precipitate from a part of the extract and the supernatant after the precipitate was removed.


Results and Discussion


Survey of Solvents for Extracting Nematicidal Substances from Pangola Digitgrass Roots


The results are shown in Table 3-1. Pentane extracted no substances that caused significant mortality of eggs or of hatched larvae. The acetone extract contained substances that caused slight mortality of eggs, but larvae that successfully hatched were not killed. The methanol extract caused egg mortality but did not kill hatched larvae. Water extracts following each of the pentane, acetone, and methanol extractions contained active nematicidal substances that caused high mortality of eggs and hatched larvae. This experiment shows that water is the best solvent for the nematicidal substance(s) in the

roots, but methanol appears to extract some small quantity. Water was chosen for future work.





17


Dose-response Relationship with Aqueous Extract


The results are shown in Table 3-2. There was a significant difference between all treatments in the percentage of eggs hatched, but no significant differences occurred in larval survival in extracts with 1.0 GE versus 0.5 GE of roots. Also no differences occurred between treatments containing 0.1 GE of roots and the water control; the former two were different from the latter two, however. Although more data would be desirable, the results do indicate a dose-response relationship, as might be expected if a chemical substance(s) is responsible for the nematicidal activity.


Successive Water Extractions of Pangola Digitgrass Roots


Few larvae hatched in assays of the first, second, or third extraction, and all that hatched died (Table 3-3). These data show that active material remained in the roots at least for three successive extractions.


Effect of Heat, Autoclaving, and Cold Storage on the Active Principle of Pangola Digitgrass Roots


The results are shown in Table 3-4. Cold storage (4*C) of

extracts had little or no effect upon the active principle as indicated by the fact that only 9% of the eggs hatched and only 29% of the larvae survived. When the extract was heated at 900C, some of the active principle was destroyed as indicated by a 43'C egg hatch and a 54% larval survival. Autoclaving the extract destroyed most of the active principle as indicated by an 86% egg hatch and 100% larval survival.





18


Although heating apparently destroyed some of the active principle in extracts, it did represent a convenient method for concentrating extracts of large volume and was often used in later experiments to reduce the volume of extracts that often started at 2 liters or more.


Chromatographic Study of Extracts


Following development the thin layer chromatogram (TLC) sheet

was dried and examined under ultraviolet (UV) light. No spots were evident. The sheet then was divided into zones for elution and bioassay of any nematicidal substances that may have moved during the development of the chromatogram. Beginning at the origin, the TLC sheet was divided into six zones of 2 cm width each, with zone 1 being the region from the origin to 2 cm above the origin. Subsequent zones were numbered accordingly. The plastic backing and each zone was cut

with a scissors into small pieces and placed in 5 ml of sterile water. The beakers were covered and placed in a refrigerator for 24 hours. Then the solution from each beaker was centrifuged and passed through a micropore filter. Two 2 ml aliquots of each solution were placed in

3.5 cm diameter petri dishes and 100 eggs of M. incognita added for bioassay of the number of eggs that hatched after 10 days. A 2 cm

strip of the TLC sheet not treated with extract but immersed in the same solvent for five minutes was used as a control zone. The results

are shown in Table 3-5.

No eggs hatched in the solution from zone 1 near the origin.

Solutions from all zones caused less hatch than the control. These data suggested that most of the nematicidal principle did not move from the origin or that it moved less than 2 cm from the origin. Weak





19


biological activity in all zones does suggest some movement but probably more as a streak than as discrete spots. This solvent system and/or chromatography medium did not seem to be suitable and silica gel was abandoned.

The paper chromatograms seemed more promising. They were removed, dried, and also viewed under UV light, but again no spots were visible. The chromatogram then was divided horizontally into four zones: Zone 1 was 3 cm wide (1.5 cm above and below the extract application point). Zones 2 and 3 were each 5 cm wide, and Zone 4 was 6 cm wide. Squares (5 cm x 5 cm) were cut from a separate paper, dipped into the solvent and used as controls. The original extract was tested also as a control. The zones used in the egg hatch bioassay were cut into small pieces, soaked 24 hours in sterile water, and tested, as described in the TLC test except that 500 eggs of M. incognita were used in each of five replicates. Table 3-6 shows that zones 1, 2, and

3 from the TLC sheet developed in isopropanol :water (10:3) had greatest nematicidal activity, while zone 4 had no more activity than the control zone. When zones 1 to 4 were recombined from a second

chromatogram into one bioassay, much greater nematicidal activity was present than that in any of the zones. This is suggestive, but not conclusive, that more than one active compound is present in the extract. If the migration of one active compound was so poor as to produce a

streak along the chromatography path instead of a discrete spot, then each zone would be expected to show activity, and recombining all zones would be expected to produce greatest kill in the bioassay.

The results of tests of chromatograms with a variety of spray reagents are presented in Table 3-7. Only three reagents gave a





20


positive test on paper chromatograms of the precipitate from Pangola digitgrass roots, and two of these, 3N H2S04 and iodine vapor, are indicative only in a general way of the presence of an organic substance. Diphenylcarbazone gavea positive test, and it is known to react with certain heavy metals to produce blue to purple colors. This may mean that such a metal ion is a part of the structure of the nematicidal compound(s) in the extract. Some compounds that can chelate or complex a heavy metal (such as uric acid, xanthine, hypoxanthine, and some carbonyl containing compounds) can be detected by first spraying with a solution of mercuric chloride, followed by diphenylcarbazone and ammonia. Inasmuch as these chromatograms were not sprayed with mercuric chloride or any other heavy metal, the positive reaction with diphenylcarbazone might be indicative of a group that can chelate a small amount of heavy metal already in the extract solution prior to spraying with diphenylcarbazone. Such a metal ion might or might not contribute to the nematicidal action of the compound(s). Detection with diphenylcarbazone thus may be a fortuitous occurrence with little predictive value as to structure of the active substance. It does nevertheless provide a convenient way to follow subsequent manipulations of extracts through chromatography and other isolation procedures. Stability to Acid and Alkali


The results, shown in Table 3-8, indicate that neither hydrochloric acid nor potassium hydroxide destroyed the active principle in extracts from roots of Pangola digitgrass as measured by egg hatch. Egg hatch in extract subjected to strong acid or alkali was not





21


significantly different from that in an untreated aqueous extract, while hatch in the control was high. Dose-response Relationship with the Precipitate from Pangola Diqitgrass Root Extracts


A dose-response relationship to the redissolved precipitate is

very clearly evident in the data of Figure 3-1 and Table 3-9. Fiftyfour percent of the larvae were killed by exposure to 1 mg of the precipitate, while 26% of the larvae were killed when they were exposed to 0.5 mg of the precipitate, and only 16% were killed by 0.3 mg of precipitate. At 0.1 mg of precipitate 5% were killed. Based on these results the LD 50 for 10 day exposure of M. incognita larvae is about 1 mg of precipitate.


Determination of Completeness of Precipitation of Nematicidal Compound(s) from an Aqueous Extract of Pangola Digitgrass Roots


The results presented in Tables 3-10, 3-11, and 3-12 and in

Figures 3-2, 3-3, and 3-4 show that precipitation does not remove all of the nematicidal substance(s) from an extract. The supernatant from which the precipitate was removed still had good nematicidal activity. The estimate of the LD 50 value shows that 1 GE of the original extract is equivalent in effectiveness to 2.6 GE of precipitate and to about 3 GE of supernatant. It thus appears that about 50% of the nematicidal principle remains in the supernatant while half precipitates. This might indicate the presence of multiple compounds, some of which will not precipitate, or it may mean that only about half of a single toxic compound will precipitate under the. conditions used.






22


These bioassays, to obtain the data in Figures 3-2, 3-3, and 3-4, were made over a three-week period; the aqueous extract, supernatant, and precipitate were each tested with a different group of M. incognita. In order to test the reliability of LD50 values obtained, an experiment was designed to produce an expected 50% kill of the test nematodes by using 1 GE of aqueous extract, 2.6 GE of precipitate, and 5.2 GE of supernatant. The results, shown in Table 3-13, show that the actual values of percent kill were very close to the predicted 50% level. This close agreement of observed with predicted toxicity shows the reliability of the bioassay method and technique and suggests that this last bioassay technique requiring only 48 hr. for evaluation should be adopted for further work in characterizing and identifying the toxic compound(s) in the Pangola digitgrass roots. The technique seems to offer a semiquantitative assay for nematicidal activity of extracts based upon the observation that 1 mg of dry precipitate gives 50% kill. Since 1.2 GE of aqueous extract (Fig. 3-2) produces 50% kill, then 1 gram of roots should contain about 0.8 g of the nematicidal compound if only one compound is involved. However, it must be remembered that water probably does not extract all of the

- toxic compound, so the actual concentration may be higher.


Conclusion


An aqueous extract of Pangola digitgrass roots (20 weeks old) was tested against M. incognita larvae in an immersion assay.

The active material in extracts is stable to heat at 90C for

48 hr., but when the extract was autoclaved for 20 min. at 121'C, the





23


biological activity was destroyed. The nematicidal activity was not soluble in nonpolar solvents such as pentane and only very slightly soluble in acetone and methanol. When the extracts were chromatographed on thin layer chromatography (TLC) silica gel sheets in chloroform:ethyl acetate:formic acid (75:60:15 v/v), the active material remained at the origin. The active material was precipitated from concentrated aqueous extracts by adjusting to pH 10 with dilute KOH. The dried precipitate produced about 50% mortality of M. incognita larvae in a laboratory bioassay at a dose of 1 mg precipitate. The redissolved precipitate chromatographs as a single spot on paper chromatograms (isopronanol:H20) (10:3) and can be detected by a spray of 1% diphenyl-carbazone in ethanol.






24


Table 3-1.


Test of increasing solvent polarity for efficacy in extracting nematicidal substances from Pangola dig tgrass roots. In each bioassay 0.5 gram equivalent of Pangola digitgrass root extract was tested.


After 10 days Root extraction medium % Hatch2 % Live larvae


Pentane Acetone

Methanol


Water after pentane Water after acetone Water after methanol Water only (control)


91 a 85 a 89 a 12 c


92 a3 75 b 39 c 11 e 20 d 20 d 87 a


6 d


56 b 97 a


1One gram equivalent equals 1 gram of water (or other solvent in this particular


roots extracted with 10 ml experiment).


2Approximately 400 eggs of M. incognita were added to each replicate.
3Means in columns followed by the same letter are not significantly different at the 5% level according to Analysis of Variance and Duncan's Multiple Range Test. (All the tables in this chapter followed the same rule.)





25


Table 3-2.


Dose-response relationship between gram equivalents of roots in an aqueous extract and egg hatch and mortality of larvae of Meloidogyne incognita.


Gram equivalent of % Larval
root tissue % Egg hatch2 survival3

1.0 20 d 29 b

0.5 49 c 29 b

0.1 82 a 82 a

0 63 b 80 a


IOne gram equivalent
10 ml of water.


equals 1 gram of roots extracted with


2Two hundred eggs were added to each dish, and the egg hatch determined daily for 10 days.
3Hatched larvae were removed daily, placed in separate dishes, and the number of live and dead 10 days later determined.





26


vi


Table 3-3. Successive water extractions of Pangola digitgrass roots.



Extraction1 Effect on M. incognita2
% Egg hatch % Live larvae

1 0.006 0

2 1.6 0

3 2.6 0

Control 72 100


1Four hundred eggs were added to each of the four replicates.
2Egg hatch and mortality of larvae were determined after 10 days.





27


Table 3-4.


Effects of heat, cold storage, and autoclaving upon the active principle in aqueous extracts of Pangola digitgrass roots.


Gram
Treatment equivalents % Hatch* % Live larvae
tested after 10 days after 10 days


Stored 2 months--40C 0.5 9 d 29 c

Heated 90C for 7 hr. 1.75 43 c 54 b

Autoclaved 250*F
for 20 min. 0.3 86 b 100 a

Control (water) 98 a 99a

*
Approximately 600 eggs were added to each of four replicates.













Table 3-5.


Bioassay of silica gel thin layer chromatogram of an aqueous extract of Pangola digitgrass roots.


TLC zone2 % Egg hatch3


1 0 f

2 37 de

3 25 e

4 40 cd

5 51 bc

6 57 b

Control 86 a


1Solvent:chloroform:ethylacetate:formic acid (5:4:1 v/v).
2Zone 1 included origin to 2 cm above origin, and each successive zone included the next successive 2 cm above the previous zone.
3One hundred eggs in each replicate, hatch determined after 10 days.


4





29


Table 3-6.


Bioassay of Whatman 3M paper chromatograml of an aqueous extract of Pangola digitgrass roots.


Zone2 % Egg hatch3


1 22 b

2 18 b

3 23 b

4 57 a

Recombined zones 8 c

1-4 control zone 59 a


1Solvent:isopropanol:H20 (10:3).
2Zone 1 included area from 1.5 cm below origin to above origin; zone 2 next 5 cm; zone 3 next 5 cm; zone 6 cm; control zone was 5 cm strip of paper dipped into and dried.


1.5 cm
4 next sol vent


3Approximately 500 eggs were added to each replicate, and hatch was determined after 10 days.





30


Table 3-7. Tests of chromatograms of Pangola digitgrass root precipitate with spray reagents for indicating functional
groups.



Spray reagent Reaction (+) or (-)


1. Ninhydrin

2. 2,7 dichlorofluorescein

3. ultraviolet light

4. UV light before reagent r2

5. UV light after reagent #2 6. 0.1 N AgNO3:5N NH40H (1:1)

7. Phenol red

8. Dimethylaminobenzaldehyde

9. 3 N H2So4 followed by heat and UV light +

10. Ferric chloride-Potassium ferricyanide 11. Anthrone

12. Iodine vapor +

13. Trichloroacetic acid in ethanol followed
by UV light

14. Phosphomolybdic acid-tungstic acid 15. Phosphomolybdic acid 16. Diphenylcarbazone followed by ammonia +





31


K'


Table 3-8.


Effect of potassium hydroxide and hydrochloric acid the active principle in roots of Pangola digitgrass measured by egg hatch of Meloidogyne incognita.


Treatment


on as


Egg hatch2


Potassium hydroxide + Pangola
grass extract 5 b

Hydrochloric acid + Pangola
grass extract 4 b

Extract alone 2 b

Sterilized water 176 a


1All treatment solutions and each bioassay tested each of roots.


were adjusted to pH 6 before bioassay, treatment at a final level of 7.85 GE


21'Iean of four replicates with 300 eggs per replicate.





32


Table 3-9.


Dose-response relationship between concentration of precipitate from Pangola digitgrass root extract and mortality of Meloidogyne incognita larvae.


Dried % of dead larvaeI Corrected % mortality2
precipitate after 10 days (Abbott's formula)
(mg)

1 55 a 54

0.5 27 b 26

0.3 18 c 16

0.1 7 d 5

Control 2 e


1Two hundred and fifty larvae were replicates.
2Abbott's formula x ~ y x 100 =
x


added to each of the three % corrected


x = % of living in control y = % of living in treatment.





33


Table 3-10.


Determination of the LD50 value for mortality of Meloidoqyne incognita larvae in a 43 hr bioassay with an aqueous extract.


Gram equivalents of aqueous extract


5.2 2.6 1.6 0.5


% Mortality


Corrected % mortality
(Abbott's formula)


85 A 70 B 60 C 32 B


84 69

58 29


Water (control)


4 E


1Approximately 400 larvae were added to each of three replicates.





34


lj


Table 3-11


Determination of the LD50 value for mortality of Meloidogyne incognita larvae in a 48 hr bioassay of the supernatant after removal by centrifugation of pH induced precipitate from the aqueous extract of Table 3-10.


Gram equivalents I Corrected % mortality
of supernatant % Mortality (Abbott's formula)

5.2 55 A 53

2.6 51 A 49

1.6 26 A 23

0.5 6 B 2

Water 4 B


1Approximately 400 larvae were added to each of three replicates.


I















Table 3-12.


Determination of the LD50 value for mortality of Meloidogyne incognita larvae in a 48 hr bioassay of the dry precipitate obtained from the aqueous extract of Table 3-10.


Treatment % Mortality Corrected % mortality
(Abbott's formula)


5.2 GE 62 A 60

2.6 GE 56 AB 54

1.6 GE 41 B 38

0.5 GE 19 C 15

Water control 5D

1Approximately 400 larvae were added to each of three replicates.





36


Table 3-13.


Confirmation of gram-equivalents of aqueous extract, and of supernatant and precipitate obtained from the aqueous extract required to give 50% mortality of a test population of Meloidogyne incognita larvae in a 48 hr bioassay.


Treatment1 % Mortality2


1 GE aqueous extract 47

5.2 GE supernatant 48

2.6 GE precipitate 57


1Gram equivalents of each treatment expected to give 50%
mortality were estimated from data in Tables 3-10, 3-11, and 3-12, respectively.
2Percent mortality was not significantly different among
treatment means (three replicatesO in an Analysis of Variance and Duncan's Multiple Range Test.


41


I

























Figure 3-1. The probits method of determining the LD50 of different concentrations of a
precipitate (mg/ml) from Pangola digitgrass root extract.













7

9') "4)

6
80 /O LA)
mIA)



00


'1 0
4
10






0l 0.3 0.5 1.0
MILLIGRAMS





















Figure 3-2. The probits method of determining the LD50 of different concentrations of the
original extract (GE/ml) from Pangola digitgrass root.














7



'2


6
80






93.
-n 5







110






3 I I I H I I I I I
0.5 1 2 5 10

GRAM FOUIVALENTS



























Figure 3-3. The probits method of determining the LD50 of different concentrations of
supernatant (GE/ml) from Pangola digitgrass root extract.
















































, , , m, , , i , , ,


1 2


. , , I


, , , , i , L


5 10


GRAM EQUIVALENTS


7


51-


C a-


4






3


95 90 80 70

60m 50

40

30

20 10

5

2


0.5


D




















Figure 3-4. The probits method of determining the LD50 of different concentrations of
precipitate (GE/ml) from Pangola digitgrass root-extract.













7- 98


95 90
6
80 70

e- -60 as 5 -.50

-40

-30

-20 4

10

5

3[a 2
0.5 1 2 5 10

GRAM EQUIVALENTS














CHAPTER 4

THE EFFECT OF TRANSVALA DIGITGRASS ON
MELOIDOGYNE INCOGNITA AND BELO:iOLAIMUS LONGICAUDATUS Introduction


Transvala digitgrass (Digitaria decumbens Stent P.I. 299601) is a cultivar named for its native habitat, the province of Transvaal in South Africa. The grass was first brought to the U.S.A. from Africa in 1964 in a collection of plant introductions by Dr. A. J. Oakes, Jr., U.S.D.A., Beltsville, Maryland. Boyd and Perry (1969) found Transvala digitgrass to be nearly immune to damage by the sting nematode, Belonolaimus longicaudatus. There are no other known reports on the effects of nematodes on this grass.

The purpose of this investigation was to determine the effects of Transvala digitgrass on Meloidogyne incognita.


Materials and Methods


Effect of Transvala Digitgrass on M. incognita


Twenty pots were filled with autoclaved soil. Five of the pots

were planted with one five-week-old rooted Transvala digitgrass cutting,

five with one five-week-old tomato seedling, five with one five-weekold Pangola digitgrass cutting, and five were left fallow as controls. The soil in each pot was infested with 20 egg masses of H. incognita. Three months later the plants were harvested, the roots washed, and


45





46


the number of galls and egg masses on the roots counted. All the soil in each pot was processed and the larvae extracted and counted. Transvala Digitgrass Interplanted with Tomato


Ten pots were filled with autoclaved soil and infested with 20 egg masses of M. incognita. Five of the pots were interplanted each with one five-week-old rooted Transvala digitgrass cutting and one five-week-old tomato seedling. Five control pots were planted with one five-week-old tomato seedling. The experiment was terminated three months later.


Larval Penetration and Development


Forty-two styrofoam cups were filled with autoclaved builders sand. Twenty-one of these cups were planted with one five-week-old Transvala digitgrass cutting and 21 with one five-week-old tomato seeding. Five hundred eggs of M. incognita, separated from egg masses by Hussey and.Barker method (1973), were added to each replicate. The experiment was carried out in an incubator at 25'C.

Every four days, for twenty-eight days, plants from three pots of Transvala digitgrass and three pots of tomato were harvested. The roots were washed, stained with acid fuchsin in lactophenol for 24 hours, destained in lactophenol for 48 hours, mounted on slides, and examined for the presence of larvae. Effect of Transvala Digitgrass on B. lonqicaudatus


Fifteen pots were filled with autoclaved soil; five were planted with one five-week-old Pangola digitgrass cutting, five with one





47


five-week-old Transvala digitgrass cutting, and five with one fiveweek-old tomato seedling to serve as controls. The soil in each pot was infested with 200 mature B. lonqicaudatus. After three months the plants were harvested, the number of B. longicaudatus in the soil counted, and the roots and top weights determined.


Results


Effect of Transvala Digitgrass on M. incognita


Three months after this experiment was established, Transvala digitgrass roots contained an average of 26 galls per root system, and there was an average of 1210 larvae in the soil of each treatment pot (Table 4-1). By comparison, Pangola digitgrass roots were not galled and not many larvae were in the soil. Tomato plant roots were heavily galled and an average of 8767 larvae were in each pot. In pots left fallow, an average of 50 larvae were in the soil. Galls on Transvala digitgrass roots were small compared with galls on tomato roots.


Transvala Digitgrass Interplanted with Tomato


More galls and egg masses were present on tomato when interplanted with Transvala digitgrass than when tomato was planted alone (Fig. 4-1, Table 4-2). The number of larvae recovered from soil planted to tomato alone was fewer than from soil with the two interplanted.








Larval Penetration and Development


Four days after this experiment was initiated, second stage larvae were present in Transvala digitgrass roots but not as many as were present in tomato roots (Fig. 4-2). Third and fourth stage larvae first developed in tomato roots after 8 days and in Transvala digitgrass after 12 days. After 24 days, adult females were present in both with egg masses present in tomato after 24 days and in Transvala after 28 days. At any given time more individuals of any given life stage were present in tomato roots than in Transvala digitgrass roots. The life stages of M. incognita in roots of Transvala digitgrass are shown in Figure 4-3 a, b, c, d, and e. Effect of Transvala Digitgrass on B. longicaudatus


Three months after this experiment was established, B. longicaudatus had reproduced on Transvala digitgrass but at a level that maintained the population at about the inoculum level. On the known hosts, Pangola digitgrass and tomato, populations had increased some

6 and 25 times, respectively (Table 4-3).


Discussion


While the population of M. incognita increased on Transvala

digitgrass, it did so at about one seventh the rate of increase on tomato. When Transvala digitgrass and tomato were interplanted, the nematode population increased a little more than on tomato alone. These results indicate that Transvala digitgrass is a satisfactory host for Ml. incognita but not an excellent host. It certainly can






49


maintain populations of the nematode and thus could not be used in a rotation system to reduce population levels of M. incognita. Since the root system of the grass did not grow well, it is apparent that the grass will not yield as much forage when grown in the presence of the nematode.

Conversely, populations of B. longicaudatus remained at about the inoculum level, indicating that the grass is a poor host for it. Also, the roots and tops grew well, indicating that the grass sustained little, if any, damage.

Therefore, it can be concluded that Transvala digitgrass could be grown successfully in areas where B. longicaudatus is present and that the nematode populations likely would not increase. It would be damaged by M. incognita, however, and would permit the nematode populations to increase also.












Table 4-1. Effects of Transvala digilgrass, compared with Pangola digitgrass, tomato and fallow,
on Meloidogyne incognita.




Galls/root No. Wt. (g) No. larvae No. egg
Treatment system larvae/pot2 root system in 10 g root masses/per
root system

Transvala 26 b3 1210 b 7.9 271 b 6 b

Pangola -- 34 c 20.3 -- -TomaLo 195 a 8767 a 9.1 644 a 213 a

Fallow -- 50 c -- -- -ISoil in each treatment was infested with 20 egg masses; the experiment was terminated after 90 days.
2Approximately 1200 cm3 of soil; average of five replicates.
3Data in vertical columns followed by the same letter are not significant at the 5% level, according to Duncan's multiple range test.











Table 4-2. Effect of Transvala digitgrass interplanted with Rutgers tomato on Meloidogyne
incognita.


No. galls/ No. egg masses/
Treatment1 No. larvae/ root system root system
pot
Tomato Transvala Tomato Transvala


Tomato +
Transvala
digitgrass 13,114aj 172a 48 166a 17

Toma to
alone 11,683b 134b -- 122b -ISoil in each treatment was infested with eight egg masses; experiments were terminated after 90 days.
2Approximately 1200 cm3 of soil per pot; average of five replicates.
3Data in vertical columns followed by the same letter are not significant at the 5% level according to Duncan's multiple range test.


(n





52


Table 4-3.


Effects of Transvala digitgrass, compared with Pangola digitgrass and tomato, on Belonolaimus longicaudatus.


Treatment Nematode per pot2 Weight (q)
Roots Tops

Pangola 3
digitgrass 1203b 2.4 4.1

Transvala
digitgrass 262c 27.7 41.3

Rutgers
tomato 4965a 1.9 2.3


Each treatment received 200 mature ment was terminated after 90 days.
2Mean of four pots (1200 cm3 soil).


nematodes; the experi-


3Data in vertical column followed by the same letter are not significantly different at the 5% level according to Duncan's
multiple range test.


4y

























Figure 4-1. Effect of Transvala digitgrass interplanted with tomato on relative mass of the
tomato plants. Left: tomato planted alone; right: tomato (right) interplanted
with Transvala digitgrass (left).
























f



"' vi






















Figure 4-2. Development of Meloidogyne incognita in roots of Transvala digitgrass and tomato.
















TRANSVALA DIGITGRASS


A


* SECOND STAGE LARVAE (L2) V] L3 AND L,
ADULT FEMALES
EGGS


= I


02
0










0
cU U



z


4 8 12 16
DAYS


20 24


220 180 140 100 60

20 220 180 140 100 60

20


B RUTGERS TOMATO






- --


28


el a C


























Figure 4-3. Life stages of Meloidoqyne incognita in roots of
Transvala digitgrass (a) invading second stage
larvae, (b) late second stage larva, (c) third or fourth stage larva, (d) mature female, (e) eggs.


k-






















Nr\,


a


4 -l




0


A
A
S

4 t
A
V,4F. *'~'
- -~ ~ .*,



C - .




r


7


Le~

9


Il



















p.


LV.


























- 40


t\~4 p. E%


6~ ~

'li
snob's. 1'ANN,

P&













CHAPTER 5

EFFECTS OF ROOT LEACHATE FROM PANGOLA AND TRANSVALA
DIGITGRASSES ON MELOIDOGYNE INCOGNITA AND BELONOLAIMUS LONGICAUDATUS


Introduction


Some plants which are antagonistic to nematodes have been shown to produce root secretions that are toxic. Oostenbrink (1950) showed that when Tagetes patula was grown, Pratylenchus populations in the

soil were reduced 90%. Rohde and Jenkins (1958) found that asparagus was resistant to Trichodorus christiei and that populations declined more rapidly in soil containing asparagus roots than in soil without roots. A toxic compound recovered from the soil was shown to

have originated in the asparagus roots where it occurred in high concentrations. The compound was identified as a glycoside with a low molecular weight aglycone.

Investigations by Triffitt (1929) and Morgan (1925) showed that

diffusates from roots of Mustard, Brassica campestris L., were antagonistic to potato, Solanum tuberosum L., and did not stimulate potato cyst nematode, Heterodera rostochiensis Wollenweber 1923. Christie (1959) reported that rutabagas, Brassica napobrassica L. Mill., secrete a compound into the soil that made the burrowing nematode, Radopholus similis, unable to find roots of corn, Zea mays L.


61





62


This investigation was initiated to determine if root diffusates from Pangola and Transvala digitgrasses would affect egg hatch and larval survival of M. incognita and survival of B. longicaudatus.


Materials and Methods


The Leachate


To collect root leachates a drainage hole was made in the bottom of each of 16 styrofoam cups (32 oz.) and a length of sterile tubing inserted into the hole. Cheesecloth was placed over the end of the tube to serve as a filter. The cups were filled with autoclaved soil. Treatments were cuttings of Pangola digitgrass, cuttings of Transvala digitgrass, tomato seed, and fallow. Each treatment was replicated four times. All cuttings first were surface sterilized by immersing the cutting in one part Clorox� to nine parts water for three minutes, then rinsing in three separate changes of sterile water to remove the Clorox�. The cups were planted and placed in a growth chamber at 25*C. All treatments were fertilized with 200 ml of Nutrisof solution (12-10-12 fertilizer). Beginning four weeks after the test was initiated, weekly for 10 weeks leachate was collected by pouring 300 ml of sterile water into each cup. The water percolated through the soil, and the excess passed through the tubing and was collected in sterilized flasks. One hour after the water was poured into the cups, the flasks containing the leachate were removed, and the leachate from each flask was passed through a micropore filter and stored in a freezer at -84'C.





63


In order to quantify the relative concentration of the leachate from each plant, when the last leachate sample was collected, the plants were removed, the roots washed, and weighed fresh.


Effect of Root Leachates on Egg Hatch and Larval Survival of M. incognita


The experiment was conducted using 5 ml of leachate and 500 eggs of M. incognita in 10 ml autoclavable tubes. The number of eggs that hatched and the number of live and dead larvae were determined after six days. The experiment was carried out at room temperature. Leachates were from Pangola and Transvala digitgrasses, tomato, and fallow soil. Each treatment for each of the 10 plant ages was replicated four times.


Effect of Root Leachates on Survival of Adult B. longicaudatus


This experiment was conducted similarly to that with M. incognita except that 50 ml of leachate and 50 adult B. longicaudatus in 100 ml sterilized flasks were used. The number of live and dead nematodes was determined after 48 hours.


Results

The Leachates


The amount of leachate collected each week from each treatment ranged from 180 to 250 ml. Over the entire 10 weeks, the amounts collected from each plant were relatively uniform and less than from the fallow soil cups. At the last week of the experiment, the ratio of fresh root weight to leachate was 8 ml per gram of root






64


for Pangola digitgrass, 9 ml per gram of root for Transvala digitgrass, and 12 ml per gram of root for tomato (Table 5-1). While such ratios are not known for earlier weeks, we can assume that the root weight increased each week and, therefore, that the concentration of any substance secreted by the roots would have increased each week as the plants grew larger.


Effect of Root Leachates on Egg Hatch and Larval Survival of M. incognita


In leachate from Pangola digitgrass four weeks old, 54% of the 500 eggs hatched. The percentage of hatch generally decreased until in leachate from 13-week-old plants, 2% hatched.

In leachate from Transvala digitgrass four weeks old, 26% of

the eggs hatched. The percentage of hatch generally increased, except at week five, until in leachate from 13-week-old plants, 38% of the eggs hatched.

In the leachate from tomato and from fallow soil, the percentage of hatch over the 10 weeks was about the same. The hatch in leachate from four-week-old tomato plants was 52% and that from fallow soil, 47%. In leachate from 13-week-old tomato plants hatch was 44% and from fallow soil, 49% (Fig. 5-1, Table 5-2).

The percentage of larval survival was generally very high in all weeks under tomato and fallow treatment. In leachate from Transvala digitgrass through week seven, 100% of the larvae were alive; after week seven a few larvae died, with a maximum of 32% dead larvae in week 11.





65


In leachate from Pangola digitgrass, 100% of the larvae were live through week six. In leachate from week 8 through week 13 a few eggs hatched, and most of the larvae died. One hundred percent, 95%, and 100% of the larvae were dead when they were exposed to root leachates from weeks 11, 12, and 14, respectively (Table 5-2). Effect of Root Leachates on Survival of Adult B. longicaudatus


In leachates from Transvala digitgrass plants 8 through 13 weeks

old, except for week 11, survival of B. longicaudatus was significantly less than in other leachates from corresponding age plants and from fallow soil. Also, survival from week 8 through week 13 was significantly less than during the first four weeks. Survival at week 12 was significantly less than at previous weeks, and survival at week 13 was significantly less than at week 12. In leachate from Pangola digitgrass survival was not affected except at week 13. Survival was not significantly different in leachates from tomato and fallow soil at any age (Table 5-3, Fig. 5-2).


Discussion

It is not surprising that leachate from plants up through seven weeks old had no effects on egg hatch and larval survival of M. incognita and survival of B. longicaudatus because at those ages the quantity of roots must have been rather low and the concentration of root secretions thus rather dilute. However, these results confirm previous work by Haroon (1979) who showed that root extract of equal concentration from Pangola digitgrass plants 4 to 10 weeks old had no effect on survival of M. incognita until the plants were 11 weeks old. Additional research needs to be conducted, however, to determine





66


whether equal concentrations of root extracts of Transvala digitgrass are not effective until the plants are at least eight weeks old. Also, additional research should be conducted to determine the effects of different dilutions of root leachates on B. longicaudatus.

Leachate from Transvala digitgrass plants, 4, 5, and 12 weeks old inhibited hatch of eggs of M. incognita when compared to leachate from other age roots, while leachates from Pangola digitgrass plants four through seven weeks old stimulated egg hatch compared to leachate from older plants. In leachate from tomato roots and from fallow soil some 40-52% of the eggs hatched in leachate at all ages indicating no effects. Therefore, a significant percentage of egg hatch above 52% would indicate a stimulation to hatching and below 40% an inhibition to hatching. Based on that range, Pangola digitgrass root leachate did not stimulate hatch at any age but inhibited hatch when plants were 8 to 13 weeks old. Transvala digitgrass root leachate inhibited hatch at 4, 5, and 12 weeks of age but had no effects at other ages. Most

of the hatched larvae in leachate from Transvala digitgrass, tomato, and fallow soil were alive, while in Pangola digitgrass root leachate most of the larvae died in leachate from plants 8-13 weeks old.

The fact that leachate from Pangola digitgrass (except at week 13), tomato, and fallow soil had no effect on B. longicaudatus at any plant age is not surprising since both plants are hosts for the nematode. Since Transvala digitgrass is reported to be resistant to B. longicaudatus, it is somewhat surprising that effects of the leachate on survival did not occur until plants were eight weeks old. Perhaps this was due to the concentration of the leachate--a point that needs investigation.






67


Table 5-1.


Leachate collected from Pangola digitgrass, Transvala digitgrass, and tomato roots at week 13 and its relative concentration per gram of roots.


Treatment Weight (g)1 Leachate at week 13
root system ml ml/root

Pangola digitgrass 23 192 8

Transvala digitgrass 21 185 9

Tomato 16 195 12

Fallow -- 235.5 -IAverage of four replicates.






Table 5-2.


Average number of hatched eggs and dead larvae of M. incognita when 500 eggs were exposed to leachate from different ages of Pangola and Transvala digitgrasses, tomato, and fallow soil.


TreatmentI Pangola Transvala Tomato Fallow

No. of No. of No. of No. of
Plant age Eggs Dead Eggs Dead Eggs Dead Eggs Dead
(weeks) hatched larvae hatched larvae hatched larvae hatched larvae

4 273(a)2a3 0 133(c)b 0 261(a)a 2 234(a)a 0
5 195(b)a 0 45(d)b 0 207(a)a 0 207(a)a 0
6 175(b)b 0 211(a)a 0 225(b)a 4 201(a)a 0
7 214(b)b 3 214(a)b 0 211(b)b 8 258(a)a 4

8 16(d)c 15 182(b)b 21 220(b)a 0 258(a)a 2

9 96(c)b 79 226(a)a 50 236(a)a 3 258(a)a 0
10 14(d)b 11 268(a)a 44 244(a)a 3 253(a)a 1
11 27(d)b 27 223(a)a 71 213(b)a 2 235(a)a 3

12 59(c)d 56 147(c)c 37 275(a)a 2 210(a)b 0
13 11(d)b 11 193(b)a 33 225(b)a 0 248(a)a 0

1Average of four replicates.
2Data in vertical columns followed by the same letter in parentheses are not significant at the 5% level according to Duncan's multiple range test.
3Data in horizontal rows followed by the same letter are not significant at the 5% level according to Duncan's multiple range test.


0c1









Table 5-3. The number of live Belonolaimus longicaudatus after exposure for 48 hours to root leachate
from Pangola and Transvala digitgrasses, tomato, and fallow soil.


Weeks
Leachate
source 4 5 6 7 8 9 10 11 12 13
No. of live nematodes2

Pangola 50a(a)3 50a(a) 50a(a) 48a(a) 47a(a) 47a(a) 46a(a) 41a(a) 44a(a) 35a(b)

Transvala 49a(a)4 50a(a) 48a(a) 47a(a) 35b(b) 38b(b) 37b(b) 37a(b) 20b(c) 7b(d)

Tomato 50a 49a 50a 49a 50a 50a 49a 48a 50a 49a

Fallow 50a 50a 50a 50a 50a 50a 49a 48a 48a 48a


1Average of four replicates per treatment.
2Fifty adult nematodes were added for each replicate.
3Data in vertical column followed by the same letter are not significantly different at the 5% level according to Duncan's Multiple Range Test.
4Data in horizontal rows followed by the same letter in parentheses are not significantly different at the 5% level according to Duncan's multiple range test. There was no significant difference amont tomato and fallow treatments.

























Figure 5-1. The influence of root leachates from Pangola and Transvala digitgrass and tomato
from plants 4 to 13 weeks old and from fallow soil on egg hatch of Meloidogyne
incognita.


































280
0xx


FALLOW
240 -' /
x TOMATO


0 200 . --' I
L- * / I TRANSVALA
I *3. '
o 1 -El S

160 " ID

LU I
El' I
o 120

ma I

z 80 - .


ID

40 -


* Z)PANGOLA
0 1 -1 1 a A
4 5 6 7 8 9 10 11 12 13
AGE OF PLANTS (Weeks)

























Figure 5-2. The influence of root leachate from Pangola and Transvala digitgrass plants from
4 to 13 weeks old on the survival of Belonolaimus longicaudatus over a 48 hour
period.








50


040



z30

.- PANGOLA
20- A--ATRANSVALA


z
10 A


4 5 6 7 8 9 10 11 12 13
AGE OF PLANTS (Weeks)













CHAPTER 6

THE EFFECTS OF VARIOUS COMBINATIONS OF PANGOLA DIGITGRASS,
TRANSVALA DIGITGRASS AND TO'1ATO ON MELOIDOGYNE
INCOGNITA AND BELONOLAIMUS LONGICAUDATUS


Introduction


Plants which have a detrimental effect on nematodes greater than that of nonhost plants are said to be antagonistic. Some plants known to have antagonistic properties against certain nematodes are marigolds, Tagetes spp., (Tyler 1938, Steiner 1941, and Suatmadji 1969), crotalaria, Crotalaria spectabilis (Good et al. 1965), hairy indigo, Indigofera hirsuta L. (Ruehle and Christie, 1958) and castor bean, Ricinus communis (Lear and Miyagua 1966).

Pangola digitgrass, Digitaria decumbens Stent (P.I. 111110), and Transvala digitgrass, Digitaria decumbens Stent (P.I. 200601), have antagonistic properties to certain species of nematodes. Pangola digitgrass is antagonistic to at least four species of Meloidogyne (Chapter 2) but is a good host of the sting nematode, Belonolaimus longicaudatus. Transvala digitgrass is resistant to B. longicaudatus but is a host of Meloidogyne incognita. Experiments reported here were designed to determine whether interplanting Pangola and Transvala digitgrasses in soil infested with B. longicaudatus and N. incognita would mutually protect each of these grasses from its nematode pathogen; also, whether the two grasses interplanted with Rutgers tomato, a good

host of both nematodes, would protect tomato from injury.


74





75


Materials and Methods


The experiment consisted of 12 treatments (Table 6-1) with each

replicated five times. Treatments were 1) Pangola and Transvala digitgrass interplanted in soil infested with Meloidogyne incognita; 2) Pangola and Transvala digitgrass interplanted in soil infested with B. longicaudatus; 3) Pangola and Transvala digitgrasses interplanted in soil infested with both M. incognita and B. longicaudatus; 4) Pangola and Transvala digitgrasses interplanted and uninoculated (control); 5) Transvala digitgrass in soil infested with both M. incognita and B. longicaudatus; 6) Pangola digitgrass in soil infested with both M. incognita and B. longicaudatus; 7) Rutgers tomato in soil infested with M. incognita and B. longicaudatus; 8) Transvala and Pangola digitgrasses interplanted with tomato in soil infested with M. incognita and B. longicaudatus; 9) Transvala digitgrass interplanted with-tomato in soil infested with M. incognita and B. longicaudatus; 10) Pangola digitgrass interplanted with tomato in soil infested with M. incognita and B. longicaudatus; 11) Pangola digitgrass uninoculated (control); and 12) Transvala digitgrass uninoculated (control).

Sixty plastic trays (60 x 16 x 24.5 cm) were filled with 2000 cm3 of autoclaved soil. Unrooted digitgrass cuttings and tomato seed were used to initiate the experiment. Each tray contained a total of six plants; when two or three different plants were interplanted, they were alternated in two or three rows. All treatments were arranged randomly on a greenhouse bench. Temperature was 25 2'C. Plants were watered as needed, and once a week each tray received about 100 ml of a fertilizer solution made up with one gram per liter of Nutrisol�





76


12-10-12 analysis. Nematode inoculum was added five weeks after the experiment was initiated. Trays inoculated with M. incognita received 2,000 second stage larvae hatched no more than 24 hours previously. Trays inoculated with B. longicaudatus received 400 nematodes of mixed life stages and sexes. The experiment was terminated 12 weeks after inoculation.


Results


When Pangola and Transvala digitgrasses were interplanted and

inoculated with M. incognita, the population was reduced to very low

levels (Table 6-1). Neither grass showed any root galls or egg masses. Some second stage larvae were present in the roots. Root and top weights were not significantly different than when Pangola and Transvala digitgrasses were interplanted and not inoculated.

When Pangola and Transvala digitgrasses were interplanted and inoculated with B. longicaudatus, soil populations of the nematode were reduced to zero. Roots and top weights were not significantly different than when the two grasses were interplanted and not inoculated.

When Pangola and Transvala digitgrasses were interplanted and inoculated with both M. incognita and B. longicaudatus, the soil population of B. longicaudatus was reduced to five per 100 cm3 of soil and that of M. incognita to zero. There were no galls or egg masses of M. incognita on the roots, and weights of plants did not differ from the controls (compare Figs. 6-1 and 6-2).

When Transvala was planted alone and inoculated with M.

incognita and B. longicaudatus, the soil population of B. longicaudatus





77


was reduced to three per 100 cm3 of soil and that of M. incognita to 28. Ninety-three larvae of M. incognita were recovered from the root systems, and galls and egg masses were present. Root growth was less than half that of controls and top growth was limited but not quite so much (Table 6-1, Fig. 6-3).

When Pangola digitgrass was planted alone and inoculated with M. incognita and B. longicaudatus, no M. incognita was found in the soil or roots, but an average of 694 B. longicaudatus per 100 cm3 of soil was recovered. There was a significant retardation in growth of the root system. Roots were injured and weighed significantly less than those of uninoculated plants (Table 6-1, Fig. 6-4).

When tomato was planted alone and inoculated with M. incognita and B. longicaudatus, high populations of both nematodes were recovered from the soil with an average of 367 B. longicaudatus and 1,810 M. incognita per 100 cm3 of soil (Table 6-1, Fig. 6-5). An average of 14,820 second larvae of M. incognita were recovered from 10 grams of tomato roots. A large number of galls and egg masses were observed on the root system. Root and top weights of the tomato plants were significantly less than the controls (Table 6-1).

When Pangola and Transvala digitgrasses were interplanted with tomato and inoculated with M. incognita and B. longicaudatus, the root systems of Pangola and Transvala digitgrasses were damaged severely with roots of Pangola digitgrass showing symptoms of B. longicaudatus damage. Tomato was not damaged as much as when planted alone nor were the number of M. incognita and B. longicaudatus as high when the three were interplanted as when





78


tomato was planted alone. However, all three plants were damaged (compare Figs. 6-5 and 6-6).

When Transvala digitgrass was interplanted with tomato and

inoculated with M. incognita and B. longicaudatus, the soil population level of B. longicaudatus was reduced to six per 100 cm3 of soil, while an average of 386 second stage larvae of 1A. incognita were recovered from the soil (Table 6-1, Fig. 6-7).

Conversely, when Pangola digitgrass was interplanted with tomato

and inoculated with both nematodes, an average of 198 B. longicaudatus and 9 M. incognita per 100 cm3 of soil were recovered (Fig. 6-8).


Discussion


Pangola digitgrass reduces soil and root populations of M. incognita but allows populations of B. longicaudatus to increase. Transvala digitgrass reduces populations of B. longicaudatus but allows populations of M. incognita to increase. The two grasses interplanted reduce populations of both nematodes and provide mutual protection to each other against the nematodes. In addition, the two grasses interplanted with tomato provide some protection to tomato, but the presence of tomato, a host for both nematodes, allows populations of

both nematodes to increase and damage the two grasses as well as tomato. Therefore, one could not expect to interplant Pangola

and Transvala digitgrasses with a crop susceptible to both Meloidogyne spp. and B. longicaudatus and provide significant protection to that crop. However, it would seem logical to interplant Pangola and Transvala digitgrasses in pastures where both Meloidogyne spp. and B. longicaudatus are present. Better growth of





79


each grass may be expected and populations of both nematodes should be reduced. In a rotation system, protection would be provided to the following crop. However, as shown by the host range study in Chapter 7, each grass is susceptible to attack by additional plant parasitic nematodes. If those nematodes are present, some damage may still occur, but it should not be of the same magnitude as caused by the presence of Meloidogyne spp. and B. longicaudatus.







Table 6-1.


Influence of Pangola digitgrass, Tra svala digitgrass, tomato and com inations of the three on populations of Meloidogyne incognita and Belonolaimus longicaudatus.


Treatment/ Nematode Fresh weight 13.1. P-. i. Gls Egmse
plant Roots Tops In soil Second stage larvae Galls Egg masses
(g) In soil In roots


Pangola + Transvala Pangola + Transvala Pangola + Transvala Pangola + Transvala Transvala


Pangola Tomato


Pangola + Transvala + Tomato


M. incognita


B. longicaudatus


M. incognita + B. longicaudatus

Uninoculated Control

M. incognita + B_. longicaudatus M. incognita + B. longicaudatus M. incognita + B. longicaudatus M. incognita + B. longicaudatus


42 27


68
48


29 78 31 13 54 61 26 62 35 65 36 56 12 31


0


5


3


6 35 694


3 5 367


1
4 17


19 10 65


338


26
41


13


0


11


0


0


28


93


0O CD


0


11


25


14,820


1810 313


99


2
112
550


84


0.3 118


12
139







Table 6-1. Continued.


Treatment/ Nematode Fresh weight B.l . M. I. Gls Egmse
plant Roots Tops In soil Second stage larvae Galls Egg masses
(g) In soil In roots

Transvala + M. incognita + 11 15 6 386 113 2 1
Tomato B. longicaudatus 4 56 1175 20 14

Pangola + M. incognita + 8 70 198 9 24 --- --Tomato B. longicaudatus 8 39 62 85 58

Pangola Control 46 49 --- --- --- --- --Transvala Control 29 43 --- --- --- ---


1Inoculum
(20/100 ci3 of inoculation.


of M. incognita was 2000 larvae (100/100 cm3 of soil); of B. longicaudatus, 400 nematodes soi~) of mixed sexes and life stages. The experiment was terminated 90 days after


2Nematodes per 100 cm3 of soil.
310 g of roots.


E




















Figure 6-1. Figure 6-2.


Pangola digitgrass interplanted with Transvala digitgrass and inoculated with Meloidogyne incognita and Belonolaimus longicaudatus.
























Pangola digitgrass interplanted with Transvala digitgrass and left uninoculated.
























PANGOLA & TRANSVAL AROOT KNOT & STING PANGOLA B RA ALA


PANGOLA '-.

PANGOLA &
NO IN


T

)


. 7r



TRANSVALA RANSVALA CULUM



















Figure 6-3. Transvala digitgrass inoculated with Meloidogyne
incognita and Belonolaimus longicaudatus.


























Figure 6-4. Pangola digitgrass inoculated with Meloidogyne
incognita and Belonolaimus longicaudatus.




















TRANSVALA
ROOT KNOT & STING r ~



If


ell





PANGOLA
ROOT KNOT& STING























Figure 6-5. Rutgers tomato inoculated with Meloidogyne incognita
and Belonolaimus longicaudatus.

























Figure 6-6. Pangola digitgrass interplanted with Transvala
digitgrass and tomato and inoculated with
Meloidogyne incognita and Belonolaimus
longicaudatus.























TOMATO
ROOT KNOT & STING


~4~" ~PANGOAL.
r' TRANS & PANGOLA TOMAl 0 & TOMATO
ROOT KNOT & STING TRANSVALA




















Figure 6-7. Transvala digitgrass interplanted with tomato and
inoculated with Meloidogyne incognita and
Belonolaimus longicaudatus.

























Figure 6-8. Pangola digitgrass interplanted with tomato and
inoculated with Meloidogyne incognita and
Belonolaimus longicaudatus.






















TOMATO TRANSVALA

TOMATO & TRANSVALA
ROOT KNOT & STING















NGOL A TOMATO



TOMATO & PANGOLA
ROOT KNOT & STING




Full Text

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AN EVALUATION OF TWO CULTIVARS OF DIGITARIA DECUMBENS AS BIOLOGICAL CONTROL AGENTS OF NEMATODES WITH EMPHASIS ON MELOIDOGYNE INCOGNITA AND BELONOLAIMUS LONGICAUDATUS BY SANAA A. HAROON 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 1982

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Dedicated to the memory of my father, the love of my mother, my devoted husband, Samir, and my son, Ahmed, and My professors Dr. G. C. Smart, Jr. , and Dr. R. A. Dunn

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ACKNOWLEDGMENTS I extend special thanks to Dr. G. C. Smart, Jr., chairman of the supervisory committee, for the supervision of this research since its beginning. Deep appreciation is extended, also, to him for his friendly attitude, patience, time-consuming assistance, encouragement, and suggestions. I express my appreciation to Dr. J. L. Nation, cochairman of the supervisory committee, for providing guidance and supervision of this research. Also, I express gratitude to Mrs. A. J. Overman, Dr. H. L. Rhoades, and Dr. 0. C. Ruel ke, members of my graduate committee, for their valuable assistance in conducting this study and for their critical reading of the manuscript. Special appreciation goes to Dr. R. A. Dunn for encouragement, inspiration, and friendship and for providing me with an assistantship during my graduate program. Appreciation is extended also to the American Association of University Women for providing a scholarship during 1980. Finally, to my husband, Dr. Samir El-Agamy, and to my son, Ahmed, I convey sincere appreciation for their understanding and cooperation during this period of my studies.

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TABLE OF CONTENTS PAGE ACKNOWLEDGMENTS iii LIST OF TABLES vi LIST OF FIGURES viii ABSTRACT x CHAPTER 1 INTRODUCTION 1 CHAPTER 2 EFFECTS OF PANGOLA DIGITGRASS ON MELOIDOGYNE ARENARIA , M. JAVANICA , AND M. HAPLA 4 Introduction 4 General Methods 4 Materials and Methods 5 Results . . .......... 6 Discussion 6 CHAPTER 3 ISOLATION, PURIFICATION, AND CHARACTERIZATION OF A CHEMICAL SUBSTANCE FROM PANGOLA DIGITGRASS THAT IS TOXIC TO MELOIDOGYNE INCOGNITA 8 Introduction 8 Materials and Methods 9 Results and Discussion 16 Conclusion 22 CHAPTER 4 THE EFFECT OF TRANSVALA DIGITGRASS ON MELOIDOGYNE INCOGNITA AND BELONOLAIMUS LOGICAUDATUS 45 Introduction 45 Materials and Methods 45 Results 47 Discussion 48 CHAPTER 5 EFFECTS OF ROOT LEACHATE FROM PANGOLA AND TRANSVALA DIGITGRASSES ON MELOIDOGYNE INCOGNITA AND BELONOLAIMUS LONGICAUDATUS 61 Introduction 61 Materials and Methods 62 iv

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PAGE Results 63 Discussion 65 CHAPTER 6 THE EFFECTS OF VARIOUS COMBINATIONS OF PANGOLA DIGITGRASS, TRANSVALA DIGITGRASS AND TOMATO ON MELOIDOGYNE INCOGNITA AND BELONOLAIMUS LONGICAUDATUS 74 Introduction 74 Materials and Methods 75 Results 76 Discussion 78 CHAPTER 7 EFFECT OF TWO DIGITGRASS CULTIVARS OF PI GITARIA DECUMBENS ON EIGHT SPECIES OF NEMATODES 90 Introduction 90 Materials and Methods 90 Results 91 Discussion 92 CHAPTER 8 DISCUSSION AND CONCLUSIONS 101 REFERENCES 105 BIOGRAPHICAL SKETCH .". . -.' .... 108 v

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LIST OF TABLES TABLE PAGE 21 Effect of Pangola digitgrass on Meloidoqyne arenaria , M. hapla , M. javanica , and M. incognita 7 31 Test of increasing solvent polarity for efficacy in extracting nematicidal substances from Pangola digitgrass roots. In each bioassay 0.5 gram equivalent of Pangola digitgrass root extract was tested 24 3-2 Dose-response relationship between gram equivalents of roots in an aqueous extract and egg hatch and mortality of larvae of Meloidogyne incognita 25 3-3 Successive water extractions of Pangola digitgrass roots 26 3-4 Effect of heat, cold storage, and autoclaving upon the active principle in aqueous extracts of Pangola digitgrass roots. 27 3-5 Bioassay of silica gel thin layer chromatogram of an aqueous extract of Pangola digitgrass roots 23 3-6 Bioassay of Whatman 3H paper chromatogram of an aqueous extract of Pangola digitgrass roots 29 3-7 Tests of chromatograms of Pangola digitgrass root precipitate with spray reagents for indicating functional groups 30 3-8 Effect of potassium hydroxide and hydrochloric acid on the active principle in roots of Pangola digitgrass as measured by egg hatch of Meloidogyne incognita 31 3-9 Dose-response relationship between concentration of precipitate from Pangola digitgrass root extract and mortality of Meloidogyne incognita larvae 32 3-10 Determination of the LD50 value for mortality of Meloidogyne incognita larvae in a 48 hr. bioassay with an aqueous extract 33 vi

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TABLE PAGE 3-11 Determination of the LD50 value for mortality of Mel oidogyne incognita larvae in a 48 nr. bioassay of the supernatant after removal by centrifugation of pH induced precipitate from the aqueous extract of Table 3-10 34 3-12 Determination of the LD50 value for mortality of Mel oidogyne incognita larvae in a 48 hr. bioassay of the dry precipitate obtained from the aqueous extract of Table 3-10 35 313 Confirmation of gram-equivalents of aqueous extract, and of supernatant and precipitate obtained from the aqueous extract required to give 50% mortality of a test population of Meloidogyne incognita larvae in a 48 hr. bioassay 36 41 Effects of Transvala digitgrass, compared with Pangola digitgrass, tomato and fallow, on Meloidogyne incognita . . 50 4-2 Effect of Transvala digitgrass interplanted with Rutgers tomato on Meloidogyne incognita 51 43 Effects of Transvala digitgrass, compared with Pangola digi tgrass and tomato , on Belonolaimus longicaudatus . ... 52 51 Fresh weight of Pangola digitgrass, Transvala digitgrass, and tomato roots after 14 weeks 67 5-2 Average number of hatched eggs and dead larvae of Meloidogyne incognita when 500 eggs were exposed to leachate from different ages of Pangola and Transvala digitgrasses , tomato, and fallow soil 68 53 The number of live Belonolaimus longicaudatus after exposure for 48 hours to root leachate from Pangola and Transvala digitgrasses, tomato, and fallow soil 69 61 Influence of Pangola digitgrass, Transvala digitgrass, tomato and combinations of the three on populations of Meloidogyne incognita and Belonolaimus longicaudatus ... 80 71 Effect of eight genera of plant parasitic nematodes on Pangola and Transvala digitgrasses 93 vi i

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LIST OF FIGURES FIGURE PAGE 3-1 The probits method of determining the LD^q of different concentrations of a precipitate (mg/ml ) from Pangola digitgrass root extract 38 3-2 The probits method of determining the LD50 of different concentrations of the original extract (GE/ml ) from Pangola digitgrass root 40 3-3 The probits method of determining the LD50 of different combinations of supernatant (GE/ml) from Pangola digitgrass root extract 42 34 The probits method of determining the LDcq of different concentrations of precipitate (GE/ml) from Pangola digitgrass root-extract 44 41 Effect of Transvala digitgrass interplanted with tomato on relative mass of the tomato plants . ..... . . 54 4-2 Development of Meloidoqyne incognita in roots of Transvala digitgrass and tomato 56 43 Life stages of Meloidoqyne incognita in roots of Transvala digitgrass (a) invading second stage larva, (b) late second stage larva, (c) third or fourth stage larva, (d) mature female, (e) eggs 58 51 The influence of root leachates from Pangola and Transvala digitgrass and tomato from plants 4 to 13 weeks old and from fallow soil of egg hatch of Meloidogyne incognita . ' 71 52 The influence of root leachate from two digitgrass plants from 4 to 13 weeks old on the survival of Bel onolaimus longicaudatus over a 48 hour period 73 61 Pangola digitgrass interplanted with Transvala digitgrass and inoculated with Meloidoqyne incognita and Belonolaimus longicaudatus 83 6-2 Pangola digitgrass interplanted with Transvala digitgrass and left uninoculated 83 v i i i

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FIGURE PAGE 6-3 Transvala digitgrass inoculated with Meloidogyne incognita and Belonolaimus 1 ongicaudatus 85 6-4 Pangola digitgrass inoculated with Meloidogyne incognita and Belonolaimus 1 ongicaudatus 85 6-5 Rutgers tomato inoculated with Meloidogyne incognita and Belonolaimus longicaudatus 87 6-6 Pangola digitgrass interplanted with Transvala digitgrass and tomato and inoculated with Meloidogyne incognita and Belonolaimus 1 ongicaudatus 87 6-7 Transvala digitgrass interplanted with tomato and inoculated with Meloidogyne incognita and Belonolaimus longicaudatus 89 68 Pangola digitgrass interplanted with tomato and inoculated with Meloidogyne incognita and Belonolaimus longicaudatus 89 71 Effect of eight genera of plant parasitic nematodes on Pangola and Transvala digitgrasses when each was inoculated with (a) Pratylenchus brachyurus , (b) Hel icotyl enchus erythrinae , (c) Trichodorus christiei , (d) Xiphinema americanum , (e) Tylenchorhynchus martini , (f ) Hemicycl iophora parvana , and (g) Heterodera glycines 95 7-2 The effect of Pangola and Transvala digitgrass on populations of eight genera of plant parasitic nematode . . 100 ix

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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 AN EVALUATION OF TWO CULTIVARS OF DIGITARIA DECUMBENS AS BIOLOGICAL CONTROL AGENTS OF NEMATODES WITH EMPHASIS ON MELOIDOGYNE INCOGNITA AND BELONOLAIMUS LONGICAUDATUS By Sanaa A. Haroon December 1982 Chairman: G. C. Smart, Jr. Cochairman: J. L. Nation Major Department: Entomology and Nematology Pangola and Transvala digitgrasses are cultivars of Digitaria decumbens Stent. , which originated in Africa and have been released in Florida. Pangola digitgrass is a host for Belonolaimus longicaudatus but is antagonistic to Meloidogyne incognita ; some larvae entered the roots but none developed beyond the late second stage. The roots of Transvala digitgrass were entered by larvae of four Meloidogyne spp., and some developed to maturity and produced eggs; root galls were small. Transvala was antagonistic to B_. longicaudatus . When soil was infested with both ft. longicaudatus and M. incognita and interplanted with Pangola digitgrass and Transvala digitgrass, populations of both nematodes decl ined. An aqueous root extract of Pangola digitgrass (20 weeks old) killed many larvae of M. incognita prior to hatching and reduced survival of those that did hatch.

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The active material is stable after heating for 7 hours at 90°C but not after heating for 20 minutes at 121°C. It is not soluble in nonpolar solvents. The active material was precipitated from concentrated aqueous extracts by adjusting to pH 10 with dilute NaoH. The dried precipitate at a dose of 1.0 mg precipitate/ml water produced about 50% mortality of M. incognita larvae in a laboratory bioassay. The active material migrates as one spot in several paper chromatographic systems and is detectable with a spray of 1% di phenyl carbazone reagent. Root leachate from Pangola digitgrass three months old or older limits egg hatch and kills larvae of M. incognita , while that from Transvala digitgrass kills B_. longicaudatus . Pangola digitgrass and Transvala digitgrass were hosts to and damaged by Trichodorus christiei , Xi phinema ameri canum , Hoplolaimus galeatus , and Tylenchorhynchus martini . Transvala digitgrass was a poor host for Hemicycl iophora parvana , He] j cotyl enchus erythrinae , and Pratylenchus brachyurus , but Pangola digitgrass was an excellent host for all three. Transvala digitgrass was a good host for Heterodera glycines , but Pangola digitgrass was a poor host. xi

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CHAPTER 1 INTRODUCTION Root-knot nematodes, Meloidoq.yne spp., and the sting nematode, Belonolaimus longicaudatus Rau 1958, are economically important parasites of plants. Meloidoq.yne spp. affect many species of plants, including most of the major crops of the world such as tobacco, peanut, cotton, rice, wheat, soybean, and vegetables. Although Belonolaimus longicaudatus severely damages many crops including corn, cotton, vegetable crops, turf grasses, and pasture grasses, the nematode is restricted to sandy soil, mostly in the southeastern United States. Nematicides are used to control both nematodes; however, environmental considerations and costs of nematicides dictate that other methods of control be investigated. One alternative method is the use of antagonistic plants in rotation with or interplanted with crop plants. Antagonistic plants reduce populations of plant parasitic nematodes to a greater extent than do nonhost plants or fallow. Digitaria decumbens Stent cv. Pangola (P.I. 111110) and cv. Transvala (P.I. 299601) are two forage digitgrasses that originated in Africa and have been introduced to Florida. Winchester (1962A) and h'aroon (1 979) reported a rapid reduction of established populations of Meloidogyne incognita (Kofoid and White 1919) Chitwood 1949 (reported as Meloidogyne 1

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2 incognita acrita Chitwood and Oteifa 1952 by Winchester and Hayslip (1960) under Pangola digitgrass. Additionally, Haroon (1979) showed that although second stage larvae entered the roots of Pangola digitgrass, they failed to develop beyond the late second stage. The plants did not develop giant cells (nurse cells) on which the sedentary larvae and adult females feed. That could account for lack of development in plants, but some other mechanism is involved also, since larvae in the soil are killed and larvae die quickly in root extract from plants 11 weeks old and older. In extract from younger plants eggs hatch sooner than in extract from older plants or in water (Haroon, 1979). Although Pangola digitgrass is antagonistic to Meloidogyne incognita , it is a host for Belonolaimus 1 ongicaudatus and some other nematodes (Overman 1961). Boyd and Perry (1969), while testing various grasses for their suitability in Florida, discovered that the related cultivar, Transvala digitgrass, is resistant to EL longicaudatus . In order to learn more about the influence of Pangola and Transvala digitgrasses on nematodes, the following research was conducted to: 1. Determine whether Pangola digitgrass is antagonistic to Meloidogyne arenaria (Neal 1889) Chitwood 1949, M. ha pi a Chitwood 1949, and M. javanica (Treub 1885) Chitwood 1949; 2. To isolate, purify and characterize the chemical substance or substances from Pangola digitgrass which is toxic to M. i ncognita ; 3. Determine the effects of Transvala digitgrass on M. incognita and B_. longicaudatus ;

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Determine the effect of root leachates from Pangola and Transvala digitgrasses on hatch and survival of M. incognita and survival of B. lonqicaudatus ; Determine the effects of various combinations of Transvala and Pangola digitgrasses on M. incognita and B. lonqicaudatus ; Determine the effects of Pangola and Transvala digitgrasses on some other nematodes.

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CHAPTER 2 EFFECTS OF PANGOLA DIGITGRASS ON MELOIDOSYNE ARENARIA , M. JAVANICA , AND M. HAPLA Introduction Pangola digitgrass is antagonistic to Meloidogyne incognita (Winchester and Hayslip 1960, Haroon 1979), but its effect on other species of Meloidogyne has not been tested. The four most common and widespread species of Meloidogyne in the world are M. arenaria , M. hapla , M. incognita , and M. javanica (Taylor and Sasser 1978). All four species are present in tropical and subtropical regions of the world (M. hapla at high altitudes) which might be suitable for production of the digitgrasses. Since Pangola digitgrass is known to be antagonistic to M. incognita , experiments were conducted to determine whether it is antagonistic to the three other primary species of Meloidogyne . General Methods The population of Meloidogyne incognita used in these investigations was increased on tomato, Lycopersicon esculentum Mill, cv. Rutgers. The following were common to all experiments. The soil type used was Arredondo fine sand (90.6% sand, 3.9% silt, 5.5% clay, and 1.9% organic matter). It was autoclaved for 15 min. under pressure 4

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5 of 1.41 kg/cm (15 psi). All containers used were 15-cm diameter clay pots unless otherwise stated. All experiments were conducted in a greenhouse with temperatures at approximately 25°C ±3. The plants were watered as needed and once a week each received about 100 ml of solution made up with lg/1 of Nutrisol® fertilizer 12-10-12 analysis. Pangola and Transvala digitgrasses were obtained from cuttings that were rooted for five weeks before inoculation unless otherwise stated. A randomized block design was used in all experiments except those reported in Chapter 6 where a split plot design was used. All experiments were statistically analyzed by analysis of variance (ANOVA) and Duncan's Multiple Range Test (Steel and Torrie 1960). Materials and Methods Forty pots were filled with autoclaved soil, 20 of them planted with one unrooted cutting of Pangola digitgrass and 20 with one tomato seed to serve as a control . Treatments were M. arenaria , M. ha pi a , M. javanica , and M. incognita (standard) and tomato (control). Each treatment was replicated five times. Five weeks later the soil in each treatment was infested with 15 egg masses. All pots were placed in a greenhouse at about 25°C and watered as needed. The experiment was terminated after 90 days. The plants were removed from the soil, the tops removed and weighed, the roots washed, weighed, and the number of galls and egg masses determined. Then the roots were stained with acid fuchsin in lactophenol, destained in lactophenol , mounted on slides, and examined for the presence of different life stages of Meloidogyne . The population of second stage larvae in the

PAGE 17

soil in each pot was determined by removing larvae from a 100 cm aliquot of soil by a centrifugation-flotation technique (Hussey and Barker 1973). Results After 90 days, the soil populations of second stage larvae of all four species of Meloidogyne in pots were low ranging from 3 to 36. Soil populations in the tomato controls ranged from 2,412 to 12,180 (Table 2-1). In roots of Pangola digitgrass, the number of second stage larvae was low ranging from 9 to 43. Only one third or fourth stage larva was found in one replicate of M. arenaria . No other life stages were present in Pangola digitgrass roots nor were the roots galled or egg masses present (Table 2-1). In the tomato controls all life stages were present for all four species of Meloidogyne and galls and egg masses were on the roots. Discussion Pangola digitgrass exhibits the same antagonistic traits to M. arenaria , M. hapla , and M. javanica as to M. incognita as shown by the failure of any species to develop and mature in the roots and the small soil populations of second stage larvae. Thus, Pangola digitgrass should be a suitable crop to use wherever it can be grown for pasture or forage or as an antagonisitc plant to control the four major species of Meloidogyne before a desired crop is planted. It is probable that the grass is antagonistic to other species of Meloidogyne , but experiments must be conducted before one could be certain.

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CHAPTER 3 ISOLATION, PURIFICATION, AND CHARACTERIZATION OF A CHEMICAL SUBSTANCE FROM PANGOLA DIGITGRASS THAT IS TOXIC TO MELOIDOGYNE INCOGNITA Introduction Plant parasitic nematodes feed by piercing plant cells and ingesting the cell contents. Some nematodes feed as ectoparasites and others as endoparasites--either migratory or sedentary. Meloidogyne incognita is a sedentary endoparasite that spends most of its life embedded in the roots of the host, and host plants respond to infection by developing specialized cells referred to as giant cells or nurse cells on which the nematode feeds. Nonhost plants usually do not develop nurse cells and thus the nematode cannot develop. Antagonistic plants not only do not produce nurse cells but also produce substances that are toxic to nematodes. Tyler (1938) and Steiner (1941) reported resistance by Tagetes spp. (Marigolds) to Meloidogyne . Suatmadji (1969) tested several species and hybrids of Tagetes for nematicidal properties and found Tagetes patula var. "Golden Harmony" to be most effective in reducing populations of certain nematodes. Uhlenbroek and Bijloo (1959) identified the nematicidal compounds terthienyl and dithienyl from Tagetes . The structure of terthienyl was confirmed by Horn and Lamberton (1963). Thus, the first naturally occurring chemical compound which had nematicidal properties against the endoparasites 8

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9 Pratylenchus penetrans (Cobb, 1917) Filipjev, Schuurmans-Stekhoven, 1941, M. hapla , M. incognita , and M. javanica was reported. Asparagus, Asparagus officinalis var. "Mary Washington," effectively reduced populations of Paratrichodorus teres; an unidentified nematicidal compound was isolated from the roots (Rohde and Jenkins 1958). Spraying leaves of tomato with asparagus root extract indicated that the material acted systematically and was related to an acetylcholinesterase effect as described by Rohde (1960). Schaffer et al . (1962) found catechol as a nematicidal compound in roots of Eragrostis curvula . Taylor and Murant (1966) noted nematicidal activity of aqueous extracts of raspberry roots and canes. Winchester (1962B) reported that an aqueous extract of mature roots of Pangola digitgrass reduced larval emergence of M. incognita acrita while extract from young roots increased emergence compared to a water control. Haroon (1979) reported that eggs of M. incognita hatch faster in root extract from Pangola digitgrass plants up to 10 weeks old than in extract from older plants. Tests with leachates (Chapter 5) showed that a nematicidal substance (or substances) were present in Pangola digitgrass growing in pots killed M. incognita . This investigation was initiated to further characterize and to isolate and purify the nematicidal substance in roots of Pangola digitgrass. Material s and Methods General Methods All investigations reported in this chapter were conducted with roots of 20-week-old plants of digitgrass, Digitaria decumbens Stent.

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10 cv. Pangola. The roots were surface sterilized by immersing them in one part Clorox to 9 parts water for 3 minutes and rinsing them in three separate changes of sterile water. Extracts were prepared by comminuting a weighed portion of roots in a food blender for 30 seconds in sterile water, and the solution was passed through a micropore filter. Extracts were stored at -87°C. The test nematode used was — • incognita. All eggs used in these tests were extracted from egg masses using the method of Hussey and Barker (1973) but modified by shaking the egg masses for 40 seconds instead of 4 minutes, and a 10% Clorox solution was used instead of a 20% Clorox® solution. In order to compare the nematicidal activity of extracts prepared in different ways or subjected to various experimental treatments, a procedure was adopted that characterized an extract on the basis of the number of grams of Pangola digitgrass roots contained in the extract. Thus, 1 gram equivalent (1 GE) was defined as 1 gram of roots extracted with 10 ml of water. One milliliter of such an extract would contain 0.1 GE of roots. A method of computing the effectiveness of the nematicide in the root extract was calculated by using Abbott's formula (1925). Survey of Solvents for Ex tracting Nematicidal Substa nces from Pangola Digitgrass Roots ~ The objective of this experiment was to determine the best extraction medium for the nematicidal principle by using a series of solvents of increasing polarity, including pentane (nonpolar), acetone, methanol, and water (most-polar). Ten grams of roots of Pangola digitgrass (20 weeks old) were surface sterilized and comminuted for 60 seconds in 100 ml pentane in

PAGE 22

11 a food blender. The solution was passed through a micropore filter and designated as the pentane extract. The remaining root mass was blotted dry and found to weigh 7.32 grams. Sterile water (73.2 ml in order to keep the roots:water ratio equal to 1:10) was added to the root fragments, and they were extracted again in the food blender for 60 seconds. This extract, designated as the "water after pentane" extract, was also passed through the micropore filter. A second sample of 10 grams of roots was extracted in a similar manner with acetone and then with water after acetone. A third sample of 10 g of roots was extracted with methanol and then water after methanol. The final solutions of pentane, acetone, and methanol were evaporated to dryness under vacuum on a rotary bath (temp, about 40°C), and then 100 ml of water was added to dissolve the residue before assay for nematicidal activity. The bioassay of extracts was run by placing 400 eggs of M. incognita in 5 ml of each treatment solution, with each replicated four times. Sterile water was used as a control. Egg hatch and larval survival were measured each day for 10 days. Dose-Repsponse Relationship with Aqueous Extract An extract was prepared by comminuting 10 g of Pangola digitgrass roots in 100 ml of sterile water to produce an extract with 10 GE of roots. Aliquots of the extract were diluted to give test solutions containing 1 GE, 0.5 GE, and 0.1 GE of roots in a final assay volume of 10 ml, and each assay was replicated three times. Two hundred eggs of M. incognita were placed into each treatment solution contained in 5.5-cm diameter petri dishes. The number of eggs that hatched each 24 hours was recorded daily for 10 days. The larvae that emerged

PAGE 23

12 during each 24 hour period were removed and placed in a new dish containing extract with the same GE of roots in which they had hatched to determine their survival over a period of 10 days. Successive Extraction of Roots by Water The purpose of this experiment was to determine whether all of the active factor(s) in the roots of Pangola digitgrass could be removed with three successive extractions in water. For the first extraction, 100 grams of roots were placed in a food blender with 100 ml water and comminuted for 60 seconds. The solution was filtered as described earlier. The remaining root residue weighed 75 grams. To keep the same ratio of roots to water, 75 ml water was added and the above steps repeated. The remaining root residue weighed 62 g; 62 ml water was added and the above steps repeated. The three extracts were tested for activity by diluting aliquots to give 0.1 GE of roots in a final assay volume of 5 ml in 3.5-cm diameter petri dishes containing 500 eggs of M. incognita . The number of eggs that hatched after 10 days and the number of dead and live larvae were determined. Each treatment was replicated four times. Effect of Heat, Autoclavinq, and Cold Storage on the Active Principle in Pangola Digitgrass Roots This experiment was conducted to determine if the active principle(s) in Pangola digitgrass root extract is heat stable and if it remains stable in cold storage at 4°C. An extract containing 10 GE of roots in 100 ml of water was divided so that 70 ml (7 GE) were placed

PAGE 24

13 in an oven at 90°C for 7 hr. until all water had evaporated. The residue was dissolved in 20 ml sterile water that was further divided in four replicate bioassay volumes of 5 ml each (1.75 GE/bioassay volume) . Portions of the original extract were also stored at 4°C for two months or were autoclaved at 1.41 kg/cm 2 per square inch (15 psi) at 121°C (250°F) for 20 min. in a laboratory autoclave. Each of the extracts subjected to the three treatments, and a water control, were bioassayed with 600 eggs of M. incognita in each of four replicates. Number of eggs that hatched and mortality of larvae were noted after 10 days. Chromatographic Study of Extracts An aqueous solution of root extract was concentrated by heating at 90°C until it contained 10 GE of roots/ml. A total of 50 yl was placed in each of 10 spots at the origin of a 20 x 20 cm sheet of TLC silica gel on plastic. The chromatograms were developed in chloroform:ethyl acetate :formic acid (75 ml: 60 ml: 15 ml) or in isopropanol :water (10:3). Chromatograms on Whatman 3M paper were also prepared with 50 ul of the extract in each of 10 spots across the origin, and the chromatograms were developed in isopropanol :water (10:3). The paper was suspended above the solvent for 3-1/2 hours to saturate the paper with solvent vapor and then developed for 13 hours. Paper chromatograms developed in isopropanol rwater (10:3) were sprayed or dipped in spray reagents prepared according to Stahl (1965) in order to look for a detection reagent for the nematicidal compound in extracts.

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14 Stability of Nematicidal Principle in Extract to Acid and Alkali Two milliliters of root extract (31.4 GE) were added to 4 ml of 1 N hydrochloric acid (HC1), the mixture was heated at 90°C until dry, and 5 ml of sterile water was added to dissolve the residue. The pH of the solution was neutralized to pH 7.0 by adding 0.5 M potassium hydroxide (KOH) dropwise. The final volume was adjusted to give 1.57 GE/ml by adding sterile water. The solution was divided into four equal portions and placed in 5.5 cm petri dishes for bioassay. Each bioassay dish contained 7.85 GE of roots in a final volume of 5 ml . Effect of alkali was tested by adding 5 ml of 0.1 N KOH to 2 ml of extract (31.4 GE) to produce a strongly alkaline pH. The solution was neutralized with HC1 . Without evaporating this solution to dryness the volume was adjusted to 20 ml and 5 ml was added to each of four petri dishes. The bioassay of both acid and alkali treated extract was determined with 300 eggs per dish. Hatch of eggs only was tested. In the tests of stability of the nematicidal compound(s) to acid and alkali, a brown precipitate occurred when KOH was added to the water extract. The precipitate redissolved when the pH was adjusted to about 6. In other tests it was found that the precipitate could also be obtained from aqueous solutions without using KOH by adjusting the pH to 8 or greater with buffer (for example, 0.1 M TRIS buffer pH 8), and also by adding excess acetone to aqueous solutions. It thus appeared that the precipitated material was not very soluble at alkaline pH's and that it could be forced out of solution also by a solvent that was miscible with water but in which the precipitated

PAGE 26

15 material was not soluble, such as acetone. The precipitate obtained in either way was readily redissolved in weakly acidic water (i.e., final pH of solution was 6). These observations on solubility of the precipitated substance suggested that weak acid might be a better extracting medium for roots than water. To test this idea 284 g of roots that previously had been extracted in a blender with sterile water were reextracted with water acidified to pH 3 with hydrochloric acid. The final volume of extract was 1550 ml with a final pH of 4. The extract was concentrated at 90°C to 60 ml during 24 hours of heating. The 60 ml were divided into two 30 ml portions. To one 30 ml portion 120 ml of acetone was added and the mixture was shaken and allowed to sit in the refrigerator for about 30 minutes. The precipitate that formed was removed by centrifugation, washed by acetone, dried, and found to weigh 74.6 mg. To the second 30 ml portion, 4 N K0H was added to give a pH of 10 or greater. A precipitate formed immediately and was removed by centrifugation, washed by acetone, and dried. It weighed 78.3 mg. Thus, essentially the same weight of precipitated material was obtained by each method for inducing precipitation. The precipitate obtained in each way was dissolved in water that was acidified to pH 6 with 1 N HC1 and bioassayed. Each precipitate was active and killed the test nematodes. Dose-response Relationship with the Precipitate from Panqola Digitgrass Root Extracts The purpose of this experiment was to test the precipitate and also to determine a dose-response relationship. Each treatment level

PAGE 27

16 was replicated three times. The final volume of solution was 5 ml into which 250 larvae were placed. The treatment levels were 1 mg, 0.5 mg, 0.3 mg, and 0.1 mg precipitate per bioassay dish. The control consisted of sterile water. Determination of Completeness of Precipitation of Nematicidal Compound(s) from an Aqueous Extract of Pangola Digitgrass Roots In order to determine if precipitation resulted in all nematicidal activity in the precipitate, four different experiments were conducted to determine the relative killing effect of an aqueous extract (412 g/1236 ml water--evaporated to 80 ml), the precipitate from a part of the extract and the supernatant after the precipitate was removed. Results and Discussion Survey of Solvents for Extracting Nematicidal Substances from Pangola Digitgrass Root s The results are shown in Table 3-1. Pentane extracted no substances that caused significant mortality of eggs or of hatched larvae. The acetone extract contained substances that caused slight mortality of eggs, but larvae that successfully hatched were not killed. The methanol extract caused egg mortality but did not kill hatched larvae. Water extracts following each of the pentane, acetone, and methanol extractions contained active nematicidal substances that caused high mortality of eggs and hatched larvae. This experiment shows that water is the best solvent for the nematicidal substance(s) in the roots, but methanol appears to extract some small quantity. Water was chosen for future work.

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17 Dose-response Relationship with Aqueous Extract The results are shown in Table 3-2. There was a significant dif ference between all treatments in the percentage of eggs hatched, but no significant differences occurred in larval survival in extracts with 1.0 GE versus 0.5 GE of roots. Also no differences occurred between treatments containing 0.1 GE of roots and the water control; the former two were different from the latter two, however. Although more data would be desirable, the results do indicate a dose-response relationship, as might be expected if a chemical substance(s) is responsible for the nematicidal activity. Successive Water Extractions of Pangola Digitgrass Roots Few larvae hatched in assays of the first, second, or third extrac tion, and all that hatched died (Table 3-3). These data show that active material remained in the roots at least for three successive extractions. Effect of Heat, Autoclavinq, and Cold Storage on the Active Principle of Pangola Digitgrass Roots The results are shown in Table 3-4. Cold storage (4°C) of extracts had little or no effect upon the active principle as indicated by the fact that only 9% of the eggs hatched and only 29% of the larvae survived. When the extract was heated at 90°C, some of the active principle was destroyed as indicated by a 43% egg hatch and a 54% larval survival. Autoclaving the extract destroyed most of the active principle as indicated by an 86% egg hatch and 100% larval survival .

PAGE 29

18 Although heating apparently destroyed some of the active principle in extracts, it did represent a convenient method for concentrating extracts of large volume and was often used in later experiments to reduce the volume of extracts that often started at 2 liters or more. Chromatographic Study of Extracts Following development the thin layer chromatogram (TLC) sheet was dried and examined under ultraviolet (UV) light. No spots were evident. The sheet then was divided into zones for elution and bioassay of any nematicidal substances that may have moved during the development of the chromatogram. Beginning at the origin, the TLC sheet was divided into six zones of 2 cm width each, with zone 1 being the region from the origin to 2 cm above the origin. Subsequent zones were numbered accordingly. The plastic backing and each zone was cut with a scissors into small pieces and placed in 5 ml of sterile water. The beakers were covered and placed in a refrigerator for 24 hours. Then the solution from each beaker was centrifuged and passed through a micropore filter. Two 2 ml aliquots of each solution were placed in 3.5 cm diameter petri dishes and 100 eggs of M. incognita added for bioassay of the number of eggs that hatched after 10 days. A 2 cm strip of the TLC sheet not treated with extract but immersed in the same solvent for five minutes was used as a control zone. The results are shown in Table 3-5. No eggs hatched in the solution from zone 1 near the origin. Solutions from all zones caused less hatch than the control. These data suggested that most of the nematicidal principle did not move from the origin or that it moved less than 2 cm from the origin. Weak

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19 biological activity in all zones does suggest some movement but probably more as a streak than as discrete spots. This solvent system and/or chromatography medium did not seem to be suitable and silica gel was abandoned. The paper chromatograms seemed more promising. They were removed, dried, and also viewed under UV light, but again no spots were visible. The chromatogram then was divided horizontally into four zones: Zone 1 was 3 cm wide (1.5 cm above and below the extract application point). Zones 2 and 3 were each 5 cm wide, and Zone 4 was 6 cm wide. Squares (5 cm x 5 cm) were cut from a separate paper, dipped into the solvent and used as controls. The original extract was tested also as a control. The zones used in the egg hatch bioassay were cut into small pieces, soaked 24 hours in sterile water, and tested, as described in the TLC test except that 500 eggs of M. incognita were used in each of five replicates. Table 3-6 shows that zones 1, 2, and 3 from the TLC sheet developed in i sopropanol :water (10:3) had greatest nematicidal activity, while zone 4 had no more activity than the control zone. When zones 1 to 4 were recombined from a second chromatogram into one bioassay, much greater nematicidal activity was present than that in any of the zones. This is suggestive, but not conclusive, that more than one active compound is present in the extract. If the migration of one active compound was so poor as to produce a streak along the chromatography path instead of a discrete spot, then each zone would be expected to show activity, and recombining all zones would be expected to produce greatest kill in the bioassay. The results of tests of chromatograms with a variety of spray reagents are presented in Table 3-7. Only three reagents gave a

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20 positive test on paper chromatograms of the precipitate from Pangola digitgrass roots, and two of these, 3N H 2 S0 4 and iodine vapor, are indicative only in a general way of the presence of an organic substance. Diphenylcarbazone gave a positive test, and it is known to react with certain heavy metals to produce blue to purple colors. This may mean that such a metal ion is a part of the structure of the nematicidal compound(s) in the extract. Some compounds that can chelate or complex a heavy metal (such as uric acid, xanthine, hypoxanthine, and some carbonyl containing compounds) can be detected by first spraying with a solution of mercuric chloride, followed by diphenylcarbazone and ammonia. Inasmuch as these chromatograms were not sprayed with mercuric chloride or any other heavy metal, the positive reaction with diphenylcarbazone might be indicative of a group that can chelate a small amount of heavy metal already in the extract solution prior to spraying with diphenylcarbazone. Such a metal ion might or might not contribute to the nematicidal action of the compound(s). Detection with diphenylcarbazone thus may be a fortuitous occurrence with little predictive value as to structure of the active substance. It does nevertheless provide a convenient way to follow subsequent manipulations of extracts through chromatography and other isolation procedures. Stability to Acid and Alkali The results, shown in Table 3-8, indicate that neither hydrochloric acid nor potassium hydroxide destroyed the active principle in extracts from roots of Pangola digitgrass as measured by egg hatch. Egg hatch in extract subjected to strong acid or alkali was not

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21 significantly different from that in an untreated aqueous extract, while hatch in the control was high. Dose-response Relationship with the Precipitate from Pangola Digitgrass Root Extracts A dose-response relationship to the redissolved precipitate is very clearly evident in the data of Figure 3-1 and Table 3-9. Fiftyfour percent of the larvae were killed by exposure to 1 mg of the precipitate, while 26% of the larvae were killed when they were exposed to 0.5 mg of the precipitate, and only 16% were killed by 0.3 mg of precipitate. At 0.1 mg of precipitate 5% were killed. Based on these results the LD 50 for 10 day exposure of M. incognita larvae is about 1 mg of precipitate. Determination of Completeness of Precipitation of Nematicidal Compound(s) from an Aqueous Extract of Pangola Digitgrass Roots The results presented in Tables 3-10, 3-11, and 3-12 and in Figures 3-2, 3-3, and 3-4 show that precipitation does not remove all of the nematicidal substance(s) from an extract. The supernatant from which the precipitate was removed still had good nematicidal activity. The estimate of the LD 50 value shows that 1 GE of the original extract is equivalent in effectiveness to 2.6 GE of precipitate and to about 3 GE of supernatant. It thus appears that about 50% of the nematicidal principle remains in the supernatant while half precipitates. This might indicate the presence of multiple compounds, some of which will not precipitate, or it may mean that only about half of a single toxic compound will precipitate under the conditions used.

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22 These bioassays, to obtain the data in Figures 3-2, 3-3, and 3-4, were made over a three-week period; the aqueous extract, supernatant, and precipitate were each tested with a different group of M. incognita . In order to test the reliability of LD 5Q values obtained, an experiment was designed to produce an expected 50% kill of the test nematodes by using 1 GE of aqueous extract, 2.6 GE of precipitate, and 5.2 GE of supernatant. The results, shown in Table 3-13, show that the actual values of percent kill were very close to the predicted 50% level. This close agreement of observed with predicted toxicity shows the reliability of the bioassay method and technique and suggests that this last bioassay technique requiring only 48 hr. for evaluation should be adopted for further work in characterizing and identifying the toxic compound(s) in the Pangola digitgrass roots. The technique seems to offer a semiquantitative assay for nematicidal activity of extracts based upon the observation that 1 mg of dry precipitate gives 50% kill. Since 1.2 GE of aqueous extract (Fig. 3-2) produces 50% kill, then 1 gram of roots should contain about 0.8 g of the nematicidal compound if only one compound is involved. However, it must be remembered that water probably does not extract all of the toxic compound, so the actual concentration may be higher. Conclusion An aqueous extract of Pangola digitgrass roots (20 weeks old) was tested against M. incognita larvae in an immersion assay. The active material in extracts is stable to heat at 90°C for 48 hr., but when the extract was autoclaved for 20 min. at 121°C, the

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23 biological activity was destroyed. The nematicidal activity was not soluble in nonpolar solvents such as pentane and only very slightly soluble in acetone and methanol. When the extracts were chroma tographed on thin layer chromatography (TLC) silica gel sheets in chloroform:ethyl acetate: formic acid (75:60:15 v/v), the active material remained at the origin. The active material was precipitated from concentrated aqueous extracts by adjusting to pH 10 with dilute KOH. The dried precipitate produced about 50% mortality of M. incognita larvae in a laboratory bioassay at a dose of 1 mg precipitate. The redissolved precipitate chromatographs as a single spot on paper chromatograms (isopronanol :H 2 0) (10:3) and can be detected by a spray of 15! di phenyl -carbazone in ethanol .

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24 Table 3-1 Test of increasing solvent polarity for efficacy in extracting nematicidal substances from Pangola digitgrass roots. In each bioassay 0.5 gram equivalent' of Pangola digitgrass root extract was tested. Root extraction medium 1 After 10 days % Hatch' % Live larvae Pentane 92 a 3 91 a Acetone 75 b 85 a Methanol 39 c 89 a Water after pentane 11 e 12 c Water after acetone 20 d 6 d Water after methanol 20 d 56 b Water only (control ) 87 a 97 a One gram equivalent equals 1 gram of roots extracted with 10 ml water (or other solvent in this particular experiment). Approximately 400 eggs of M. incognita were added to each rep! icate. 3 Means in columns followed by the same letter are not significantly different at the 5% level according to Analysis of Variance and Duncan's Multiple Range Test. (All the tables in this chapter followed the same rule.)

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25 Table 3-2. Dose-response relationship between gram equivalents of roots in an aqueous extract and egg hatch and mortality of larvae of Meloidoqyne incognita . Gram equivalent of % Larval root tissue 1 % Egg hatch' 1 survival 3 1.0 20 d 29 b 0.5 49 c 29 b 0.1 82 a 82 a 0 63 b 80 a One gram equivalent equals 1 gram of roots extracted with 1 0 ml of water. Two hundred eggs were added to each dish, and the egg hatch determined daily for 10 days. 3 Hatched larvae were removed daily, placed in separate dishes, and the number of live and dead 10 days later determined.

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26 Table 3-3. Successive water extractions of Pangola digitgrass roots. Extraction^ Effect on M. incognita ^ % Egg hatch % Live larvae 1 0.006 0 2 1 .6 0 3 2.6 0 Control 72 100 Four hundred eggs were added to each of the four replicates. 2 Egg hatch and mortality of larvae were determined after TO days.

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27 Table 3-4. Effects of heat, cold storage, and autoclaving upon the active principle in aqueous extracts of Pangola digitgrass roots. Treatment Gram equivalents tested % Hatch* after 10 days % Live larvae after 10 days Stored 2 months--4°C 0.5 9 d 29 c Heated 90°C for 7 hr. 1.75 43 c 54 b Autoclaved 250°F for 20 min. 0.3 86 b 100 a Control (water) 98 a 99a * Approximately 600 eggs were added to each of four replicates.

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Table 3-5. Bioassay of silica gel thin layer chromatogram 1 of an aqueous extract of Pangola digitgrass roots. TLC zone 1 2 3 4 5 6 Control % Egg hatch' 0 f 37 de 25 e 40 cd 51 be 57 b 86 a ""solvent: chloroform:ethylacetate:formic acid (5:4:1 v/v). 2 Zone 1 included origin to 2 cm above origin, and each successive zone included the next successive 2 cm above the previous zone. 3 0ne hundred eggs in each replicate, hatch determined after 10 days.

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29 Table 3-6. Bioassay of Whatman 3M paper chroma togram of an aqueous extract of Pangola digitgrass roots. 0 Zone % Egg hatch 3 1 22 b 2 18 b 3 23 b 4 57 a Recombined zones 8 c 1 -4 control zone 59 a Solvent: isopropanol :hL0 (10:3) . 2 Zone 1 included area from 1.5 cm below origin to 1.5 cm above origin; zone 2 next 5 cm; zone 3 next 5 cm; zone 4 next 6 cm; control zone was 5 cm strip of paper dipped into solvent and dried. Approximately 500 eggs were added to each replicate, and hatch was determined after 10 days.

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30 Table 3-7. Tests of chromatograms of Pangola digitgrass root precipitate with spray reagents for indicating functional groups. Spray reagent Reaction (+) or (-) 1 I . Ni nhydri n 2. 1,1 dichlorofl uorescein 3. ultraviolet 1 ight 4 . UV light before reagent #2 5. UV light after reagent #2 6. 0.1 N AgN0 3 :5N NH^OH (1 :1) _ 7. Phenol red _ 8. Dimethyl ami nobenzaldehyde 9. 3 N H 2 S0 4 followed by heat and UV light + 10. Ferric chloride-Potassium ferricyanide 11 . Anthrone 12. Iodine vapor + 13. Trichloroacetic acid in ethanol followed by UV light 14. Phosphomolybdic acid-tungstic acid 15. Phosphomolybdic acid 16. Di phenyl carbazone followed by ammonia +

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31 Table 3-8. Effect of potassium hydroxide and hydrochloric acid on the active principle in roots of Pangola digitgrass as measured by egg hatch of Mel oidogyne incognita . Treatment Egg hatch Potassium hydroxide + Pangola grass extract 5 b Hydrochloric acid + Pangola grass extract 4 b Extract alone 2 b Steri 1 ized water 176 a All treatment solutions were adjusted to pH 6 before bioassay, and each bioassay tested each treatment at a final level of 7.85 6E of roots. 2 Mean of four replicates with 300 eggs per replicate.

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32 Table 3-9. Dose-response relationship between concentration of precipitate from Pangola digitgrass root extract and mortality of Meloidogyne incognita larvae. Dried precipitate (mg) % of dead larvae 1 after 10 days Corrected % mortality^ (Abbott's formula) 1 55 a 54 0.5 27 b 26 0.3 18 c 16 0.1 7 d 5 Control 2 e Two hundred and fifty larvae were added to each of the three repl icates. Abbott's formula x " ^ x 100 = % corrected x x = % of living in control y = % of living in treatment.

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33 Table 3-10. Determination of the LD 50 value for mortality of Meloidogyne incognita larvae in a 43 hr bioassay with an aqueous extract. Gram equivalents of aqueous extract % Mortality 1 Corrected % mortality (Abbott' s formula) 5.2 85 A 84 2.6 70 B 69 1.6 60 C 53 0.5 32 B 29 Water (control ) 4 E Approximately 400 larvae were added to each of three replicates.

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34 Table 3-11 Determination of the LD 50 value for mortality of tteloidogyne incognita larvae in a 48 hr bioassay of the supernatant after removal by centrif ugation of pH induced precipitate from the aqueous extract of Table 3-10. Gram equivalents of supernatant % Mortality 1 Corrected % mortality (Abbott's formula) 5.2 55 A 53 2.6 51 A 49 1.6 26 A 23 0.5 6 B 2 Water 4 B Approximately 400 larvae were added to each of three replicates.

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35 Table 3-12. Determination of the LD 5 q value for mortality of Meloidoqyne incognita larvae in a 48 hr bioassay of the dry precipitate obtained from the aqueous extract of Table 3-10. Treatment % Mortality 1 Corrected % mortality (Abbott's formula) 5.2 GE 62 A 60 2.6 GE 56 AB 54 1 .6 GE 41 B 38 0.5 GE 19 C 15 Water control 5D Approximately 400 larvae were added to each of three replicates.

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36 Table 3-13. Confirmation of gram-equivalents of aqueous extract, and of supernatant and precipitate obtained from the aqueous extract required to give 50% mortality of a test population of Meloidogyne incognita larvae in a 48 hr bioassay. 1 ? Treatment % Mortality 1 GE aqueous extract 47 5.2 GE supernatant 48 2.6 GE precipitate 57 Gram equivalents of each treatment expected to give 50% mortality were estimated from data in Tables 3-10, 3-11, and 3-12, respectively. 2 Percent mortality was not significantly different among treatment means (three replicatesO in an Analysis of Variance and Duncan's Multiple Range Test.

PAGE 48

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PAGE 56

CHAPTER 4 THE EFFECT OF TRANSVALA DIGITGRASS ON HELOIDQGYNE INCOGNITA AND BELONOLAIMUS LONGICAUDATUS Introduction Transvala digitgrass ( Digitaria decumbens Stent P.I. 299601) is a cultivar named for its native habitat, the province of Transvaal in South Africa. The grass was first brought to the U.S.A. from Africa in 1964 in a collection of plant introductions by Dr. A. J. Oakes, Jr., U.S.D.A., Beltsville, Maryland. Boyd and Perry (1969) found Transvala digitgrass to be nearly immune to damage by the sting nematode, Belonolaimus lonqicaudatus . There are no other known reports on the effects of nematodes on this grass. The purpose of this investigation was to determine the effects of Transvala digitgrass on Meloidogyne incognita . Materials and Methods Effect of Transvala Digitgrass on M. incognita Twenty pots were filled with autoclaved soil. Five of the pots were planted with one five-week-old rooted Transvala digitgrass cutting, five with one five-week-old tomato seedling, five with one five-weekold Pangola digitgrass cutting, and five were left fallow as controls. The soil in each pot was infested with 20 egg masses of M. incognita . Three months later the plants were harvested, the roots washed, and

PAGE 57

46 the number of galls and egg masses on the roots counted. All the soil in each pot was processed and the larvae extracted and counted. Transvala Digitgrass Interplanted with Tomato Ten pots were filled with autoclaved soil and infested with 20 egg masses of M. incognita . Five of the pots were interplanted each with one five-week-old rooted Transvala digitgrass cutting and one five-week-old tomato seedling. Five control pots were planted with one five-week-old tomato seedling. The experiment was terminated three months later. Larval Penetration and Development Forty-two styrofoam cups were filled with autoclaved builders sand. Twenty-one of these cups were planted with one five-week-old Transvala digitgrass cutting and 21 with one five-week-old tomato seeding. Five hundred eggs of M. incognita , separated from egg masses by Hussey and .Barker method (1 973), were added to each replicate. The experiment was carried out in an incubator at 25°C. Every four days, for twenty-eight days, plants from three pots of Transvala digitgrass and three pots of tomato were harvested. The roots were washed, stained with acid fuchsin in lactophenol for 24 hours, destained in lactophenol for 48 hours, mounted on slides, and examined for the presence of larvae. Effect of Transvala Digitgrass on B. longicaudatus Fifteen pots were filled with autoclaved soil; five were planted with one five-week-old Pangola digitgrass cutting, five with one

PAGE 58

47 five-week-old Transvala digitgrass cutting, and five with one fiveweek-old tomato seedling to serve as controls. The soil in each pot was infested with 200 mature B. lonqicaudatus . After three months the plants were harvested, the number of B_. lonqicaudatus in the soil counted, and the roots and top weights determined. Resul ts Effect of Transvala Digitgrass on M. incognita Three months after this experiment was established, Transvala digitgrass roots contained an average of 26 galls per root system, and there was an average of 1210 larvae in the soil of each treatment pot (Table 4-1). By comparison, Pangola digitgrass roots were not galled and not many larvae were in the soil. Tomato plant roots were heavily galled and an average of 8767 larvae were in each pot. In pots left fallow, an average of 50 larvae were in the soil. Galls on Transvala digitgrass roots were small compared with galls on tomato roots. Transvala Digitgrass Interplanted with Tomato More galls and egg masses were present on tomato when interplanted with Transvala digitgrass than when tomato was planted alone (Fig. 4-1, Table 4-2). The number of larvae recovered from soil planted to tomato alone was fewer than from soil with the two interpl anted.

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48 Larval Penetration and Development Four days after this experiment was initiated, second stage larvae were present in Transvala digitgrass roots but not as many as were present in tomato roots (Fig. 4-2). Third and fourth stage larvae first developed in tomato roots after 8 days and in Transvala digitgrass after 12 days. After 24 days, adult females were present in both with egg masses present in tomato after 24 days and in Transvala after 28 days. At any given time more individuals of any given life stage were present in tomato roots than in Transvala digitgrass roots. The life stages of M. incognita in roots of Transvala digitgrass are shown in Figure 4-3 a, b, c, d, and e. Effect of Transvala Digitgrass on B. longicaudatus Three months after this experiment was established, B_. longi caudatus had reproduced on Transvala digitgrass but at a level that maintained the population at about the inoculum level. On the known hosts, Pangola digitgrass and tomato, populations had increased some 6 and 25 times, respectively (Table 4-3). i Discussion While the population of M. incognita increased on Transvala digitgrass, it did so at about one seventh the rate of increase on tomato. When Transvala digitgrass and tomato were interplanted, the nematode population increased a little more than on tomato alone. These results indicate that Transvala digitgrass is a satisfactory host for M. incognita but not an excellent host. It certainly can

PAGE 60

49 maintain populations of the nematode and thus could not be used in a rotation system to reduce population levels of M. incognita . Since the root system of the grass did not grow well, it is apparent that the grass will not yield as much forage when grown in the presence of the nematode. Conversely, populations of B. 1 ongicaudatus remained at about the inoculum level, indicating that the grass is a poor host for it. Also, the roots and tops grew well, indicating that the grass sustained little, if any, damage. Therefore, it can be concluded that Transvala digitgrass could be grown successfully in areas where B_. lonqicaudatus is present and that the nematode populations likely would not increase. It would be damaged by M. incognita , however, and would permit the nematode populations to increase also.

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50 •1 — cn fO •1 — 4^ ~a C ro C7 o ro u > c LO r— C (0 CJ 5C H >l cr 4o o -o o +J o a co re 4— 4c LjJ o QJ ai +-> ro *4 L.O •r— E 00 E S_ JC sa> a> +J Ol 0J +J +-> cn Q. to -M QJ >> to ro CO tO ro ro • QJ 4^ O to -t-> uo ro i c z 10 o i i +-> ro fO o CnJ c o E SCD 'f— E 4• » — i — Sc QJ cn CL X to O) co o +J (13 o aj . O > i. it JC to c +-> QJ ro Cn i i i 4^ QJ O i i rc S 00 cn a) cn > CO •i — QJ CD tO 4E — >> o ro to cn ro ( C\J 4to i i o 4-> +-> o i JC QJ 3 O C\J +-> 0J JC o +J Sro >> • "O cu -O +-> a> > to CN1 4-> ro "O QJ +-> to QJ +-> o ai • #» O-O (_) ro u If, — . O QJ Cr I i — cn o qj O r-» o o i — sc s: ro cn IT) to o ro > OvJ r\ to 4SSCO ro 410 o to QJ C i — +j ro E Q3 "' — CO o , — 4-) O i — +-> +-> o CJ Ci o CO ro o O E CO r\j S0) s_ ro to ^.+-> i Lf) i +J u to to C\J i cr> i •rc r— >> jc +-> ro r— to o QJ scj ro ro +-> QJ C CD CO ro > 3 E Q c l/l C •r— >) X io It o +-> — Sro 4-> ro CL +-> cn sr o o Oro c a; ro ro CO r> o 3 CNJ ro "O +-> to C _o O s_ Jro c — CJ o a> ro C o s_ iro 5 ro 4u I— htiI— u_ ro ro QJ >

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52 Table 4-3. Effects of Transvala digitgrass, compared with Pangola digitgrass and tomato, on Belonolaimus longicaudatus . Treatment 2 Nematode per pot Weiqht (q) Roots Tops Pangola digitgrass 1203b 3 2.4 4.1 Transvala digitgrass 262c 27.7 41 .3 Rutgers tomato 4965a 1.9 2.3 Each treatment received 200 mature nematodes; the experiment was terminated after 90 days. 2 Mean of four pots (1200 cm 3 soil). 3 Data in vertical column followed by the same letter are not significantly different at the 5% level according to Duncan's multiple range test.

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Figure 4-3. Life stages of Heloidogyne incognita in roots of Transvala digitgrass (a) invading second stage larvae, (b) late second stage larva, (c) third or fourth stage larva, (d) mature female, (e) eggs.

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1 I H I

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CHAPTER 5 EFFECTS OF ROOT LEACHATE FROM PANGOLA AND TRANSVALA DIGITGRASSES ON MELOI DOGYNE INCOGNITA AND BELONOLAIMUS LONGICAUDATUS Introduction Some plants which are antagonistic to nematodes have been shown to produce root secretions that are toxic. Oostenbrink (1950) showed that when Tagetes patula was grown, Pratyl enchus populations in the soil were reduced 90%. Rohde and Jenkins (1958) found that asparagus was resistant to Trichodorus christiei and that populations declined more rapidly in soil containing asparagus roots than in soil without roots. A toxic compound recovered from the soil was shown to have originated in the asparagus roots where it occurred in high concentrations. The compound was identified as a glycoside with a low molecular weight aglycone. Investigations by Triffitt (1929) and Morgan (1925) showed that diffusates from roots of Mustard, Brassica campestris L. , were antagon istic to potato, Solanum tuberosum L., and did not stimulate potato cyst nematode, Heterodera rostochiensis Wollenweber 1923. Christie (1959) reported that rutabagas, Brassica napobrassica L. Mill., secrete a compound into the soil that made the burrowing nematode, Radopholus simil is , unable to find roots of corn, Zea_ mays L. 61

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62 This investigation was initiated to determine if root diffusates from Pangola and Transvala digitgrasses would affect egg hatch and larval survival of M. incognita and survival of B. 1 ongicaudatus . Materials and Methods The Leachate To collect root leachates a drainage hole was made in the bottom of each of 16 styrofoam cups (32 oz.) and a length of sterile tubing inserted into the hole. Cheesecloth was placed over the end of the tube to serve as a filter. The cups were filled with autoclaved soil. Treatments were cuttings of Pangola digitgrass, cuttings of Transvala digitgrass, tomato seed, and fallow. Each treatment was replicated four times. All cuttings first were surface sterilized by immersing the cutting in one part Clorox to nine parts water for three minutes, then rinsing in three separate changes of sterile water to remove the ® Clorox . The cups were planted and placed in a growth chamber at 25°C. All treatments were fertilized with 200 ml of Nutri sof sol ution (12-10-12 fertilizer). Beginning four weeks after the test was initiated, weekly for 10 weeks leachate was collected by pouring 300 ml of sterile water into each cup. The water percolated through the soil, and the excess passed through the tubing and was collected in sterilized flasks. One hour after the water was poured into the cups, the flasks containing the leachate were removed, and the leachate from each flask was passed through a micropore filter and stored in a freezer at -84°C.

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63 In order to quantify the relative concentration of the leachate from each plant, when the last leachate sample was collected, the plants were removed, the roots washed, and weighed fresh. Effect of Root Leachates on Egg Hatch and Larval Survival of M. incognita The experiment was conducted using 5 ml of leachate and 500 eggs of M. incognita in 10 ml autoclavable tubes. The number of eggs that hatched and the number of live and dead larvae were determined after six days. The experiment was carried out at room temperature. Leachates were from Pangola and Transvala digitgrasses, tomato, and fallow soil. Each treatment for each of the 10 plant ages was replicated four times. Effect of Root Leachates on Survival of Adult B. lonqicaudatus This experiment was conducted similarly to that with M. incognita except that 50 ml of leachate and 50 adult B_. longicaudatus in 100 ml sterilized flasks were used. The number of live and dead nematodes was determined after 48 hours. Resul ts The Leachates The amount of leachate collected each week from each treatment ranged from 180 to 250 ml. Over the entire 10 weeks, the amounts collected from each plant were relatively uniform and less than from the fallow soil cups. At the last week of the experiment, the ratio of fresh root weight to leachate was 8 ml per gram of root

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for Pangola digitgrass, 9 ml per gram of root for Transvala digitgrass, and 12 ml per gram of root for tomato (Table 5-1). While such ratios are not known for earlier weeks, we can assume that the root weight increased each week and, therefore, that the concentration of any substance secreted by the roots would have increased each week as the plants grew larger. Effect of Root Leachates on Egg Hatch and Larval Survival of M. incognita In leachate from Pangola digitgrass four weeks old, 54% of the 500 eggs hatched. The percentage of hatch generally decreased until in leachate from 13-week-old plants, 2% hatched. In leachate from Transvala digitgrass four weeks old, 26% of the eggs hatched. The percentage of hatch generally increased, except at week five, until in leachate from 13-week-old plants, 38% of the eggs hatched. In the leachate from tomato and from fallow soil, the percentage of hatch over the 10 weeks was about the same. The hatch in leachate from four-week-old tomato plants was 52% and that from fallow soil, 47%. In leachate from 13-week-old tomato plants hatch was 44% and from fallow soil, 49% (Fig. 5-1, Table 5-2). The percentage of larval survival was generally very high in all weeks under tomato and fallow treatment. In leachate from Transvala digitgrass through week seven, 100% of the larvae were alive; after week seven a few larvae died, with a maximum of 32% dead larvae in week 1 1 .

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65 In leachate from Pangola digitgrass, 100% of the larvae were live through week six. In leachate from week 8 through week 13 a few eggs hatched, and most of the larvae died. One hundred percent, 95%, and 100% of the larvae were dead when they were exposed to root leachates from weeks 11, 12, and 14, respectively (Table 5-2). Effect of Root Leachates on Survival of Adult B. longicaudatus In leachates from Transvala digitgrass plants 8 through 13 weeks old, except for week 11, survival of B. longicaudatus was significantly less than in other leachates from corresponding age plants and from fallow soil. Also, survival from week 8 through week 13 was significantly less than during the first four weeks. Survival at week 12 was significantly less than at previous weeks, and survival at week 13 was significantly less than at week 12. In leachate from Pangola digitgrass survival was not affected except at week 13. Survival was not significantly different in leachates from tomato and fallow soil at any age (Table 5-3, Fig. 5-2). Discussion It is not surprising that leachate from plants up through seven weeks old had no effects on egg hatch and larval survival of M. incognita and survival of B_. 1 ongicaudatus because at those ages the quantity of roots must have been rather low and the concentration of root secretions thus rather dilute. However, these results confirm previous work by Haroon (1979) who showed that root extract of equal concentration from Pangola digitgrass plants 4 to 10 weeks old had no effect on survival of M. incognita until the plants were 11 weeks old. Additional research needs to be conducted, however, to determine

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66 whether equal concentrations of root extracts of Transvala digitgrass are not effective until the plants are at least eight weeks old. Also, additional research should be conducted to determine the effects of different dilutions of root leachates on B_. longicaudatus . Leachate from Transvala digitgrass plants, 4, 5, and 12 weeks old inhibited hatch of eggs of M. incognita when compared to leachate from other age roots, while leachates from Pangola digitgrass plants four through seven weeks old stimulated egg hatch compared to leachate from older plants. In leachate from tomato roots and from fallow soil some 40-52% of the eggs hatched in leachate at all ages indicating no effects. Therefore, a significant percentage of egg hatch above 52% would indicate a stimulation to hatching and below 40% an inhibition to hatching. Based on that range, Pangola digitgrass root leachate did not stimulate hatch at any age but inhibited hatch when plants were 8 to 13 weeks old. Transvala digitgrass root leachate inhibited hatch at 4, 5, and 12 weeks of age but had no effects at other ages. Most of the hatched larvae in leachate from Transvala digitgrass, tomato, and fallow soil were alive, while in Pangola digitgrass root leachate most of the larvae died in leachate from plants 8-13 weeks old. The fact that leachate from Pangola digitgrass (except at week 13), tomato, and fallow soil had no effect on B_. longicaudatus at any plant age is not surprising since both plants are hosts for the nematode. Since Transvala digitgrass is reported to be resistant to B. longi caudatus , it is somewhat surprising that effects of the leachate on survival did not occur until plants were eight weeks old. Perhaps this was due to the concentration of the leachate--a point that needs investigation.

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67 Table 5-1. Leachate collected from Pangola digitgrass, Transvala digitgrass, and tomato roots at week 13 and its relative concentration per gram of roots. Treatment Weight (g) 1 root system Leachate ml at week 13 ml /root Pangola digitgrass 23 192 8 Transvala digitgrass 21 185 9 Tomato 16 195 12 Fallow 235.5 Average of four replicates.

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CHAPTER 6 THE EFFECTS OF VARIOUS COMBINATIONS OF PANGOLA DIGITGRASS , TRANSVALA DIGITGRASS AND TOMATO ON MELOIDOGYNE INCOGNITA AND BELONOLAIMUS LQNGICAUDATUS Introduction Plants which have a detrimental effect on nematodes greater than that of nonhost plants are said to be antagonistic. Some plants known to have antagonistic properties against certain nematodes are marigolds, Taqetes spp., (Tyler 1938, Steiner 1941, and Suatmadji 1969), crotalaria, Crotalaria spectabil is (Good et al . 1965), hairy indigo, Indigofera hirsuta L. (Ruehle and Christie, 1958) and castor bean, Ricinus communis (Lear and Miyagua 1966). Pangola digitgrass, Digitaria decumbens Stent (P.I. 111110), and Transvala digitgrass, Digitaria decumbens Stent (P.I. 200601), have antagonistic properties to certain species of nematodes. Pangola digitgrass is antagonistic to at least four species of Meloidogyne (Chapter 2) but is a good host of the sting nematode, Belonolaimus longicaudatus . Transvala digitgrass is resistant to B_. longicaudatus but is a host of Meloidogyne incognita . Experiments reported here were designed to determine whether interpl anti ng Pangola and Transvala digitgrasses in soil infested with B. longicaudatus and M. incognita would mutually protect each of these grasses from its nematode pathogen also, whether the two grasses interpl anted with Rutgers tomato, a good host of both nematodes, would protect tomato from injury. 74

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75 Material s and Methods The experiment consisted of 12 treatments (Table 6-1) with each replicated five times. Treatments were 1) Pangola and Transvala digitgrass interplanted in soil infested with Meloidogyne incognita ; 2) Pangola and Transvala digitgrass interplanted in soil infested with B_. longicaudatus ; 3) Pangola and Transvala digitgrasses interplanted in soil infested with both M. incognita and B_. longicaudatus ; 4) Pangola and Transvala digitgrasses interplanted and uninoculated (control); 5) Transvala digitgrass in soil infested with both M. incognita and IB. longicaudatus ; 6) Pangola digitgrass in soil infested with both M. incognita and B_. longicaudatus ; 7) Rutgers tomato in soil infested with M. incognita and B_. longicaudatus ; 8) Transvala and Pangola digitgrasses interplanted with tomato in soil infested with M. incognita and B. longicaudatus ; 9) Transvala digitgrass interplanted with tomato in soil infested with M. incognita and B_. longicaudatus ; 10) Pangola digitgrass interplanted with tomato in soil infested with M. incognita and B_. longicaudatus ; 11) Pangola digitgrass uninoculated (control); and 12) Transvala digitgrass uninoculated (control). Sixty plastic trays (60 x 16 x 24.5 cm) were filled with-2000 cm 3 of autoclaved soil . Unrooted digitgrass cuttings and tomato seed were used to initiate the experiment. Each tray contained a total of six plants; when two or three different plants were interplanted, they were alternated in two or three rows. All treatments were arranged randomly on a greenhouse bench. Temperature was 25 ±2°C. Plants were watered as needed, and once a week each tray received about 100 ml of a fertilizer solution made up with one gram per liter of Nutrisol®

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76 12-10-12 analysis. Nematode inoculum was added five weeks after the experiment was initiated. Trays inoculated with M. incognita received 2,000 second stage larvae hatched no more than 24 hours previously. Trays inoculated with EL lonqicaudatus received 400 nematodes of mixed life stages and sexes. The experiment was terminated 12 weeks after inoculation. Resul ts When Pangola and Transvala digitgrasses were interplanted and inoculated with M. incognita , the population was reduced to very low levels (Table 6-1). Neither grass showed any root galls or egg masses. Some second stage larvae were present in the roots. Root and top weights were not significantly different than when Pangola and Transvala digitgrasses were interplanted and not inoculated. When Pangola and Transvala digitgrasses were interplanted and inoculated with EL longicaudatus , soil populations of the nematode were reduced to zero. Roots and top weights were not significantly different than when the two grasses were interplanted and not inoculated. When Pangola and Transvala digitgrasses were interplanted and inoculated with both M. incognita and _B. longicaudatus , the soil population of _EL lonqicaudatus was reduced to five per 100 cm 3 of soil and that of M. incognita to zero. There were no galls or egg masses of M. incognita on the roots, and weights of plants did not differ from the controls (compare Figs. 6-1 and 6-2). When Transvala was planted alone and inoculated with M. incognita and B. longicaudatus , the soil population of B. longicaudatus

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77 3 was reduced to three per 100 cm of soil and that of M. incognita to 28. Ninety-three larvae of M. incognita were recovered from the root systems, and galls and egg masses were present. Root growth was less than half that of controls and top growth was limited but not quite so much (Table 6-1, Fig. 6-3). When Pangola digitgrass was planted alone and inoculated with M. incognita and B_. longicaudatus , no M. incognita was found in the 3 soil or roots, but an average of 694 B. longicaudatus per 100 cm of soil was recovered. There was a significant retardation in growth of the root system. Roots were injured and weighed significantly less than those of uninoculated plants (Table 6-1, Fig. 6-4). When tomato was planted alone and inoculated with M. incognita and B_. longicaudatus , high populations of both nematodes were recovered from the soil with an average of 367 B_. longicaudatu s and 1 ,810 M. incognita per 100 cm 3 of soil (Table 6-1 , Fig. 6-5). An average of 14,820 second larvae of M. incognita were recovered from 10 grams of tomato roots. A large number of galls and egg masses were observed on the root system. Root and top weights of the tomato plants were significantly less than the controls (Table 6-1). When Pangola and Transvala digitgrasses were interplanted with tomato and inoculated with M. incognita and B_. longicaudatus , the root systems of Pangola and Transvala digitgrasses were damaged severely with roots of Pangola digitgrass showing symptoms of EL longicaudatus damage. Tomato was not damaged as much as when planted alone nor were the number of H. incognita and B_. longicaudatus as high when the three were interplanted as when

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78 tomato was planted alone. However, all three plants were damaged (compare Figs. 6-5 and 6-6). When Transvala digitgrass was interplanted with tomato and inoculated with M. incognita and B_. longicaudatus , the soil popula3 tion level of EL longicaudatus was reduced to six per 100 cm of soil, while an average of 386 second stage larvae of M. incognita were recovered from the soil (Table 6-1, Fig. 6-7). Conversely, when Pangola digitgrass was interplanted with tomato and inoculated with both nematodes, an average of 198 B_. longicaudatus and 9 M. incognita per 100 cm of soil were recovered (Fig. 6-8). Discussion Pangola digitgrass reduces soil and root populations of M. incog nita but allows populations of B_. longicaudatus to increase. Transvala digitgrass reduces populations of B_. longicaudatus but allows populations of M. incognita to increase. The two grasses interplanted reduce populations of both nematodes and provide mutual protection to each other against the nematodes. In addition, the two grasses interplanted with tomato provide some protection to tomato, but the presence of tomato, a host for both nematodes, allows populations of both nematodes to increase and damage the two grasses as well as tomato. Therefore, one could not expect to interplant Pangola and Transvala digitgrasses with a crop susceptible to both Meloidogyne spp. and B_. longicaudatus and provide significant protection to that crop. However, it would seem logical to interplant Pangola and Transvala digitgrasses in pastures where both Meloidogyne spp. and B_. longicaudatus are present. Better growth of

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79 each grass may be expected and populations of both nematodes should be reduced. In a rotation system, protection would be provided to the following crop. However, as shown by the host range study in Chapter 7, each grass is susceptible to attack by additional plant parasitic nematodes. If those nematodes are present, some damage may still occur, but it should not be of the same magnitude as caused by the presence of Meloidoqyne spp. and B_. longicaudatus .

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Figure 6-1. Pangola digitgrass interplanted with Transvala digitgrass and inoculated with Meloidoqyne incognita and Belonolaimus longicaudatus . Figure 6-2. Pangola digitgrass interplanted with Transvala digitgrass and left uninoculated.

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Figure 6-3. Transvala digitgrass inoculated with Meloidogyne incognita and Belonolaimus longicaudatus . Figure 6-4. Pangola digitgrass inoculated with Meloidogyne incognita and Belonolaimus longicaudatus .

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Figure 6-5. Rutgers tomato inoculated with Meloidogy ne incognita and Belonolaimus lon^icautetus/ ^ ° gn Figure 6-6. Pangola digUgrass interplanted with Transvala digitgrass and tomato and inoculated with neloT_doq^ne incognita and Belonolaimus longicaudatus.

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Figure 6-7. Transvala digitgrass interplanted with tomato and inoculated with Meloidoqyne incognita and Belonolaimus lonqicaudatus . Figure 6-8. Pangola digitgrass interplanted with tomato and inoculated with Mel oidogyne incognita and Belonolaimus lonqicaudatus .

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CHAPTER 7 EFFECT OF TWO DIGITGRASS CULTIVARS OF DIGITARIA DECUMBENS ON EIGHT SPECIES OF NEMATODES Introduction It was shown in Chapter 2 that the four major species of Meloidogyne do not reproduce on Pangola digitgrass but are highly susceptible to B_. longicaudatus , Criconemoides spp., He! icotylenchus erythrinae Cobb, 1892, Hemic.ycl iophora parvana Tarjan, 1952, Hoplolaimus coronatus Cobb, 1923, Pratylenchus penetrans (Cobb) Filipjev, Schuurmans-Stekhoven 1941, and Scute! lonema christiei (Golden) Taylor, 1956 (Overman 1961). Boyd and Perry (1969) showed that Transvala digitgrass is resistant to B_. longicaudatus , but no other reports of its effects on nematodes were found. Thus, research was initiated to study the effects of Transvala digitgrass on eight species of nematodes. Materials and Methods Seventy-two pots were filled with autoclaved soil, and 36 were planted with one Pangola digitgrass cutting each and 36 with one Transvala digitgrass cutting each. All the pots were placed in a greenhouse at 25°C, watered as needed and fertilized every week with ® Nutrisol . When cuttings had been planted for five weeks, four pots of each grass were inoculated with 100 specimens of one of eight nematode species as follows: Pratylenchus brachyurus Godfrey, 1929 90

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91 Filipjev, Schuurmans-Stekhoven , 1941; He! icotyl enchus erythrinae , Trichodorus christiei All en , 1 957 ; Heterodera glycines Ichi nohe, 1 952; Tylenchorhychus martini Fielding, 1 956; Hem i eye 1 iophora parvana , Tarjan, 1952; Xiphinema americanum Cobb, 1913; and Hoplolaimus galeatus (Cobb 1913), Filipjev, Schuurmans-Stekhoven, 1941. Four pots of each grass served as noninoculated controls. Six months later the plants were harvested, the tops removed from the roots and weighed, the roots washed, blotted dry, and weighed, and the soil population of nematodes determined. Results In no case were nematode population levels lower after six months than the original inoculum level. However, with three nematode species there were significant differences between population levels on the two grasses. Populations of Hel icotyl enchus erythrinae and Hemicycl iophora parvana were significantly lower on Transvala digitgrass than on Pangola digitgrass, but populations of Heterodera glycines were significantly higher on Transvala digitgrass than on Pangola digitgrass (Table 7-1). Since Pangola digitgrass was shown to be antagonistic to Meloidogyne incognita , which is in the same family, Heterodendae, as H. glycines , it would appear that the antagonistic principle applies to some extent to H. glycines . The fact that Transvala digitgrass is a good host for both M. incognita and H. glycines also shows that the two grasses may react similarly to both nematodes. With the exceptions noted above, in all other cases the grasses had no adverse effects on the nematodes--in fact the reverse was

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92 true. The plants were damaged by all other nematodes (Fig. 7-1 a to g) even when populations levels increased very little (Fig. 7-2). Pi scussion Since H. glycines reproduced very little on Pangola digitgrass compared to Transvala digitgrass, it would appear that the antagonistic principle that Pangola digitgrass has to Meloidogyne spp. applies at least to a degree to H. glycines which is in the same family--Heteroderidae. Also populations of H. glycines increased on Transvala digitgrass similar to the increase shown by M. incognita on that plant. Thus, it would appear that the two grasses are consistent in showing opposite reactions to members of Heteroderidae. With all other nematodes except Hel icotyl enchus erythrinae and Hemicycl iophora parvana on Transvala digitgrass the plants. were . damaged by the nematodes. Transvala digitgrass could be grown without damage when populations of H. erythrinae and H. parvana are present but would be damaged by the other nematodes tested especially by Hoplolaimus galeatus , Pratyl enchus brachyurus , Xiphinema americanum , Trichodorus christiei , and Tyl enchorhynchus martini .

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Figure 7-1. Effect of eight genera of plant parasitic nematodes on Pangola and Transvala digitgrasses when each was inoculated with (a) Pratylenchus brachyurus , (b) Hel icotyl enchus erythrinae , (c) Trichodorus christiei , (d) Xiphinema americanum , (e) Tylenchorhynchus martini , (f) Hemicycl iophora " parvana , and (g) Hiterodera glycines .

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TYLENCHORHYNC HUS MARTINI

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NEMAT0DE8 PER POT

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CHAPTER 8 DISCUSSION AND CONCLUSIONS Earlier researchers (Winchester 1962, Overman 1961, Haroon 1979) showed that Pangola digitgrass was antagonistic to Meloidogyne incognita but a host for Belonolaimus longicaudatus (Overman 1961 , Haroon 1979). Research reported in this paper showed that the grass is antagonistic also to M. arenaria , M. hapla , and M. javanica . Since it is known to be antagonistic to the four major species of Meloidogyne , it also may be antagonistic to some or all other species of the genus. A rapid and reliable laboratory immersion bioassay method was developed to assay the nematicidal activity of Pangola digitgrass roots. The initial bioassay method required observation of the number of eggs of M. incognita that hatched over 10 days in the test solution plus the survival of larvae for 10 days after they hatched. After several of these tests, it was determined that egg hatch alone over a 10 day period was a reliable assay, and this required much less labor. Finally, the need to have a quicker assay led to development of a 48 hr. test in which survival of newly hatched larvae of M. incognita was measured. This bioassay proved to be very reproducible, and it is recommended for future work. The nematicidal substance(s) in Pangola digitgrass roots and in aqueous extracts of roots has not been chemically identified, but at least one chemical compound has been isolated from extracts by causing a precipitate to form. The precipitated material was redissolved in 101

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102 water by addition of IN HC1 to a final pH of 9 and found, in the 48 nr. bioassay with larvae, to have an LD 5Q value of about 1 mg/ml of assay solution. Only about one-half of the nematicidal activity of an aqueous extract can be removed by precipitation. The biological activity that remains in solution may be due to a different chemical compound than that which precipitates. The precise quantity of nematicidally active compound(s) in root extracts has not been determined, but preliminary assays indicate that at least 1 mg of active material is present in 1 g of roots. The precipitated substance can be detected on paper or TLC chromatograms with ]% diphenlcarbazone in ethyl alcohol reagent. While providing a suitable means of detection, di phenyl carbazone gives no real clue to the structure of the compound. Numerous detection reagents more specific for functional . groups in a molecule have failed to react with the precipitated material on chromatograms. Nematicidal activity can be extracted from Pangola digitgrass roots only with aqueous solvents. The best extraction medium found thus far is water that has been acidified to pH 3 with HC1 . The chemical nature of the nematicidal activity seems to be that of a very polar compound, and possibly slightly alkaline in reaction. Thus the activity in Pangola digitgrass roots seems to be very different chemically from any of the previously isolated plant products with nematicidal activity, all of which were soluble in relatively nonpolar organic solvents. The Pangola digitgrass activity is relatively heat stable and extracts can be concentrated by evaporation of water under low heat. In this work 90°C was used, but somewhat

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103 lower temperatures (perhaps about 70°C) in combination with a fan may be better, because some biological activity was lost at 90°C. Boyd and Perry (1969) found that Transvala digitgrass, a cultivar of Digitaria decumbens , was resistant to Belonolaimus 1 ongicaudatus , and this research shows that it is a host for M. incognita . Interplanting the two digitgrasses reduced combined populations of M. incognita and B. longicaudatus to zero or nearly zero. It would appear that the nematicidal principle in the roots of each grass is transferred to the soil, probably in the form of root secretions. Experiments with leachates from both grasses showed that the leachates were nematicidal. Hence, the nematicidal principle seems to be secreted by the roots into the soil where it kills soil populations of the nematodes and mutually protects each grass from damage by its nematode parasite. Thus, whenever one of the digitgrasses is planted in areas infested by both Meloidogyne spp. and B. longicaudatus , it would be damaged. However, if the two grasses were interpl anted, the root secretions of each grass should protect the other from damage. Growers should be advised to interplant the two grasses for best growth and yields. When the effects of the two grasses were tested against eight different species of nematodes, the most important finding was that Pangola digitgrass allowed little reproduction of Heterodera glycines , and that Transvala digitgrass was an excellent host for it. Since the nematode is in the same fami ly--Heteroderidae--as Meloidogyne , it may be that the two grasses will show the same pattern of responses to other members of that nematode family. No other patterns of reactions were obvious, but it was noted that the two grasses had

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104 opposite reactions, i.e., host-nonhost to He! icotyl enchus erythrinae and Hemicycl iophora parvana both of which reproduced well on Pangola digitgrass but little on Transvala digitgrass.

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REFERENCES Abbott, W.S. 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol . 18:265-267. Berkeley, M.J. 1855. Vibrio forming escrescences on the roots of cucumber plants. Chronicle, Apr., 1855, p. 220. Boyd, F.T., and V.G. Perry. 1969. The effect of sting nematodes on establishment, yield, and growth of forage grasses on Florida sandy soils. Soil Crop Sci. Soc. Fla. Proc. 24:288-300. Boyd, F.T., S.C. Schank, R.L. Smith, E.M. Hodges, S.H. West, A.E. Kretschmer, Jr., J.B. Brolmann, and J.E. Moore. 1973. Transvala digitgrass, a tropical forage resistant to 1. sting nematode. 2. pangola stunt virus. Fla. Agr. Exp. Sta. Circ. S-222. Chitwood, B.G. 1949. Root-knot nematodes. Part 1. A revision of the genus Meloidogyne Goeldi, 1887. Proc. Helminthol. Soc. Wash. 16:90-104. Christie, J.R. 1959. Plant Nematodes, Their Bionomics and Control . Fla. Agr. Exp. Sta., Gainesville, Fla. Gommers, F.J. 1981. Biochemical interactions between nematodes and plants and their relevance to control. Commonwealth Institute of Helminthology 50(1 ) :9-24. Good, J.M., N.A. Minton, and C.A. Jaworski. 1965. Relative susceptibility of selected cover crops and coastal bermudagrass to plant nematodes. Phytopathology 55:1026-30. Haroon, S.A. 1979. Effect of Pangola grass on the nematode Meloidogyne incognita . M.S. Thesis, Univ. of Florida, Gainesville 47 p. Hayslip, N.C., E.M. Hodges, D.W. Jones, and A.E. Krechmer, Jr. 1964. Tomato and pangola digitgrass rotation for sandy soils of south Florida. Fla. Agr. Exp. Sta. Circ. S-153:l-24. Hodges, E.M., G.B. Killinger, J.E. McCaleb, O.C. Ruelke, R.J. Allen, Jr., S.C. Schank, and A.E. Kretschmer. 1975. Pangola digitgrass. Agr. Exp. Sta. Bull. 318. 105

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106 Horn, D.H.S., and J. A. Lamberton. 1963. The nematicidal principles of Tagetes roots. Australian J. Chem. 16:475-479. Hussey, R.S., and K.R. Barker. 1973. A comparison of methods of collecting inocula of Meloidogyne spp. , including a new technique. Plant. Dis. Reptr. 57:1025-1028. Lear, B. , and S.T. Miyagawa. 1966. Influence of cropping to castor beans on populations of root knot nematodes in soil. Plant Dis. Reptr. 50(2) : 1 32-1 33. Morgan, D.O. 1 925. Investigations on eel worm in potatoes in south Lincolnshire. J. Helminthol. 3:185-192. Oostenbrink, M. 1950. Het adrdappelaattje ( Heterodera rostochiensis Wallenweber) een gevaarlijke parasiet voor de eenjijdige , aardappelcultuum. Vers!. Rpziekt Dienst Wageningen 115:144-150. Overman, A.J. 1961. Nematodes associated with pangola grass pastures. Proc. Fla. State Hort. Soc. 74:201-204. Rohde, R.A. 1960. Acetylcholinesterase in plant parasitic nematodes and anticholinesterase from asparagus. Proc. Helminthol. Soc. Wash. 27:121-123. Rohde, R.A. 1972. Expression of resistance in plants to nematodes. Annu. Rev. Phytopathol . 10:233-252. Rohde, R.A., and W.R. Jenkins. 1958. Bases for resistance of Asparagus officinialis var. a! til is L to the stubby root nematode Trichodorus ch ristiei ; Allen, 1957. Bull. Md. Agr. Expt. Sta. A-97, p. 19. Ruehle, J.L., and J.R. Christie. 1958. Feeding and reproduction of the nematode Hemicycl iophora parvana . Proc. Biol. Soc. Wash. 54:31-34. Scheffer, F., R. Kickuth, J.H. Visser. 1962. Die wirzelansscheidungen von Eragrostic curvula (Schrad.). Nees und ihr Einfluss auf Wurzel -Knoten-nematoden. Zeitschrift fur Pflanjenunahrung und Bodenkunde 98:114-120. Stahl , E. 1965. Thin Layer Chromatography, A Laboratory Handbook . Academic Press, Inc., New York. pp. 485-502. Steel, G.D., and J.H. Torrie. 1960. Principles and Procedures of Statistics . McGraw-Hill Book Co., New York. p. 107-109. Steiner, G. 1941. Nematodes parasitic on and associated with roots of marigolds ( Tagetes hybrids). Proc. Biol. Soc. Wash. 54:31-34.

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107 Suatmadj i , R.W. 1969. Studies on the effect of Tagetes species on plant parasitic nematodes. Dissertation, Agricultural University, Wageningen. 133 pp. Taylor, A.L., and J.N. Sasser. 1978. Biology, Identification, and Control of Root-knot Nematodes (Meloidogyne species ) . N.C. State Univ. Graphics, Raleigh, North Carolina. Ill pp. Taylor, C.E., and A.F. Murant. 1966. Nematicidal activity of aqueous extracts from raspberry canes and roots. Nematologica 12: 488:494. Triffitt, M.J. 1929. Preliminary research on mustard as a factor in inhibiting cyst formation in Heterodera schachtii . J. Helminthol. 7:81-92. Tyler, J. 1938. Proceedings of the root-knot nematode conference held in Atlanta, Georgia, February 4, 1938. Plant Dis. Rep. 104 (Suppl.) :133-1 51. Uhlenbroek, J.H., and J.D. Bijloo. 1958. Investigations of nematicides. I. Isolation and structure of a nematicidal principle occurring in Tagetes roots. Recueil des Travaux Chimiques des Pays-Bas 77:1004-1008. Uhlenbroek, J.H., and J.D. Bijloo. 1959. Investigations of nematicides. II. Structure of a second nematicidal principle isolated from Tagetes roots. Recueil des Travaux Chimiques des Pay-Bas 78:382-392. Winchester, J. A. 1962A. The effect of pangola digit grass Diqitaria decumbens Stent, on the cotton root-knot nematode Meloidogyne incognita acrita populations. Proc. Soil Crop Sci. Soc. Fla. 20:178-182. Winchester, J. A. 1962B. The effect of pangola grass Diqitaria decumbens Stent, on the cotton root knot nematode Meloidogyne incognita acrita Chitwood. Ph.D. Diss., Univ. Fla., Gainesville. 67 p. Winchester, J. A., and N.C. Hayslip. 1960. The effect of land management practices on the root-knot nematode Meloidogyne incognita acrita in south Florida. Proc. Fla. Sta. Hort. Soc. 73:100-104.

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BIOGRAPHICAL SKETCH Sanaa A. Haroon was born in Giza, Egypt, on March 21, 1951. After completing her diploma at Orman High School in Giza, Egypt, in 1969, she entered Tanta University. She received the Bachelor of Science degree in June 1973. From 1973 to 1975 she served with the government in the Common Service Project teaching poor elementary school children in her home for no monetary compensation. In December 1975 she travelled to Florida, U.S.A., and married her fiance, Samir El Agamy, who was studying for the Ph.D. degree in the Fruit Crops Department financed by an Egyptian scholarship. In September 1977 Sanaa entered the nematology graduate program. In 1979 she received her M.S. degree. Sanaa and Samir have one son, Ahmed, born on 19 August 1977. 108

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I certify tl 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. G. C. Smart, Jr., Channia-riA' Professor of Entomology £md 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. /3. L. Nation, Cochairman /'Professor of Entomology and 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. A. 0. Overman Professor of Entomology and Nematology

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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. H. L. Rhoades Professor of Entomology and 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. • 0. C. Ruellce. Professor of Agronomy This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. December 1982 {cuk 7^A Dean/College of Agriculture Dean for Graduate Studies and Research


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