THE EFFECTS OF SOIL SOLARIZATION, ORGANIC AMENDMENT, AND FUMIGANT NEMATICIDES ON PASTEURIA PENETRANS AND ITS INFECTIVITY

TO MELOIDOGYNE ARENARIA RACE 1 IN TOMATO

By

LEANDRO GRASSI DE FREITAS

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

UNIVERSITY OF FLORIDA

1997 I dedicate this work to all the people who care and try their best to improve human living conditions while respecting the environment. ACKNOWLEDGMENTS

I would like to thank Arlete and Lucas, my dear wife and son, for the love, for understanding my absence and for the support; and to Honorio, Dirce, and Carla, my parents and sister, for love and support.

I am indebted to the Conselho Nacional de Desenvolvimento Cientifico (CNPq), to Florida-Brazil Institute, to Dr. David Mitchell and to the Universidade Federal de

Vicosa for financial support.

Thanks are expressed to my commitee members, Dr. D. J. Mitchell (Chairman),

Dr. D. W. Dickson, Dr. R. McSorley, Dr. R. Charudattan, and Dr. D. Hopkins for their

suggestions and kindness; special thanks go to Dr. D. W. Dickson for technical support,

for helping with field experiments, and for hosting me in his lab; to Dr. McSorley for

helping with statistical analyses and for allowing me to use his students' office; and to

Dr. David J. Mitchell, my father in science, for unconditional support, inspiration, and

sincere friendship.

I would like to thank Dr. Sal Locascio and Mike Aligood for helping with field

experiments.

Thanks to Dr. Daniel Chellemi, Reggie, Dale, Miguel Quesada, and Mahendra for

the friendship and for helping with field experiments.

Thanks to Mario Serracin, Georgina Robinson, Tom Hewlett, Claudia Riegel,

Billy Crow, and Ann Landry for the great sense of humor, which made my journey much

easier.

iii I am grateful to Uilson Lopes for statistical guidance and friendship.

I would like to thank all my friends who, each in his or her own way, and perhaps without realizing it, contributed to my personal growth during my years in Gainesville, and thus for the realization of this work.

iv TABLE OF CONTENTS

ACKNOWLEDGMENTS iii

ABSTRACT vii

CHAPTERS

1. INTRODUCTION 1

Root-Knot Nematodes (Meloidogyne spp.) 2 Historical Background 2 Life Cycle 3 Meloidogyne spp. in Tomato 5 Management of Meloidogyne spp 6 Resistance 6 Chemical Control 7 Nematicides and Groundwater 14 Physical and Cultural Methods of Nematode Control 15 Biological Control 20 Biological Control of Nematodes with Fungi 20 Biological Control of Nematodes with Pasteuria spp 26

2. EFFECTS OF TEMPERATURE ON THE ATTACHMENT OF PASTEURIA PENETRANS ENDOSPORES TO SECOND-STAGE

JUVENILES OF MELOIDOGYNE ARENARIA RACE 1 37

Introduction 37 Material and Methods 39 Results 43 Discussion 4g

3. EFFECTS OF SOIL SOLARIZATION AND ORGANIC AMENDMENT ON PASTEURIA PENETRANS AND ON ITS INFECTIVITY TO

MELOIDOGYNE ARENARIA RACE 1 IN TOMATO 55

Introduction 55 Material and Methods 5g Results 65 Discussion gg

v 4. THE EFFECTS OF FUMIGANT NEMATICIDES ON PASTEURIA PENETRANS AND ITS INFECTIVITY TO MELOIDOGYNE ARENARIA

RACE 1 IN TOMATO 97

Introduction 97 Material and Methods 99 Results 106 Discussion 112

5. SUPPRESSION OF MELOIDOGYNE ARENARIA BY PASTEURIA PENETRANS IN THE FIELD 117

Introduction 117

Material and Methods 1 19 Results 123 Discussion 126

6. SUMMARY AND CONCLUSIONS 134

LITERATURE CITED 139

BIOGRAPHICAL SKETCH 155

vi Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

THE EFFECTS OF SOIL SOLARIZATION, ORGANIC AMENDMENT, AND FUMIGANT NEMATICIDES ON PASTEURIA PENETRANS AND ITS INFECTIVITY

TO MELOIDOGYNE ARENARIA RACE 1 IN TOMATO

By

Leandro Grassi de Freitas

May, 1997

Chairman: Dr. David J. Mitchell Major Department: Plant Pathology

Management of nematodes with biological control, soil solarization, organic

amendments, environmentally safe nematicides, and the integration of these methods is of

great interest to the agricultural industry. Pasteuria penetrans is a Gram-positive

bacterium that prevents Meloidogyne spp. from reproducing and diminishes their ability

to penetrate roots. In this study, P. penetrans greatly suppressed Meloidogyne arenaria

in tomato under field and greenhouse conditions. In a field naturally infested with P.

5 penetrans, the density of the bacterium was determined to be 1.27 x 10 endospores/g of

soil.

When endospores were maintained in soil to simulate solarization periods of 30 to

70 °C for 5 hours a day over 10 days, reproduction of P. penetrans and its attachment to nematodes were reduced with increasing temperature of incubation above 60 °C. Heating juveniles to sublethal temperatures (35 to 40 °C) decreased endospore attachment. When nematode juveniles were incubated in soil infested with P. penetrans, the highest

vii attachment occurred at 20 to 30 °C. In another study, incubating endospores in soil with cabbage amendment at 50 °C for 5 hours a day over 10 days reduced subsequent bacterial endospore formation in females. Volatiles released from cabbage decomposition appeared to be toxic to the bacterium.

The average maximum daily temperature reached by solarization in field soil at the depth of 20 cm was 41 °C. This temperature did not affect endospore attachment, number ofjuveniles in soil, or number of galls on tomato roots.

Chloropicrin and methyl bromide + 33% chloropicrin were highly detrimental to

P. penetrans in field and greenhouse experiments. Treatment with 1,3-Dichloropropene

(1,3-D) + 17% chloropicrin, 1,3-D + 25% chloropicrin, and 1,3-D + 35% chloropicrin had moderate effects on the bacterium. Metham sodium did not have a deleterious effect on the bacterium. Overall, the results of this work indicate that solarization may be used in combination with P. penetrans, but the use of chloropicrin and methyl bromide + 33% chloropicrin in integrated management of nematodes with P. penetrans should be avoided.

viii CHAPTER 1 INTRODUCTION

Plant-parasitic nematodes cause estimated annual yield losses of 12% in the

world's 40 major crops (Sasser and Freckman, 1987). Approximately 9% of losses occur

in developed countries and 15% in developing countries. Losses have exceeded 21% in

tomato (Sasser and Freckman, 1987). Monetary losses due to nematodes on 21 crops, 15

of which are life sustaining crops, were estimated at 77 billion dollars annually based on

1984 production figures and prices (Anonymous, 1995b). The United States' portion of these losses was 5.8 billion dollars. If all crops are considered, the estimate exceeds 100 billion dollars annually (Sasser and Freckman, 1987).

According to a questionnaire responded to by 371 nematologists from 75 countries, Meloidogyne was considered the most important genus of plant-parasitic nematodes worldwide (Sasser and Freckman, 1987). Meloidogyne spp. parasitize more than 2,000 plant species and their extensive host range includes monocotyledons and dicotyledons, and herbaceous and woody plants (Hussey, 1985).

1 2

Root-Knot Nematodes (Meloidoevne Spp.)

Historical Background

Root knot has been known as a disease of vegetable crops since 1855, when

Berkeley in England first described the disease as "vibrios forming excrescences on

cucumber roots." The causal organism was later described as Heterodera radicicola by

Miiller in 1884. Root-knot nematodes were considered a single species for 65 years and

were referred to by a number of designations, including Anguillula marioni Cornu, 1 879,

A. arenaria Neal, 1889, A vialae Lavergne, 1901 , H. javanica Treub, 1885, Tylenchus

arenarius Cobb, 1890, Meloidogyne exigua Goeldi, 1887, Oxyurus incognita Kofoid and

White, 1919, Caconema radicicola Cobb, 1924, and Heterodera marioni (Cornu, 1879)

Marcinowski, 1909 (Thorne, 1961). Prior to 1949, evidence was accumulating that root-

knot nematodes consisted of races that differed in their host preferences and pathogenic expressions of infection (Christie, 1946; Christie and Albin, 1944). Morphological differences among the populations were noted, and root-knot nematodes were reassigned to the genus Meloidogyne (Chitwood, 1949). Meloidogyne exigua, M. javanica (Treub,

1885) Chitwood, 1949, M. arenaria (Neal, 1889) Chitwood, 1949, M. hapla Chitwood,

1949, and M. incognita (Kofoid and White, 1919) Chitwood, 1949, were recognized based on the perineal pattern Hirshmann (1985) listed 54 species and two subspecies in the genus Meloidogyne.

Root-knot nematodes are cosmopolitan organisms. They are most important in

regions where summers are long and winters, if any, are short and mild. In the tropical 3

zone, the most common Meloidogyne spp. are M. incognita, M. javanica, and M.

arenaria (Johnson and Fassuliotis, 1984).

Life Cycle

The life cycle of root-knot nematodes begins with the eggs that are usually found

in the gelatinous egg mass surrounding the posterior end of the female. The first molt

takes place within the egg, and, upon hatching, the slender second-stage juveniles infect

nearby galled roots or locate new roots of susceptible plants to continue their life cycles.

Juveniles are attracted toward roots from as far away as 75 cm (Prot and Netscher, 1979).

The second-stage juveniles usually enter just behind the root cap, where there is intense

meristematic activity, but penetration may occur in other sites. The nematodes migrate

through undifferentiated root cells in the cortex until they reach the developing vascular

system. The juveniles keep their bodies in the cortical tissue and their heads in the

periphery of vascular tissue (Hussey, 1985). Cell walls are pierced with the stylet, and

secretions from the esophageal glands are extruded through the stylet. The secretions

cause an alteration to the plant cells of the nematode feeding site. These feeding cells, usually five to six, are called giant cells, and grow to various sizes, depending on the host plant and temperature (Bird, 1972). If the juvenile faied to induce the formation of giant cells, it would either starve or migrate out of the root in search of another root (Hussey,

1985).

The nematode remains sedentary during feeding and enlarges in width but not in length (Taylor and Sasser, 1978). The cells of the genital primordium divide and develop 4

into a two-sided ovary in the female or into an elongated testis in the male. About 14

days after penetration, the juvenile molts and loses the stylet and esophageal bulb. The juvenile does not feed until after the final molt. Three molts take place in just 4 days.

The stylet and median bulb are regenerated after the fourth molt; the uterus and vagina

are formed, and a perineal pattern is visible. After the last molt, the female grows rapidly

and becomes pear shaped.

In the development of a male, the juvenile undergoes metamorphosis, becomes

vermiform and coils within the third cuticle. It undergoes the fourth and final molt and

emerges from the root as an adult male. In amphimictic species, males are often found

within the gelatinous matrix secreted by the female.

Gelatinous matrix is produced by the female from six rectal glands, and 500-1,000

eggs are extruded into the matrix. Eggs are deposited in the one-cell stage, and mitosis

begins a few hours later. Embryos of M. javcmica developed in 46 to 48 days at 15 °C,

16 to 48 days at 20 °C, 1 1 to 13 days at 25 °C, and 9 to 10 days at 30 °C (Bird, 1974).

Unlike some species of the Globodera and Heterodera, there is no apparent hatching factor necessary to induce juveniles to hatch from the eggs. Usually, hatching of eggs of

Meloidogyne spp. depends only on temperature and moisture, but, in some instances, root diffusates do stimulate egg hatching (Viglierchio and Lownsbery, 1960).

Gall formation is the usual phenotypic response to parasitism by root-knot nematodes. It is a separate phenomenon from the giant cell response. The size and shape of the galls are influenced by both the host plant and nematode parasite (Dropkin, 1969a).

Galls are formed by cell hypertrophy that starts within a few hours after penetration. 5

Galls are induced by growth regulators introduced from the subventral glands of the

infective-stage juvenile or by auxins, which are formed when tryptophane is released

upon hydrolysis of plant proteins by the juvenile and reacts with endogenous phenolic

acids (Bird, 1978).

Meloidogyne spp. in Tomato

Tomato is the most valuable vegetable grown in Florida. The area planted to

tomato in Florida during 1992 and 1993 was 19,600 ha, which represented an on-farm

value of 626 million dollars. The crop is grown intensely with the use of broad-spectrum

fumigants, polyethylene mulch, relatively high rates of fertilizers, irrigation, and with

frequent use of pesticides to control foliar insects and pathogens. Soilborne organisms

that attack or compete with tomato include nematodes, fungi, bacteria, insects, and

weeds.

Nematodes are an important part of the root problems that affect tomato

production. The rhizosphere of the tomato plant supports plant parasitic nematodes, which vary in virulence, as well as saprophytic nematodes that play an important role in the decomposition of organic matter and subsequent soil fertility (Overman, 1991). Over

60 species representing 19 genera of plant-parasitic nematodes have been associated with tomato cultivation. Of these, root-knot (Meloidogyne spp.), root lesion (Pratylenchus spp.), and reniform (Rotylenchulus reniformis Linford and Oliveira) nematodes have been associated with major damage to vegetable crops (Johnson and Fassuliotis, 1984). The most important root-knot nematode species associated with tomato

production are M. incognita, M. javanica, M. arenaria, and M. hapla. All species of root-

knot nematodes have basically the same life cycle, and it can be influenced by soil

temperature and host (Johnson and Fassuliotis, 1984). According to Netcher and Sikora

(1990), the tolerance limit of tomato to M. arenaria and M. incognita is 2 to 100

3 juveniles per 100 cm of soil. Yield losses in tomato cultivated in the tropics due to root-

knot nematodes were estimated to be 24% to 38% (Sasser, 1979b).

Management of Meloidosvne spp.

The practices of management of plant-parasitic nematodes may involve reduction of nematode populations, or making them less infective than they would be otherwise

(Johnson and Fassuliotis, 1984). The classical practices of nematode management include crop resistance, crop rotation, chemical control, and cultural practices such as flooding, fallowing, organic amendment, and trap crops. Few examples of effective

biological control of plant nematodes in the field exist, and methodologies are still under study by researchers worldwide. Recent approaches in the management of plant-parasitic nematodes focus on integrated pest management involving the minimal use of chemicals and maximum use of cultural methods.

Resistance

Resistance in tomato to M. incognita, M. javanica, and M. arenaria was

developed by crossing Lycopersicon esculentum with the wild species L. peruvianun. 7

About 30 years of crosses, selections, and backcrosses were necessary before commercial

varieties with desirable horticultural characteristics and resistance against root-knot

nematodes were released (Sasser, 1979a). The Mi gene is responsible for conferring

resistance to tomato, but it does not confer resistance to M. hapla and its expression is

inhibited above 28 C (Dropkin, 1969b). Many other factors may compromise resistance

in tomato. As an example, the reproductive rate of root-knot nematode in resistant

tomato increases progressively during selection after several years of planting the same resistant cultivar (Triantaphyllou, 1987).

Chemical Control

The development of 1,3-dicloropropene, 1 ,2-dichloropropane (D-D), 1,2- dibromoethane (EDB), and l,2-dibromo-3-chloropropane (DBCP) in 1943, 1945, and

1 954, respectively, initiated a pesticide revolution that changed management of plant pathogenic nematodes from the use of a combination of methods that reduced nematode populations to the application of effective chemicals that control the nematodes. High rates of these chemicals were injected into soil, often repeatedly, around roots of established plants. Some organophosphate and carbamate chemicals developed during the

1950s and 1960s had systemic insecticidal properties, and have been used as nematicides applied to soil (Thomason, 1987).

Nematicide treatment is considered one of the most reliable means of controlling a wide variety of nematodes. Chemicals are used mainly to control nematode populations in soil before planting annual crops. Application methods were developed for applying 8

about 25 highly effective nematicides, which are used on approximately 700,000 hectares

in the United States (Johnson and Fassuliotis, 1984).

There are two types of nematicides for control of nematodes on vegetable crops,

soil fumigants and non-fumigant nematicides. Soil fumigants are liquids that are injected

below the soil surface where they volatilize to kill nematodes. Vapors from soil

fumigants diffuse through the soil, dissolve in soil water, and penetrate the cuticle of the

nematodes. Fumigation is the most effective method of chemical plant disease control, it

can be done quickly and is thorough and long lasting, if done properly. Soil fumigation

also is one of the most costly methods, with application costs of sometimes more than

2,000 dollars per hectare, which limits its use to high value crops (Maloy, 1993).

Nonfumigants and systemic nematicides are water soluble and are distributed through the

soil by percolation of water. They also enter nematode bodies through the cuticle. The

newest types of nematicides are systemic. They can be taken up by plants through the

roots after application to the soil or through foliage after spray application. After

translocation to roots, nematodes feeding on the plants are killed.

According to Hague and Gowen (1987), nematicides also may be classified

according to their following chemical compositions: halogenated aliphatic hydrocarbons

(methyl bromide, EDB, DBCP, chloropicrin, and 1,3-dichloropropene mixtures),

methylisothiocyanate precursors (dazomet, metham sodium and methylisothiocyanate

mixtures), organophosphates (fenamiphos, ethoprophos, and thionazin), and oxyme-

carbamates (aldicarb, oxamyl, and carbofuran). The first two groups include fumigants that kill nematodes and their eggs, and the third and fourth groups are non-fumigants 9

known as nematostats. Nematostats do not kill nematodes directly but do affect their

behavior (Wright, 1981).

Methyl bromide is a highly effective fumigant used to control a broad spectrum of

organisms, such as nematodes, insects, weeds, bacteria, fungi, and rodents. It has been on

the market for more than 50 years and, unlike virtually any other pesticide of its

generation, is still widely used. Fumigation with methyl bromide remains the only

generally accepted method of eradicating pathogens in many countries. It is used as a

pre-plant soil fumigant in greenhouse soils and in field situations on high value cash

crops (Minerbi, 1992). The agricultural community uses it for over 100 crops. The

production of methyl bromide in the United States in 1993 was valued at over 27 million

dollars. Eighty percent of the production is used for soil fumigation (Guerrero, 1995).

Methyl bromide, as well as 1 ,3-D and chloropicrin, are alkyl halides that penetrate

the cuticle of nematodes. Moje (1960) observed a correlation between toxicity and

relative activity of alkyl halides in reactions of nucleophilic substitution (NH2 , SH, and

OH groups) on protein surfaces, but these reactions are considered slow. The rapid action

of these nematicides may be explained better by the oxidation of iron porphyrins and hemeproteins. Iron porphyrins are components of hemeprotein molecules and are found in terminal oxidases or mitochondrial oxidases. The oxidation of heme centers in the respiratory system is rapid. Many enzymes or metabolic sites are inhibited simultaneously; thus, several vital processes are stopped, which leads to a rapid death

(Wade and Castro, 1973). 10

According to the Environmental Protection Agency (EPA), methyl bromide is a

toxic substance that, depending on the concentration and duration of the exposure, can

damage the lungs, eyes, skin and, in severe cases, cause death. According to Guerrero

(1985), agricultural field workers have developed respiratory, gastrointestinal, and

neurological problems, including inflammation of nerves and organs and degeneration of

the eyes.

World scientists participating in the assessment of ozone-depleting substances,

coordinated by the United Nations Environment Program (UNEP), concluded that

emissions from human uses of methyl bromide contribute significantly to atmospheric

ozone depletion and should be controlled. Laboratory measurements indicate that

bromine, a major component of methyl bromide, is about 50 times more efficient in

destroying atmospheric ozone than the chlorine in chlorofluorocarbons (Guerrero, 1985).

To protect the ozone layer, 24 nations, including the United States, signed the Montreal

Protocol in September 1987, agreeing to place controls on and perform further assessments of major ozone-depleting substances (Guerrero, 1985). In December 1993,

EPA issued regulations under these provisions to phase out methyl bromide. The

regulations stipulated by EPA freeze the production and importation of methyl bromide at

1991 levels until 1 January 2001 ; after that date, the pesticide can no longer be produced or imported into the United States for domestic use. The Montreal Protocol has been signed by 150 countries (Guerrero, 1985).

The elimination of soil fumigation with methyl bromide for tomato production would result in an estimated 20% yield decrease (Anonymous, 1995a). In four of the 11

most important world tomato growing regions, Florida, California, Italy, and Israel, 80%

of the fresh tomato crop is grown on soil fumigated with methyl bromide to control

nematodes, Fusarium and Verticillium wilts, and corky root (Anonymous, 1995a).

The EPA, United States Department of Agriculture (USDA), and industry

representatives generally agree that chemical substitutes and other alternatives are

available today to manage many of the pathogens and pests currently controlled with

methyl bromide (Anonymous, 1995c). To fund research on alternatives to methyl

bromide, the EPA and USDA spent about 13.3 million dollars in 1995. To date, no new

chemicals and only a few new uses of existing chemicals have been submitted to EPA as potential alternatives to methyl bromide. The EPA has found potentially serious environmental or health and safety concerns for each of the alternatives identified by the

USDA. According to USDA officials, regulatory actions by the EPA to ban or limit the use of these or other pesticides could augment the economic effects of the methyl bromide phaseout by eliminating potential effective alternatives (Anonymous, 1995c).

Research is being conducted by governmental and academic institutions, as well as by the private sector, to ensure that alternative materials and methods will be proven

viable and available to the agricultural community before methyl bromide is phased out.

Various alternatives to methyl bromide are being evaluated by the USDA and other researchers for preplant and postharvest, but there are various concerns that need to be resolved before the ban goes into effect (Anonymous, 1995c). The potential alternatives for methyl bromide's preplant uses can be divided into chemical and nonchemical. The suspension of the use of D-D, DBCP, and EDB reduced the number of effective and 12

economical soil fumigants that can be used for control of nematodes on vegetable and

fruit crops. Chemical alternatives include 1,3-dichloropropene, dazomet, metham

sodium, sodium tetrathiocarbonate, formalin or formaldehyde, chloropicrin, and

nonfumigant nematicides (Anonymous, 1995c). The nonchemical alternatives include

the use of steam, soil solarization, organic amendments, crop rotation, resistant cultivars, and biological control.

A broad-spectrum liquid fumigant, 1,3-dichloropropene (1,3-D), is comparable to

methyl bromide for controlling most soil pathogens, but it is less effective for controlling weeds. It has the same mode of action as methyl bromide. Mixtures of 1,3-D and chloropicrin are used to enhance activity on soilborne fungi. These products are registered for more than 120 vegetable crops, field crops, and nursery crops in the United

States (Melichar, 1994). Use permits were previously suspended by California because of health and safety concerns, but they are currently allowed for limited use (Anonymous,

1995c).

Dazomet is a broad-spectrum granular fumigant that reacts with soil moisture to produce toxic vapors of methylisothiocyanate. Less activity can be associated with the formation of formaldehyde, carbon disulfide, hydrogen sulfide, and methylamine. After penetrating the nematode cuticle, the methylisothiocyanate reacts with amino acids, oxidases, and nucleophilic sites on proteins (Spurr, 1985). The product effectiveness depends on the physiological state of the nematode and the relative dose of the active breakdown components. Yield increases of 83% were observed with dazomet

applications in Florida tomato trials (Roman et al, 1994). This nematicide is currently 13

registered for some food crops, but approval may not be sought for alternatives to all uses of methyl bromide. Small fruit and orchard uses are restricted to the propagation of nonbearing berry, vine, fruit and nut crops, or similar nonbearing plants, according to the

EPA (Anonymous, 1995c). Concerns about potential genotoxicity have been raised by the EPA.

Metham sodium is a broad-spectrum liquid fumigant for management of some

soilborne pathogens and pests, but it may be less effective as a nematicide. Metham

sodium releases methylisothiocyanate, which is a potential groundwater contaminant.

Like dazomet, metham-sodium's effectiveness depends on soil moisture. Its efficacy is

dependent on the availability of water to ensure even distribution in the soil. The

breakdown into methylisothiocyanate occurs at temperatures higher than 1 5 °C (Hague and Gowen, 1987). Identified as a known teratogen that causes developmental malformations, methamsodium is classified by the EPA as a probable human carcinogen.

Concerns about contamination of groundwater have been expressed by the EPA and the

UNEP (Anonymous, 1995c).

Developed as a soil fungicide, chloropicrin has been used to control plant-

parasitic nematodes, such as root-knot nematodes in Hawaii, for over 50 years. Its mode

of action is similar to that of methyl bromide and 1,3-dichloropropene. The fumigant is

applied under plastic tarp to improve efficiency and to prevent the exposure of people to this irritant, tear gas. Chloropicrin is mixed with methyl bromide in the proportion of

2%, 33%, or other proportions. In addition to its role as a fungicide and nematicide, the 14

lachrymatory nature of the chloropicrin is useful to warn operators (Hague and Gowen,

1987).

The nonfumigant nematicides are applied as granules at or just before planting, as

aqueous drenches around growing plants, or in irrigation water (Hague and Gowen,

1987). Chemicals of this group are divided in organophosphates and carbamates

according to their chemical structure. The organophosphates include fenamiphos and

isazophos. The carbamates include aldicarb, oxamyl, and carbofuran (Hague and Gowen,

1987). According to Main (1980), organophosphate and carbamate nematicides penetrate the cuticle of nematodes and inhibit acetylcholinesterase, cholinesterase, and other

esterase enzymes. Hydrolysis of acetylcholine by acetylcholinesterase is a vital part of neurotransmission in the nervous system, and organophosphates irreversibly phosphorylate active sites on acetylcholinesterase. Carbamoylation of

acetylcholinesterase tends to be more reversible. Acetylcholinesterase is present in the nerve ring and associated ganglia, amphids, phasmids, and papillae on the lips and body surfaces of nematodes (Rohde, 1960).

Nematicides and Groundwater

Nematicide application technology, including application through furrow, basin, and sprinkler irrigation, has been investigated extensively. Early studies showed the

potential for water to enhance downward movement of nematicides in soil profiles

(Baines et al, 1959). The presence of nematicides in groundwater has had serious implications for the environment. Groundwater is the most important source of potable water worldwide. In the United States, more than 50% of all domestic water is

groundwater (Holden, 1986).

The contamination of the groundwater was one of the main reasons for the

suspension of EDB and DBCP, and for the loss of aldicarb to several specific agricultural

regions (Thomason, 1987). The finding of aldicarb and carbofuran in groundwater

(Holden, 1986) threatens a group of water soluble, mobile nematicides that became

increasingly important in recent years.

Significant progress has been made in characterizing the relationships among nematode numbers, plant injury, and nematicide efficacy. Using drip irrigation, low

dosage rates applied repeatedly through the first half of the growing season can give

excellent crop response, even when total amount applied is only a half to a third of the recommended rate (Van Gundy and Garabedian, 1984). These lower concentrations of

nematicides minimize the chances of chemicals being leached through a soil profile to the groundwater.

Many chemicals derived from plants and microorganisms are being tested for nematicidal efficacy by scientists. Some have potential for commercial use in specific crops or locations, but at present they do not appear to be competitive with presently

marketed nematicides (Feldmesser et al., 1985).

Physical and Cultural Methods of Nematode Control

Steam sterilization is an expensive alternative to methyl bromide fumigation for

the control of plant-parasitic nematodes in field soil. It is extremely slow, dangerous to 16

personnel, and the necessary equipment is difficult to obtain (Noling, 1992). The cost per hectare with current technology is estimated in excess of 1,000 dollars. The use of steam sterilization could decrease the usable area to be planted because of the extended time required for the soil to attain effective temperatures (Noling, 1992).

Soil solarization is a method of controlling soilborne pests and pathogens by raising the temperature of the soil through application of thin, transparent polyethylene plastic sheets to a soil surface. Solarization has been used most effectively against nematodes and other soilborne pathogens in locations with hot and relatively cloudless conditions during the solarization periods. The use of this technique generated

significant decreases in the population densities of Pratylenchus thornei (Grinstein et al,

1979; Katan et al, 1976); Ditylenchus dipsaci (Siti et al, 1982); Heterodera cajani,

Rotylenchulus reniformis, and Helicotylenchus retusus (Chauhan et al, 1988; Sharma and

Nene, 1985); Hirschmanniella mucronata (Sivakumar and Marimuthu, 1987);

Meloidogyne incognita, and Rotylenchulus reniformis (Gaur and Dhingra, 1991); and

Meloidogyne javanica (Cartia et al, 1989). However the results have been highly variable, especially with Meloidogyne spp. (Gaur and Dhingra, 1991; Katan, 1981;

Stapleton et al., 1987), probably due to differences in soil characteristics, available solar radiation, and nematode distribution in the soil (Madulu and Trudgill, 1994).

Subtropical countries are characterized by having dry, hot summers, during which crops often are not cultivated. In these countries soil solarization has proven to be effective (Greco, 1993). Investigations in Italy on onion (Greco et al, 1976; Greco et al,

1992) and Israel on garlic (Siti et al, 1982) demonstrated that a 4- to 8-week solarization 0

17

period may provide better control of nematodes and higher yields than soil fumigation

(Greco, 1993). Tzortzakakis and Gowen (1993) observed that treatments of P. penetrans

4 at 2.5x1 spores/g of soil, oxamyl applied three times at 0.18 ml a.i./plant and 50 days

of solarization, alone and in combination, significantly reduced galling, eggs per gram of

root, and juveniles in soil. Suppressiveness to plant pathogens in solarized soils may

result from a shift in microbial populations to heat-resistant antagonists (Katan et al,

1983).

Adding organic amendment to soil reduces nematode populations (Muller and

Gooch, 1982; Singh and Sitaramaiah, 1970; Vijayalakshimi et al, 1985). Katan (1981)

suggested that solarization might increase the rate of degradation of organic matter and

result in the accumulation of toxic gases in the soil atmosphere. According to Stapleton

et al. (1993), sealing and heating soil amended with organic fertilizers and crop residues

can provide levels of disinfestation greater than with either method alone. Concentrations

of biotoxic volatiles, including alcohols, aldehydes, sulfides, and isothiocyanates, were

found in low levels in field soil amended with organic fertilizers and crop residues when

analyzed by gas chromatography. Concentrations increased when soil was sealed with polyethylene film or spray tarp, and increased further when amended soil was heated.

Reductions of M. incognita and some soilborne fungi were greatest when treated with amended, heated soil (Stapleton et al, 1993).

The addition of organic matter into soil can affect nematode population density by improving soil structure and soil fertility, altering the level of plant resistance, releasing toxic compounds, and stimulating antagonistic microorganisms (Stirling, 1 99 1 ). 18

Ammonifying bacteria in the presence of organic gases, as an example, apparently

produce enough NH3 to influence nematodes. Mankau and Miteer (1962) suggested that

NH3 produced during the decomposition of a fish amendment was responsible for the decline of root-knot nematodes.

Green tissue of Brassica spp. can decrease population densities of some fungal and nematode pathogens (Mayton et al, 1995). Brassica spp. contain an abundance of sulfur-containing materials. The compounds resulting from the decomposition of cabbage include mercaptans, sulfides of various types, and isothiocyanates (Lewis and

Papavizas, 1971). According to Mayton et al. (1995), the control of plant pathogens by

Brassica spp. is attributed to their production of glucosinolates, which are sulfur compounds composed of a thioglucosate group, a variable carbon side chain (R-group), and a sulphonated oxime. Enzymatic hydrolysis of glucosinolates by membrane-bound

thioglucosidase produces numerous compounds, including isothiocyanates, nitriles, thiocyanates, epinitriles, and glucose. Some of the hydrolytic breakdown products have antimicrobial, fungicidal, and insecticidal properties (Chew, 1988).

Gray (1987) concluded that application of soil amendments resulted in the increase

of free-living nematodes and decrease of plant-parasitic nematodes due to an increase in nematophagous fungi in the soil. The enhanced activity of the nematophagous species

present in the soil decelerated the increase of the plant-parasitic nematodes in the soil, and eventually reduced their population densities. Many experiments have been conducted on the role of the organic amendments in the biological control of plant-parasitic nematodes in soil, but the results are inconsistent because different organic matter causes different effects 19

on different nematophagous fungi (Cooke, 1961, 1962a, 1962b, 1963; Duddington and

Duthoit, 1960; Duddington^ a/., 1956a, 1956b, 1961; Galperet al., 1990; Gray, 1987;

Hams and Wilkin, 1961; Hutchingson and Mai, 1954; Jaffee, 1992, 1993; Jansson, 1982;

JanssonandNordbring-Hertz,1979;Linfordand Yap, 1939;Mankau, 1961;Tarjan, 1961).

Crop rotation is one of the oldest and most important practices for management of

plant-parasitic nematodes in annual crops. According to Johnson (1982), the principle of

crop rotation is based on the resistance, susceptibility, or tolerance of crops to the predominant population of plant nematodes of a determined area. The preceding crop may prevent damage to a crop by reducing the population of the target nematode.

Damage on crops due to nematodes generally occurs after many seasons of cultivation of the same crop without rotation. Crop rotation remains an important method for limiting damage caused by Meloidogyne spp. in the southeastern United States (Johnson, 1982).

According to Rodriguez-Kabana et al. (1988, 1989), castor (Ricinus comunis L.),

American jointvetch (Aeschynomene americana L.), partridge pea {Cassiafasiculata

Michx.), and sesame (Sesamum indicum L.) did not support M. arenaria populations in

the field and were good crops for rotations. McSorley et al. (1994) studied the effects of

summer 12 crop treatments on population densities of M. arenaria race 1 and on yields of subsequent spring vegetable crops in microplots. Yields of vegetable crops following castor were approximately double the yields of the same vegetable crops following peanut. Cotton (Gossypium hirsutum L.), velvetbean (Mucuna deeringiana [Bort.] Merr.), crotalaria {Crotalaria spectabilis Roth.), and hairy indigo (Indigofera hirsuta L.) also resulted in good control of the nematode. Crops for rotation have been studied in many other countries. Economic crop rotation sequences are often constrained by lack of

available land, crop management skills, specialized equipment to grow and harvest the

crop, or by the lack of closely located processing facilities or markets (Noling, 1992).

Biological Control

Biological control of nematodes is defined by Stirling (1991) as "a reduction of nematode populations which is accomplished through the action of living organisms other than nematode-resistant host plants, which occurs naturally or through

the manipulation of the environment or the introduction of antagonisms." This process is mediated through the mechanisms of parasitism, predation, competition, and antibiosis by a diverse range of antagonistic organisms, such as fungi, bacteria, viruses, nematodes, mites, insects, protozoans, turbellarians, tardigrades, and oligochaetes (Stirling, 1991).

The array of antagonists to plant-parasitic nematodes is so diverse that a discussion of the general biology of individual species and their interactions with nematodes associated

with plants has been the subject of books, such as those by Barron (1977), Jairajpuri et al.

(1990), and Stirling (1991). Thus, only fungi and the bacteria of the genus Pasteuria will be discussed in the following review.

Biological Control of Nematodes with Fungi

Fungal endoparasites of nematodes can be easily recognized in nematodes extracted from the soil by the presence of assimilative hyphae, zoosporangia, and resting 21

spores. These fungi are often obligate parasites, and have a limited saprophytic stage

(Stirling, 1991).

According to Gray (1988), the genera of endoparasites are found in a number of

taxonomic classes, which can be loosely categorized into three groups: Group I, encysting

species of Chytridiomycetes and oomycetes; Group II, deuteromycetes producing

adhesive conidia and ingested conidia; and Group III, basiomycetes forming adhesive conidia.

Zoospores of some fungi from Group I, such as Catenaria anguillulae,

Lagenidium caudatum, Aphanomyces sp., and Leptolegnia sp., move toward chemical gradients formed by exudates from nematode orifices. Once they reach the nematode body, the zoospores encyst close to natural apertures, such as the mouth, anus, vulva, and excretory pores, and form a germ tube that penetrates through an orifice or directly penetrates through the cuticle of the nematode (Gray, 1988).

The infection process in the fungi from the Group II and III is initiated when their

conidia adhere to the cuticle of the nematode. The cuticle is penetrated by a germ tube, a

bulbous infection hypha forms within the nematode, and then assimilative hyphae fill the body cavity (Stirling, 1991). Verticillium balanoides, Drechmeria coniospora, Hirsutella rhossiliensis, Macrobiophthora vermicola, Nematoctonus leiosporus, N. concurrens, N. haptocladus, and Myzocytium humicola are examples of endoparasites with adhesive spores.

The genus Harposporium, with the exception of one species, possesses conidia that initiate infection by lodging in the buccal cavity or the gut of the host. Since plant- 22

parasitic nematodes are unable to ingest these conidia, this group of fungi is not efficient

for their biocontrol (Stirling, 1991).

Nematode-trapping fungi also are known as predatory fungi. They possess mycelium that has been modified to form organs capable of capturing nematodes.

According to Stirling (1991), this is the most wellknown group of nematophagous fungi, and their predacious organs have been studied for more than a century. There are six types of traps produced by these fungi.

Adhesive hyphae are produced by species in two genera of Zygomycetes

(Stylopage and Cystopage) and by a few species of Hyphomycetes. A modified structure, the adhesive branch, is produced only by a few species, of which Monacrosporium

cionopagum is one of the most commonly isolated.

A second type of trap is formed by erect branches of one to three cells that may anastomose to form simple loops or two-dimensional networks. A thin film of adhesive material is secreted over the entire surface of each branch.

The third type, the adhesive network trap, is an evolutionary development from adhesive branches. The traps are formed by an erect lateral branch growing from the vegetative hypha and curving to fuse with the parent hypha (Stirling, 1991). Further loops are produced on this loop or on the parent hypha, until a complex, three-

dimensional, adhesive-covered network of anastomosed loops is formed. These adhesive nets, found in almost all soils, are the most common type of trap produced by nematode- trapping fungi. Arthrobotrys oligospora is frequently encountered, and uses this trapping mechanism. 23

Adhesive knobs represent the fourth type of adhesive trapping device. Knobs

consist of adhesive cells that are either sessile on the hypha or are borne on top of a short,

erect stalk (Barron, 1977). These globose cells are usually closely spaced along the

hyphae, so that nematodes are often captured by several knobs. If the nematode escapes,

it causes the detachment of the knobs from their stalks. The knobs remain firmly attached

to the nematode and penetration will proceed normally. Adhesive knobs are one of the

most common trapping mechanisms among the Hyphomycetes, and they are also found in

the Basidiomycetes. The genus Nematoctonus has characteristic, non-detachable,

hourglass shaped knobs that are engulfed in a larger, spherical ball of adhesive material.

Non-constricting rings, the fifth type of trapping device, are produced when erect

lateral branches from vegetative hyphae thicken and curve to form a generally three-

celled ring, which then fuses to the support stalk. A nematode entering the ring becomes

captured when the ring becomes attached around its body. Although this trapping process

was previously thought to be passive, recent evidence suggests that there may be an

adhesive layer associated with the inside of the ring of at least some species (Dowsett and

Reid, 1977, 1979; Saikawa, 1985).

Constricting rings are the sixth type of trap device. They are formed in a similar

manner to non-constricting rings, but they are attached to the hyphae by a shorter stalk

(Barron, 1977). Nematodes entering a ring trigger the three ring cells to swell rapidly

inwards, so that they close around the prey and hold it securely. The ring closure takes

less than 0. 1 second once initiated, but a lag period of 2 to 3 seconds from the time the nematode first touches the ring cells until the ring begins to close may allow the prey to 24

escape. Because of the lag period, constricting rings are not efficient trapping devices, and, even when numerous rings and large nematode populations are present, only a small proportion of the nematodes are ever caught.

Female and egg parasites are represented in a diverse array of classes and niches; they range from obligate parasites to saprophytes sustained by soil organic matter. The zoosporic pathogens of females are closely related to the zoosporic species that attack vermiform nematodes. Catenaria auxiliaris appears to be a relatively common parasite of Heterodera glycines and H. avenae (Crump et al, 1983; Stirling and Kerry, 1983), and

Nematophthora gynophila is common in H. avenae-infested European soils (Kerry and

Crump, 1980) and in soil in the United States (Crump et al, 1983). In both C. auxiliaris and N. gynophila, the infection process starts when zoospores lodge on the immature female nematodes after they become exposed on the root surface.

Many other fungi have been found in association with nematode females, cysts, and eggs, but many of them are rarely encountered, and some are saprophytes rather than parasites (Morgan-Jones and Rodriguez-Kabana, 1988; Stirling, 1988; Tribe, 1977,

1979).

Rodriguez-Kabana and Morgan-Jones (1 988) reviewed the reports of fungi isolated up to 1 988 from Heteroderidae collected from Australia, Europe, and North and South

America. Since then, many more fungal species have been reported from nematodes collected in various geographic locations. The fungi most frequently isolated from females, eggs, egg masses, and cysts of Heteroderidae are limited to species of the following genera: 25

Acremonium, Alternaria, Catenaria, Cylindrocarpon, Exophiala, Fusarium, Gliocladium,

Nematophthora, Paecilomyces, Penicillium, Phoma, and Verticillium

Species of Verticillium are some of the most important nematophagous fungi.

Prior to infection of the nematode egg, V. chlamydosporium forms a branched mycelial network in close contact with the smooth egg shell (Lopez-Llorca and Duncan, 1988;

Morgan-Jones et al, 1983). Specialized perforation organs are produced, but penetration also occurs from lateral branches of the mycelium. The fungus causes disintegration of the vitelline layer of the egg shell and partial dissolution of the chitin and lipid layers due to the activity of exoenzymes. Verticillium chlamydosporium parasitizes the developing embryo or affects its development by increasing the egg shell's permeability, which

allows the entrance of toxins present in the soil environment. It has been suggested that

V. chlamydosporium itself might produce a toxin, because eggs did not hatch when the fungus grew near eggs (Meyer et al, 1990; Morgan-Jones et al, 1983). Isolates vary greatly in aggressiveness, growth, sporulation, temperature relationships, and chlamydospore production in culture (Irving and Kerry, 1986; Kerry, 1981; Kerry et al,

1986).

Paecilomyces lilacinus has cosmopolitan distribution, but it is more common in

the warmer regions of the world (Domsch et al, 1980). It was found parasitizing eggs of

Meloidogyne incognita in Peru (Jatala et al, 1979), and has since been associated with root-knot and cyst nematodes in many geographical locations (Gintis et al, 1983; Godoy et al., 1983; Morgan-Jones et al, 1984a; Morgan-Jones and Rodriguez-Kabana, 1986).

Isolates of P. lilacinus vary in their pathogenicity to nematode eggs in in vitro tests. 26

Some isolates are aggressive parasites, while other morphologically indistinguishable

isolates are nonpathogenic (Dunn et al, 1982; Freitas et al, 1995). Colonization by pathogenic strains generally occurs as a result of simple penetration of the egg by

individual hyphae (Morgan-Jones et al, 1984b), but occasionally it is associated with a specialized structure, possibly an appressorium (Dunn et al, 1982). Infection by P.

lilacinus totally destroys the egg contents. Ecologically the fungus is primarily a

saprophyte; it is able to compete for and use a wide range of common substrates in soil

(Domsch et al, 1980). Although P. lilacinus has shown promise as a biological control agent against several nematode species (Jatala 1985, 1986), isolates known to be pathogenic to nematode eggs have sometimes failed to provide any nematode control, despite being present at relatively high populations (Hewlett et al, 1988; Rodriguez-

Kabana et al, 1984). Other fungi, such as Dactylella oviparasitica and Rhopalomyces elegans, are known to attack eggs of root knot nematodes (Stirling, 1991).

Biological Control of Nematodes with Pasteuria Spp.

Pasteuria penetrans is an obligate nematode parasite that has considerable

potential as a control agent (Davies et al, 1991). This bacterium was first classified as a

protozoan by Thorne (1940), who described it as Duboscqia penetrans. The results of an ultrastructural study led Mankau (1975) to reclassify the organism as a prokaryote, specifically as a member of the genus Bacillus (Sturhan, 1985). The rediscovery of

Pasteuria ramosa Metchnikoff 1 888 by Sayre et al. (1977) and its morphological similarities to Bacillus penetrans suggested that the two bacteria shared a common 27

generic relationship. After extensive studies Sayre and Starr (1985) were led to designate the bacterial organism parasitizing Meloidogyne incognita as Pasteuria penetrans (Sayre and Starr, 1988).

A recent revision of the genus Pasteuria delineated two species, P. penetrans

Starr and Sayre, 1988, sensu stricto, and P. thornei Starr and Sayre, 1988 (Starr and

Sayre, 1988), which are parasites of the root-knot nematode, Meloidogyne incognita, and the root-lesion nematode, Pratylenchus brachyurus (Godfrey, 1929) Filipjev and

Schuurmans Stekhoven, 1941, respectively. Another possible species of the genus

Pasteuria parasitizing Heterodera elachista Oshima, 1974 was found by Nishizawa

(1984). Following his initial observation, Nishizawa (1986) associated the field presence of the bacterium with a decline in the population densities of the cyst nematode.

Confirmation of this phenomenon was noted at the Tochigi Agricultural Experiment

Station, where soybean breeding lines were being screened for cyst nematode resistance

(Nishizawa, 1987). This bacterium was later classified by Sayre et al. (1991) as P.

nishizawae Sayre, Wergin, and Starr, 1991. Several new populations of Pasteuria sp. have been observed to parasitize and suppress populations of cyst nematodes. One group, which was observed to parasitize Heterodera avenae, H. cajani, H. sorghi, and H. zeae,

follows a life-cycle similar to that of P. penetrans on root-knot nematodes (Davies et al.,

1992); but another Pasteuria sp., which parasitizes Heterodera goettingiana in Germany, completes its life-cycle in the second-stage juvenile (Sayre et al, 1990; Sturhan and

Winkelheide, 1990). 28

Life History of Pasteur ia penetrans . Upon contact, the endospores of Pasteuria

spp. attach to the cuticle of their appropriate nematode hosts. Infection by P. penetrans

may not only prevent Meloidogyne spp. from reproducing, but may also diminish their activity and the ability of the infected juveniles to penetrate roots (Sturhan, 1985). The bacterial protoplast enters the body of the nematode through a germination tube, and thallus-like vegetative microcolonies develop in the pseudocoelom of the nematode. The bacterial development is usually in synchrony with the nematode development in the root

system, and it is temperature dependent (Stirling, 1981). Terminal cells separate themselves from these microcolonies and form sporangia, and ultimately endospores, which may fill the entire body of the nematode. More than two million endospores were found in a parasitized female of a Meloidogyne sp. (Sturhan, 1985). The endospores are released into the soil by the death and decay of the host. As the endospores are nonmotile, they are dispersed primarily by percolation of water through the soil and by tillage (Sturhan, 1985). The endospores remain viable in the soil for many years; they are resistant to desiccation and high temperatures (Stirling, 1984).

Population increase of P. penetrans endospores in soil is influenced by various environmental factors. Endospore attachment to juveniles and development and reproduction of P. penetrans infecting M. javanica and M. arenaria increased with increases in temperature between 20 and 30 °C (Hatz and Dickson, 1992; Stirling, 1981;

Stirling et al, 1990), suggesting that P. penetrans would be most effective under high soil temperatures (Nakasono et al, 1993). 29

Host Specificity of Pasteuria penetrans . The nematode hosts of Pasteuria spp. include about 175 species in nearly 70 genera from 10 orders (Starr and Sayre, 1988;

Sturhan, 1985). Nematodes from each taxon have different feeding habits and life cycles that vary in duration. Therefore, each nematode species has a slightly different physiology. Selection pressure favors the bacterium whose physiology parallels that of a particular nematode host. The one-to-one relationship that exists between these bacterial parasites and their nematode hosts suggests that the genus Pasteuria contains numerous

bacterial species (Sayre et al., 1991).

Host specificity is important when differentiating parasites. Although attachment studies have shown that populations of the bacterium isolated from one nematode genus can adhere to juveniles in another genus and even colonize them, individual populations

usually exhibit a restricted host range. In only one case, when 1 1 populations of P. penetrans isolated from Meloidogyne spp. were tested against a range of different cyst and root-knot nematodes, was there a small degree of endospore attachment to a cyst nematode (Davies et al, 1988).

Endospores have been shown to differ even in their level of attachment to the same species of nematodes (Stirling, 1985). In a study of four populations of

Meloidogyne spp., Stirling (1985) concluded that endospore attachment was not related to either the species from which endospores were obtained or the species of the recipient nematode. In contrast, endospores of P. penetrans from different populations of

Meloidogyne spp. were shown by Davies et al. (1988) to adhere in greatest numbers to the species from which they had been isolated originally. Channer and Gowen (1992), in 30

a experiment using populations of root-knot nematodes from eight countries and P. penetrans from four countries, observed that Meloidogyne spp. host populations

influenced the subsequent host specificity of P. penetrans isolates. The ability of a P.

penetrans population to attach to the host nematode on which it was last cultured increased dramatically, while its ability to attach to the Meloidogyne sp. population on which the P. penetrans isolate was originally more aggressive diminished. Cultivating the same P. penetrans isolate on a different nematode population of the same species, M. incognita, had no significant effect on its subsequent spectrum of specificity. The difference in behavior between these two M. incognita populations confirms the

allegation by Stirling (1985) that the recognition process in endospore attachment is determined by characteristics which are variable within Meloidogyne spp.

According to Charmer and Gowen (1992), one explanation for the apparent switch in host specificity is that field populations of P. penetrans are genetically heterogeneous, and selection occurs when endospores that attach better to a determined nematode

population result in endospore production that is carried forward. This population of the bacterium then predominates in the next generation. An alternative explanation for switches in host specificity is that P. penetrans undergoes some form of host-induced

adaptation. This implies that P. penetrans can recognize a host type it has successfully infected and produce endospore progeny with an enhanced ability to reinfect that particular host.

Host specificity varies among individual populations or isolates of P. penetrans, and such specificity may result from differences in the amount and type of endospore surface proteins (Davies et al, 1992). Carbohydrate recognition domains may be present

on the surface of the nematode cuticle and interact with N-acetylglucosamine moieties on

the endospore surface. These in turn are linked either to proteins or peptidoglycans that

are extensions of the bacterial cell wall (Davies and Danks, 1993).

Cultivation of Pasteur ia penetrans . Pasteuria penetrans has great potential as an

economically and environmentally valuable biological control agent (Bird and Brisbane,

1988; Brown et al., 1985; Dickson et al, 1994; Stirling, 1984). The major obstacle to

realizing this potential has been the inability to culture this organism in vitro (Bishop and

Ellar, 1991). Methods of mass producing P. penetrans depend on the multiplication of

the bacterium in its nematode host on greenhouse grown plants (Stirling and Wachtel,

1980). Root systems of tomato plants containing large numbers of females of

Meloidogyne sp. parasitized by P. penetrans are air-dried and finely ground, which

produces a powder that is easy to handle and store. Juveniles become encumbered with

endospores when the powder preparation is introduced into soil (Stirling and Wachtel,

1980).

According to Williams et al. (1989), the mass production of endospores might be

improved by culturing the nematode and pathogen in excised or transformed root, but

commercial use of the pathogen will most likely require an in vitro method of culture.

Verdejo and Mankau (1986) grew P. penetrans in M. incognita in excised tomato roots.

Surface sterilized seeds were grown on modified 1% Tepfer and Tempe medium in petri

dishes, and epicotyls were removed after 1 week. A female infected with P. penetrans was crushed on a small block of agar to release the endospores, and then a single M. incognita egg mass was placed on the agar block. The hatched juveniles became

encumbered as they moved toward the roots. More than 50% of the females per plate

were infected in all cases, and 100% were infected in two plates. Some parasitized

females were able to produce a few eggs (Verdejo and Mankau, 1986).

Verdejo et al. (1987) cultivated roots transformed in vitro with Agrobacterium

rhizogenes on modified Murashige and Skoog medium. One hundred and fifty second-

stage juveniles of M. javanica were placed in Solanum tuberosum root cultures for

endospore production, and healthy and infected females were obtained after incubation at

25 °C. The endospores from infected females obtained in vitro adhered to M. javanica

juveniles. Transformed roots offer excellent opportunities to culture and study nematodes

and their parasites, since they grow rapidly, tolerate stress, and subculture easily by root

transfer.

Williams et al. (1989) tried to culture P. penetrans outside its nematode host, but

they were not successful, despite the wide range of simple and complex media they used.

These included media developed to cultivate fastidious organisms such as Spiroplasma

citri, Legionella pneumophila, and Clavibacter xyli subsp. xyli; media containing root

extract, soil extract, or crushed nematodes; media suitable for the growth of nematodes

such as Caenorhabditis briggsae; and media containing compounds such as sterols,

which are essential for nematode growth. These rich media should be able to fulfill the

nutritional requirements of most microorganisms, but the inability to germinate P. penetrans endospores may have been the limiting factor in the growth of the bacterium.

The treatment of the endospores with isozymes, alkaline solutions, polymyxin, and 33

various enzymes, and amino acids, and with physical treatments, such as heat shocking, failed to initiate germination of endospores of P. penetrans. Since P. penetrans endospores germinate after spore-encumbered juveniles enter the root and begin to feed

(Sayre and Wergin 1977), many factors could be responsible for triggering germination.

The contact of the endospore with the nematode, exudates from root cells, and changes in the physical environment that occur after the nematode enters a root are some of the factors that may affect endospore germination.

The inability to induce the germination of endospores forced Williams et al. (1989) to use vegetative mycelial bodies as a source of inoculum, but these also failed to grow when manipulated on various media. According to the authors, the removal of mycelial bodies

from the nematode host subjects them to stresses, such as osmotic shock. Thus, it becomes important to mimic more carefully the physical and chemical conditions that exist inside the nematode when trying to culture the bacterium. Pasteuria penetrans develops within the

host nematode without greatly affecting its growth. This implies that this bacterium has a relatively specialized mode of nutrition.

Bishop and Ellar (1991) failed to obtain reproduction of P. penetrans in several culture media, and lysis of the inoculated bacteria occurred rapidly. Two new semidefined media were formulated and used either alone or in combination with various supplements.

These media also provided osmotic support, pH value equivalent to that of the host, amino acids, vitamins, precursors of nucleic acids, and a variety of trace elements. One of the media maintained inoculated mycelia of P. penetrans for a month, and resulted in low yields of mature endospores. Another medium permitted a small increase, less than fourfold, in the number of inoculated mycelia, but instead of sporulation, the colonies lysed. Cell-free

extracts from other organisms were screened without success (Bishop and Ellar, 1991). The

development of in vitro culture techniques for P. penetrans may therefore depend on by

more intensive physiological and biochemical studies of the interaction between host and

parasite.

If P. penetrans could be cultivated and mass reared in vitro, the interaction with

nematodes and several aspects of biological control could be investigated more readily,

and field experiments could be conducted more readily. The commercial use of P.

penetrans then would be possible in a short period of time. It hopefully would be easier

then to cultivate other species of Pasteuria, so that their use in biological control of

various genera of nematodes could be investigated better.

Integrated Nematode Management Using Pasteuria penetrans . The use of

Pasteuria spp. in combination with other management practices, especially nematicides,

is of great interest to nematologists. Stirling (1984) claimed that infection of M. javanica

by P. penetrans after treatment in vitro was not affected by 1,3-D or DBCP. However,

his conclusion must be qualified because the author did not compare the percentage of

females infected with P. penetrans in fumigated soils with the percentage in non-

fumigated soil. Brown and Nordmeyer (1985) demonstrated a synergistic reduction of

root galling by M. javanica with carbofuran or aldicarb combined with P. penetrans. A possible explanation for the synergism is that low concentrations of carbamate

nematicides stimulate the movement and orientation of the nematodes toward host roots, rather than kill the nematodes; thus, the probability of nematode contact with bacterial 35

endospores increased. Brown and Nordmeyer (1985) did not report on the reproduction

of the bacterium in the nematicide treated soils, but it is expected that it is inferior to that

obtained in a nontreated soil, since the galling was greatly reduced in the presence of the

nematicides. Walker and Wachtel (1988) observed a reduction in parasitism of M.

javanica juveniles by P. penetrans following the use of nonfumigant nematicides.

Organophosphate and carbamate nematicides at high concentrations decreased the

mobility of nematodes, and the most likely explanation of the reduction in infection is a

decreased probability of contact between nematodes and passive endospores of the

parasite. To date, no detailed study on the direct effects of nematicides on P. penetrans

has been conducted.

The efficacy of P. penetrans may also be enhanced by soil solarization

(Tzortzakakis and Gowen, 1993). Solarization increases nematode infection by the

parasite without increasing soil population densities ofjuveniles (Walker and Wachtel,

1988). A higher incidence of endospore attachment under solarization conditions may be

explained by an increase of soil temperature, resulting in increased motility of nematodes

(Bird and Wallace, 1965) and, consequently, increased probability of contact between

nematodes and endospores (Walker and Wachtel, 1988). Stirling (1981) observed that

the length of the life cycle of P. penetrans was reduced by about 70% at 30°C, compared

with that at 20°C, and that M javanica was parasitized at an earlier stage of its

development at the higher temperature. Thus, increased temperatures in solarized soil

may increase both the rate of development of females of Meloidogyne spp. and the production of P. penetrans endospores (Walker and Wachtel, 1988). Integrated strategies may be useful for managing plant-parasitic nematodes and providing crop nutrition. Combinations of treatments may become increasingly important as pesticide usage is reduced. The effects of integrating biological control agents, such as P. penetrans, with other treatments have not been investigated thoroughly, however, and further study is warranted (Stapleton and Heald, 1991). The general objectives of this study were to evaluate the effects of soil solarization, organic

amendment, and nematicides on P. penetrans and its infectivity to M. arenaria race 1 in tomato. CHAPTER 2 EFFECTS OF TEMPERATURE ON THE ATTACHMENT OF PASTEURIA PENETRANS ENDOSPORES TO SECOND-STAGE JUVENILES OF MELOIDOGYNE

ARENARIA RACE 1

Introduction

Pasteuria penetrans is an obligate parasite of nematodes that has great potential

as a biological control agent (Bird and Brisbane, 1988; Brown et al., 1985; Stirling,

1984) . Upon contact, the endospores of Pasteuria spp. attach to the cuticle of their

appropriate nematode hosts. The adhesion of the endospores is the first step in the life

cycle of the bacterium and is essential for its reproduction. Infection by P. penetrans

may prevent Meloidogyne spp. from reproducing, and attachment of high numbers of

endospores to the juvenile diminishes its activity and ability to penetrate roots (Sturhan,

1985) .

Few studies have been conducted on the effects of temperature on the attachment

of endospores of Pasteuria spp. to the nematode cuticle. Dutky and Sayre (1978) studied

the effect of temperature on the endospores of P. penetrans by subjecting soil infested

with endospores to 40, 60, 80, 100, and 130 °C for 30 to 60 minutes and subsequently

introducing non-treated, second-stage juveniles of Meloidogyne incognita into soil to test

for attachment. There was no attachment of endospores after they were exposed to 1 30

°C for 1 hour. Attachment occurred after exposure in soil heated at 80 °C for 30 minutes, but P. penetrans did not penetrate and develop inside the nematode. Unfortunately, the

37 38

authors did not report the rate of attachment of endospores receiving treatment at the other temperatures studied. The percentage of M. javanica encumbered with endospores increased as temperature increased from 15 °C to 30 °C and as time increased from 24 to

72 hours (Sirling, 1981). Attachment of Pasteuria sp. on Heterodera cajani increased from 15 to 25 °C, but decreased at 35 °C (Singh and Dhawan, 1990). Hatz and Dickson

(1992) determined that 30 °C was the optimum temperature for attachment when M.

arenaria race 1 was incubated for 24 hours in soil infested with P. penetrans. Although

the attachment process is not well understood, it involves proteins and carbohydrates, which are temperature dependent (Davies and Danks, 1993; Persidis et al, 1991); also the increased movement of the nematode at higher temperatures (Bird and Wallace, 1965) increased the chance of contact between endospores and nematodes, which may result in increased attachment.

Pasteuria penetrans has a great potential for integration with other nematode management practices (Stapleton and Heald, 1991), including soil solarization (Stirling,

1981; Tzortzakakis and Gowen, 1993). As a preliminary study to a field solarization test, the effects of temperature on the attachment of P. penetrans endospores to juveniles of

Meloidogyne arenaria race 1 were studied at temperature ranges normally achieved

during soil solarization at different soil depths. The study was divided into three parts.

First, the influence of temperature on the receptivity of the nematode cuticle to endospore attachment was evaluated. The second part consisted of tests on the effect of

temperature on endospores incubated in water suspensions; and in the third part, the 39

effect of temperature on the attachment in an integral soil-bacterium-nematode system was evaluated.

Material and Methods

Nematode origin . Meloidogyne arenaria race 1 used in the experiments was originaly isolated from the Green Acres Research Farm of the University of Florida in

Alachua County, Florida. The nematode was cultured on 'Rutgers' tomato, and perineal patterns were examined periodically to assure populations free of contamination with other nematode species. The nematode population was allowed one generation per year on peanut (Arachis hypogea L.). Eggs of the nematode were collected from galled roots by dissolving gelatinous matrices containing eggs with 0.5% NaOCl and then collecting the dislodged eggs on a sieve with 25-um-pore openings (500-mesh) (Hussey and Barker,

1973). Second-stage juveniles were hatched from eggs in a modified Baermann funnel

(Pitcher and Flegg, 1968) for 2 to 3 days and collected on an autoclaved 500-mesh sieve.

Origin of endospores of Pasteuria penetrans . Isolate P25 of P. penetrans was

originally obtained from females of M. arenaria race 1 parasitizing roots of peanut in a field at the Green Acres Research Farm. The galled peanut roots were collected from the field and spread on greenhouse benches to dry for 3 weeks. The roots were cut into pieces 2 to 5 cm long and incubated in an 8:1 (v/v) aqueous solution of commercial

Cytolase PCL5 (Genecor International Inc., Rolling Meadows, IL) at room temperature

(approximately 24 °C). Softened roots were placed in a sieve with 600-um-pore openings (30-mesh) nested over a sieve with 180-um-pore openings (80-mesh) and 40

sprayed with a vigorous stream of tap water. The dislodged females together with root

debris were transferred to a beaker by washing the sieve from the bottom side with a jet stream of water. The females were separated from the root debris with a Pasteur pipette under a stereoscope and examined for P. penetrans infection with an inverted compound microscope. The infected females were differentiated from healthy females by their dense and opaque milky color. Endospores of P. penetrans were obtained by rinsing infected females with distilled water for 3 minutes, and then macerating their bodies in

sterile deionized water in a 1 5-ml tissue grinder (Pyrex Tenbroeck Tissue Grinder).

Endospore suspensions were stored prior to use in glass test tubes at 5 °C.

Origin of infested soil . Soil containing endospores of isolate P25 of P. penetrans was collected from a field in the Green Acres Research Farm. The soil was obtained from a depth of 0 to 30 cm from randomly selected places, mixed, and stored in a 150- liter plastic container, in the shade, until used in laboratory experiments.

Effect of temperature on juveniles . New, 1. 5-ml polypropylene centrifuge tubes were washed with detergent, and 65, 1- to 4-day-old juveniles of M. arenaria were added

to each tube in 1 ml of water. Four tubes were immersed for 10 minutes in water in each of six water-bath tanks adjusted to 25, 30, 35, 40, 45, or 50 °C. After the incubation period, the tubes were removed from the tanks, shaken and opened. Five microliters of a

6 water suspension of P. penetrans at 5.2 x 10 endospores/ml were added to each tube.

The tubes were closed and placed in an automatic shaker (Eberback Coorporation, Ann

Arbor, Michigan) for 8 hours. After this period, the tubes were maintained at 5 °C in the refrigerator until used for assessment of attachment. The numbers of endospores attached 41

per juvenile were counted for 20 juveniles per tube using a Nikon inverted microscope at a magnification of x400. The experiment was repeated once using the same methodology, except that the treatments were replicated five times in the repetition. The data were subjected to regression analyses with SAS software (SAS Institute, Cary, NC

27512).

Effect of the temperature on endospores . A suspension of endospores of isolate

6 P25 of P. penetrans was adjusted to 5.2 x 10 endospores/ml of distilled water, and 10 ml of the suspension were placed into each of 30, 15-ml, polysterene centrifuge tubes

(Falcon Blue Max Jr., Becton Dickinson and Company, NJ). Five water-bath tanks were

adjusted to a daily cycle of 5 hours at 30, 40, 50, 60, or 70 °C, followed by 19 hours at room temperature (23 °C). Five glass tubes, each with 10 ml of the endospore suspension, were heated in boiling water (100 °C) for 5 hours a day, and were then maintained at room temperature (23 °C) for the remaining 19 hours each day. After 10 days of heat treatment, all tubes were removed from the water, shaken and opened.

One hundred and eighty, 1- to 4- day-old second-stage juveniles of M. arenaria in

1 ml of water were placed in each of 30, prewashed microcentrifuge tubes (Conical- bottom tubes, Fisher Scientific, Pittsburgh, PA). Endospores subjected to heat treatment

5 were added to the nematode suspensions to produce concentrations of 1 x 10 endospores/tube. The microcentrifuge tubes, with suspensions of nematodes and endospores, were placed on a shaker (Eberback Corporation, Ann Arbor, MI) for 24

hours at 24 °C. After this period, the tubes were stored at 5 °C in the refrigerator until used for assessment of attachment. The number of endospores on 20 randomly selected juveniles per tube was counted using an inverted microscope at x400 magnification. The

data were subjected to regression analyses using SAS software (SAS Institute, Inc., Cary,

NC).

Effect of temperature on juveniles and endospores in soil . Seventy grams of dry

soil infested with endospores of P. penetrans and 7 ml of water (10 % vol:weight) were

placed into a pre-washed, 50-ml, polystyrene centrifuge tube (Blue Max MPS conical

tubes, Bencton Dickinson and Company, NJ). The water was allowed to diffuse

throughout the soil for 30 minutes before 440 second-stage juveniles in 5 ml of water

were introduced into each tube. The experiment had five treatments (incubation at 10, 20,

30, 40, or 50 °C) and five replicates per treatment. Five tubes were maintained in a cool

room with a constant temperature of 10 °C, and another five tubes were maintained in a

growth chamber at 20 °C. Three water-bath tanks were adjusted to have a daily cycle of 5

hours at 30, 40, or 50 °C, followed by 19 hours at room temperature (23 °C); and five

tubes were maintained for each temperature cycle. The juveniles in the soil were

incubated for 4 days to allow them to move throughout the soil and to come into contact

with P. penetrans. After the incubation period, the juveniles were extracted by

centrifugal flotation (Jenkins, 1964). The experiment was repeated once. The juveniles

used in inoculations in the first trial were 1 to 4 days old. Juveniles used in the second trial were from the same batch of the ones used in the first test, but were stored in 500 ml of water at 5 °C for 40 days. After this period, the nematodes in water were moved to

room temperature (23 °C) and subjected to bubbling air for 1 hour before they were used in the experiment. 43

A difference in the level of attachment of endospores occurred among juveniles stored in water for 40 days and the 1- to 4-day-old juveniles, so a bioassay was conducted to investigate if the long-term storage in water at low temperature was the cause of the difference. The experiment consisted of two treatments and four replicates. The

treatments were attachment using second-stage juveniles stored in water for 45 days at 5

°C and attachment using 1- to 4-day-old juveniles. Second-stage juveniles of M. arenaria

race 1 , originally from the same field population and kept under the same conditions in a greenhouse, were used for both treatments. Ninety juveniles were added per prewashed

microcentrifuge tube (Conical-bottom tubes, Fisher Scientific, Pittsburgh, PA) in 1 ml of water. Endospores of P. penetrans were added to the nematode suspensions to produce

5 concentrations of 1 x 10 endospores/tube. The microcentrifuge tubes, with the suspension of nematodes and endospores, were placed on a shaker (Eberback

Coorporation, Ann Arbor, MI) for 24 hours at 24 °C. The number of endospores on 20

randomly selected juveniles from each tube were counted using an inverted microscope at x400 magnification. The data were subjected to regression analyses using SAS software

(SAS Institute, Cary, NC).

Results

Effect of temperature on juveniles . Attachment of endospores to juveniles in both the first and second trials initially increased and then decreased with increasing

temperatures of incubation ofjuveniles before exposure to endospores (Fig. 2.1). Higher

numbers of endospores were attached to juveniles treated at 30 °C than those treated at 25 °C in both tests. However, the temperatures at which attachment was highest were 30 °C and 30-35 °C in first and second trials, respectively. Temperatures higher than 35 °C

were deleterious to attachment in both experiments, and little or no attachment occurred when nematodes were heated to 45 °C and above; thus, regression equations were calculated for the range of temperatures of 25 °C to 45 °C.

When juveniles of M. arenaria were evaluated for attachment after exposure to the temperature at which approximately 50 % of the juveniles were dead

(40 °C for 10 min), only 46.7 % and 71 .4 % of the attachment observed when nematodes

were treated at 25 °C occurred in the first and second experiments, respectively.

Effect of temperature on endospores. Exposure of endospores to temperatures

higher than 30 °C resulted in decreased attachment (Fig. 2.2). The attachment declined rapidly from an average of 61.1 ± 9.5 endospores per juvenile at 30 °C to 8.0 + 5.1 endospores per juvenile at 60 °C. A residual level of less than eight endospores per juvenile resulted from exposure of endospores from 60 °C to 100 °C; accordingly, regression equations were calculated for the range of temperatures of 30 °C to 60 °C.

Effect of temperature on juveniles and endospores in soil . Residual attachment occurred at 40 °C and above; therefore, regression equations were calculated for the range of temperatures of 10 °C to 40 °C. Based on the data generated from these regression

analyses (Fig. 2.3), maximum attachment occured when juveniles and endospores in soil were incubated at approximately 25 °C. Data from regression curves for both tests 45

6

• 2 2 5 . Y\ = -0.019X +1.14A-- 12.9, R ^ 0.73 P± 0.0001 = 2 Y2 -0.020 A" + 1.32 - 18.3, R 0.76 P ^0.0001

4 .

•»* • Observed 1 ft \ Observed 2 3 4 Estimated 1

• Estimated 2

1 1

1 . \

0 1 1 — 1 Ml 1 25 30 35 40 45 50

Temperature (°C)

Fig. 2.1. Regression of the number of endospores attached per second-stage juvenile of Meloidogyne arenaria race 1 to the temperature of incubation ofjuveniles of

M. arenaria for 10 minutes in water. Equation Yl and the estimated curve 1 are for the first trial, and equation Y2 and the estimated curve 2 are for the second trial. 46

Fig. 2.2. Number of endospores of Pasteuria penetrans attached to second-stage juveniles of Meloidogyne arenaria race 1 after incubation of endospores for 10 days in water at different temperatures. —

47

2 y = -0.276 X + 13.71 A- - 103.49

2 /? = 0.96, P< 0.001

ao O -a c c2

30 40 50 Temperature (°C)

2 B Y = -0.01 A- +0.53 A- - 4.40

2.5 • 2 /? = 0.70, />< 0.001

O T3

c 1

0.5

1 1 aL <

30 50 Temperature (°C)

Fig. 2.3. Endospores oiPasteuria penetrans attached per second-stage juvenile of

Meloidogyne arenaria race 1 after 4 days of incubation at different temperatures in soil infested with bacterial endospores. The curves represented by the solid lines resulted

from regression equations, and the black dots represent the attachment observed at different temperatures in the first test (A) and in the second test (B). 48

predicted that attachment was close to null in soils treated at temperatures close to 10 and

40 °C. Although both curves were similar (Fig. 2.3), the highest average of endospores

attached per juvenile in the first trial was 63.4, while the maximum was only 2.6 in the

second trial.

This difference in attachment levels led to a supplementary bioassay to determine

if long-time storage ofjuveniles in water before exposing them to endospores resulted in low attachment. One- to four-day-old juveniles had an average attachment of 58.6 endospores per juvenile, while juveniles stored for 45 days had an average of 0.2 endospores per juvenile. The difference was statistically significant at the 1% probability level.

Discussion

Effect of temperature on juveniles . Heating second-stage juveniles of M. arenaria for 10 minutes at temperatures higher than 35 °C was deleterious to attachment in both tests in this study. The intensity of the effects of temperature on juveniles of root-knot nematodes depends on the time of exposure. Different species of the same genus may have different thermal death curves (Wallace, 1963). Walker (1960) found that, at 44 °C, the times required to kill 50% of the juveniles of M. arenaria, M. javanica, M. incognita, and M. hapla were 35, 27, 18, and 8 minutes, respectively. If such variations occur among species of the same genus, variations may also occur among races of the same species or even populations of the same race. 49

Attachment was reduced by about 50% to 70% when juveniles were heated to a

temperature at which approximately 50% of the juveniles were dead when compared to

attachment to juveniles treated at 25 °C. Similarly, Stirling et al. (1986) observed that

the attachment of endospores to nematodes killed at 60 °C for 5 minutes was 66.7% of

the rate of attachment on living nematodes. Although Stirling et al. (1986) did not report

the temperature at which the attachment on living nematodes occurred, it is assumed to

have been 25 °C, since other attachment assays made in the same publication were

conducted at 25 °C. If this presumption is correct, the authors general conclusion that the

attachment of endospores on living nematodes is not different from the attachment on dead nematodes was inaccurate. In the present study attachment after treatment of juveniles at temperatures above 25 °C increased before both the endospore attachment

level and survival ofjuveniles declined with increasing temperatures. Stirling et al.

(1986) evaluated only two temperatures, and thus would not have observed initial increases and then critical decreases in endospore attachment to juveniles treated as occurred in the present study with the more extensive range of temperatures employed.

Several factors may be involved with decreased endospore attachment to juveniles exposed to high temperatures. Proteins with carbohydrate recognition domains on the surface of the nematode cuticle are involved in endospore attachment (Davies and Danks,

1993). A protein responsible for P. penetrans attachment was characterized by Davies

(1994) as a 190-kDa glycoprotein. Davies and Danks (1993) observed that attachment increased after incubation of the juveniles in cuticle denaturing reagents, 1% sodium dodecylsulphate and 5% P-mercaptoethanol, for 2 hours at room temperature; however, 50

attachment decreased about 70% when temperature and time were increased. They also

reported decreased attachment when juveniles were incubated at 100 °C for 10 minutes in

phosphate buffer, when compared to attachment to juveniles kept in the same buffer at

room temperature. The incubation ofjuveniles in glycolytic and proteolytic enzymes also

reduced attachment (Davies and Danks, 1993). The authors concluded that these

treatments caused the solubilization and denaturation of the ligants involved in

attachment. The results of the present study support the findings of Davies and Danks

(1993), and indicate that the thermal solubilization and denaturation of ligants involved in

attachment is a gradual process which is time and temperature dependent.

The evaluation of the effects of short exposures of nematodes to higher temperatures that may stimulate or inhibit attachment may help to elucidate the synergistic effects of the bacterium and solarization reported by Tzortzakakis and Gowen

(1993) and Walker and Wachtel (1988). The heat generated by solarization forms a gradient from the surface to deeper layers of the soil, causing the nematodes to migrate deeper. Juveniles that escape lethal temperatures may have their cuticle altered to be more receptive to endospore attachment. This factor, combined with the increased nematode movement at higher temperatures, may result in attachment levels higher than those expected in non-solarization conditions.

Effect of temperature on endospores . The mechanisms by which P. penetrans endospores adhere to nematode cuticle has not been elucidated completely. Davies and

Danks (1993) tested attachment after treating endospores with enzymes, lectins and carbohydrates, and concluded that N-acetylglucosamine moieties present on the surface 51

coat of endospores are the adhesins involved in attachment. The authors suggested that

these adhesins are linked either to proteins or peptidogycans, which are extensions of the

cell walls of Gram-positive bacteria. Decreased attachment resulting from exposure of

endospores to heat suggests that N-acetylglucosamine or similar moieties were

solubilized or denatured. Temperature also may denature proteins or peptidogycans in

the bacterial cell wall, or break the bonds that link N-acetylglucosamine moieties to them.

Further biochemical studies are required for the understanding of the mechanisms that

govern the decline of attachment with increasing temperature.

Dutky and Sayre (1978) did not observe attachment to juveniles after soil with

endospores was heated at 130 °C for 1 hour; however, attachment occurred after treatment

at 80 °C for 30 minutes. In the present study, attachment was close to minimum at 60 °C

or higher, but it was not completely prevented even at 100 °C for 5 hours per day over 10

days. Endospores used in this investigation were in high concentrations in water when

they were induced to attach through agitation for 24 hours at 24 °C. This method was

chosen to reduce the variation in attachment encountered in soil, but it probably forced

the endospores against the juvenile cuticle several times during the agitation period. This

may explain why attachment occurred with endospores exposed to 100 °C for 10 days in

this study, while no attachment was observed by Dutky and Sayre (1978) after soil with

endospores was heated at 130 °C for 1 hour. Although proceedures for endospore

attachment vary among the present study and those of Dutky and Sayre (1978) and Bird

et al. (1989), it is clear that time and temperature are inverse in reducing attachment of treated endospores to juveniles. 52

Effect of temperature on juveniles and endospores in soil . In soil with nematodes

and bacteria exposed to treatments together, the temperature may affect the capability of

endospores to attach to the nematode, juvenile cuticle's receptivity to endospores, and the

incidence of contact ofjuveniles with endospores. The increase in attachment observed

from 10 °C to 20 °C, followed by optimum attachment between 20 °C and 30 °C, and decreased attachment after 30 °C agreed with previous studies (Hatz and Dickson, 1992;

Singh and Dhawan, 1990; and Stirling, 1981).

Little or no attachment occurred when endospores and juveniles were exposed to

10 °C for 4 days, which was assumed to be caused by the limited movement ofjuveniles of M. arenaria in soil at that temperature. According to Thomason and Lear (1961), M. arenaria is a thermophile and does not have extended survival in soils below 10 °C.

As in the low temperature treatment, a reduction in movement of the nematode, which prevented contact among juveniles and endospores, occurred above 40 °C. In addition, about 50% of the juveniles extracted after 4 days at 40 °C were dead. Some juveniles had cuticle deformities that are characteristic of exposure to high temperature.

The absence of attachment at 50 °C may be best explained by the direct impact of the high temperature on mortality of the nematode.

Carbohydrates and proteins present on the nematode surface coat (or glycocalyx) are secreted by the nematode via excretory pore, mouth, anus, amphids, and phasmids, and spread over the nematode surface (Bird and Bird, 1991; Bird et al, 1989; and Bird and Zuckerman, 1989). These proteins have a carbohydrate recognition domain and are thought to be responsible for attachment, since they bind to N-glycosamine on the surface 53

of the P. penetrans endospores (Davies and Danks, 1993). Proteins of the surface coat of

second-stage juveniles are transitory and are released into water during incubation (Lin

and McLure, 1993). According to Spiegel and McLure (1995), the surface coat, when

removed from the juvenile cuticle, is replaced in 24 hours. These authors found that the

surface coat production is a metabolic process and is dependent on nematode age and

temperature. Since the loss of the surface coat in water has been observed (Lin and

McLure, 1993), it is expected that the juveniles used in the bioassay had lost a substantial

portion, if not the totality, of their surface coat after the long period of incubation in

water. Hence, at 5 °C the juveniles of M. arenaria were in anabiosis and, therefore, they

were incapable of restoring their lost surface coats. The attachment was probably low at

the time the juveniles were introduced into soil. This hypothesis is supported by the

differential attachment observed when 1- to 4-day-old juveniles and 45-day-old juveniles

stored at 5 °C were subjected to attachment in endospore suspensions under agitation.

The attachment on the 1- to 4-day-old juveniles was 255 times higher than that on the

juveniles stored at 5 °C. A new surface coat should have been secreted by the juveniles

during the 4 days in the soil. Although the juveniles of M. arenaria stored in water for 45

days at 5 °C recovered their motility after 1 hour at 23 °C under bubbling air, their

metabolism may have been altered by long-term storage at low temperature and resulted

in reduced surface coat rejuvenation. Indeed, juveniles extracted from soil in the first

trial were more active than the ones from the second trial.

The low temperatures during the winter in Florida caused a decrease in the nematode population in the greenhouses, and the lack ofjuveniles to conduct experiments resulted in the use of stored juveniles instead of freshly hatched ones. Although the

average of endospores attached per juvenile in the first trial was almost 25 times higher

than in the second trial, the trends of the curves for the attachment in both trials were

similar. The difference in information generated from fresh and stored inoculum should

be useful to warn other researchers of the consequences of storing juveniles in water at

low temperatures before conducting studies with P. penetrans.

In summation, temperature treatments influenced endospores of P. penetrans, juveniles of M. arenaria, and the interaction of the parasite and the nematode. The

greatest receptivity of second-stage juveniles of M. arenaria to endospore attachment

occurred when juveniles were exposed to 30 to 35 °C. Incubating P. penetrans

endospores for 10 days at 50 °C and higher resulted in a reduction in female nematodes.

Finally, the highest endospore attachment level occurred when nematode juveniles were

incubated in soil infested with endospores at 20 to 30 °C for 4 days. CHAPTER 3 EFFECTS OF SOIL SOLARIZATION AND ORGANIC AMENDMENT ON PASTEURIA PENETRANS AND ON ITS INFECTIVITY TO MELOIDOGYNE

ARENAR1A RACE 1 IN TOMATO

Introduction

Integrated strategies may be useful for managing plant-parasitic nematodes while

providing crop nutrition. Combinations of treatments may become increasingly

important as pesticide usage is reduced. The effects of integrating biological control agents, such as P. penetrans, with other treatments have not been thoroughly

investigated, however, and further study is warranted (Stapleton and Heald, 1991).

According to Tzortzakakis and Gowen (1993), the efficacy of P. penetrans may

be enhanced by soil solarization. Solarization is the process of heating moist soil under transparent plastic sheets to temperatures which are lethal to plant pathogens (Katan and

DeVay, 1991). The use of this technique generated significant decreases in the

population densities of several plant-parasitic nematodes (Chapter 1). Suppressiveness to plant pathogens in solarized soils may result from a shift in microbial populations to heat- resistant antagonists (Katan et al, 1983). Higher levels of attachment of endospores of P. penetrans to nematodes occur under solarization conditions, and may be explained by the stimulation of nematode motility with increasing soil temperature (Bird and Wallace,

1965), which consequently increases probability of contact between nematodes and

55 56

endospores (Walker and Wachtel, 1988). Solarization increases nematode infection by P. penetrans without increasing soil population densities ofjuveniles (Walker and Wachtel,

1988). Stirling (1981) found that the influence of solarization on the interaction of the

nematode and P. penetrans was not restricted to the period when higher temperatures

were experienced. He observed that the length of the life cycle of P. penetrans was

reduced by about 70% at 30 °C compared with that at 20 °C, and that M. javanica was

parasitized at an earlier stage of its development at the higher temperature. Thus,

increased temperatures in solarized soil may increase both the rate of development of

females of Meloidogyne spp. and the production of P. penetrans endospores (Walker and

Wachtel, 1988). Nakasono et al. (1993) observed a great increase in the population

density of P. penetrans in solarized soil when compared with non-solarized soil, but the authors did not report on the maximum temperature of the soil reached during solarization. According to Katan and DeVay (1991), the soil temperatures under transparent plastic mulch in tropical and subtropical countries typically range between 40 and 50 °C in the first 15-cm layer, and can reach 60 °C at a depth of 10 cm under double- layered plastic separated by 50 cm of air (Katan and DeVay, 1991). McSorley and

Parrado (1 986) recorded a soil temperature of 5 1 .6 °C at a depth of 1 5 cm under solarization in Florida.

The time and temperature to which endospores are exposed are interacting factors

(Chapter 2). Attachment of P. penetrans on M. javanica decreased from 9.3 endospores per juvenile at 100 °C for 5 min, to 0.3 endospores per juvenile at 100 °C for 60 minutes

(Williams et al., 1989). The percentage of infected females resulting from endospores 57

treated at 100 °C was reduced from 1.7 when endospores were exposed for 5 minutes to

0.6 when endospores were exposed for 60 minutes. Ninety four percent of the females

were infected by untreated endospores. In Chapter 2, attachment of endospores of P. penetrans to juveniles of M. arenaria was minimal after 10 days of exposure of

endospores to 60 °C for 5 hours/day. Thus, the effect of solarization for extended periods

on P. penetrans in the field warrants further investigation.

Management of plant-parasitic nematodes also may be achieved by adding organic amendment to soil (Muller and Gooch, 1982; Singh and Sitaramaiah, 1970;

Vijayalakshimi et al, 1985). Katan (1981) suggested that solarization might increase the rate of degradation of organic matter and result in the accumulation of toxic gases in the soil atmosphere. According to Stapleton et al. (1993), sealing and heating soil amended with organic fertilizers and crop residues can provide levels of disinfestation greater than with either method alone. Reductions of M. incognita and some soilborne fungi were greatest when soil was amended and heated. Green tissue of Brassica spp. can decrease

population densities of some fungal and nematode pathogens due to its abundant sulfur- containing materials (Mayton et al., 1995). The objectives of this study were to determe

the density of endospores of P. penetrans in field soil and to evaluate the effects of soil solarization, organic amendment, and chloropicrin on root knot of tomato, caused by M.

arenaria, in soil naturally infested with P. penetrans in the field. Material and Methods

Bioassay for determining the density of endospores of P. penetrans in field soil .

Soil samples were extracted from a field infested with the bacterium, combined, spread in

thin layers on trays, and allowed to dry at 22 °C for 3 weeks to kill the nematodes. Two

20-g subsamples were each placed in a 9-cm-diameter petri dish and kept for 3 days at

room temperature. One kilogram of soil was autoclaved twice for 30 minutes at 120 °C

to kill nematodes and P. penetrans endospores, and 20 g were distributed into each of

eight petri dishes. Two milliliters of a suspension of P. penetrans at 10-*, 10^, 10^, or

106 endospores/g of soil were added to each of two petri dishes, and the soil was allowed

to dry in all eight dishes for 3 days. After this period, the soil in the petri dishes was

mixed well and 4 ml of water were spread on the surface of the soil in each of the 10

dishes and maintained for 2 hours to provide uniform soil moisture. Two milliliters of

water containing 600 juveniles of race 1 of M. arenaria were spread on the surface of the

soil of each dish, and the dishes were maintained in the dark in an incubator at 26 °C for

3 days. The nematodes were extracted by centrifugal flotation (Jenkins, 1964) for

evaluation of the number of endospores attached per juvenile in each treatment. The number of P. penetrans endospores attached to each of 20 juveniles was recorded. The endospore density in each field was determined by comparison with soil in which known concentrations of the bacterium were added. The data were subjected to regression analyses using SAS software (SAS Institute, Cary, NC). 59

Evaluation of the effects of soil solarization, organic amendment, and chloropicrin

on root knot of tomato, caused by M. arenaria, in soil naturally infested with P.

penetrans in the field . The experiment was conducted in a field at the University of

Florida Green Acres Agronomy Farm, Alachua County, Florida. The soil type was an

Arredondo fine sand. The treatments were arranged in a randomized, complete block

design, with six replications per treatment. The plots were 1 m wide and 10 m long.

Treatments included methyl bromide plus 33% chloropicrin; chloropicrin alone;

solarization with clear, gas-permeable polyethylene plastic film (PE); organic amendment

(chopped cabbage incorporated into soil to provide approximately 0.25% in the upper 20 cm); PE with methyl bromide plus 33% chloropicrin; PE plus chloropicrin alone; PE plus organic amendment; solarization with gas-impermeable plastic film (GI), GI plus organic amendment; and the untreated control.

The field with M. arenaria and P. penetrans was deep-plowed and cultivated on

16 May, 1995. Fertilizer of the formulation 13-1 .8-1 1 (N-P-K) was broadcast over the area at 450 kg/ha. Soil from cultivated plots was sampled for the initial populations of

M. arenaria before the treatment applications on 13 June by removing and combining six soil cores (2.5 cm in diameter and 20 cm deep) from each plot. Cabbage (Brassica oleracea L) of the cultivar Constanza was grown in the field of the University of Florida

Research Station in Quincy, Florida, and brought to the Green Acres Agronomy Farm,

Alachua County, Florida, where it was chopped and spread on the surface of the

designated plots on 13 June. The chopped cabbage was allowed to dry on the soil surface for 6 days and was incorporated into the soil on 19 June. All other treatments were 60

applied 20 June. The soil was overhead irrigated for 2 hours and the excess water

allowed to percolate for 2 hours before it was covered with solarization plastic mulch or

other treatments were applied. Methyl bromide plus chloropicrin and chloropicrin alone

were applied in the beds using chisels spaced 30 cm apart and 25 cm deep over preformed

beds, which were covered immediately with opaque-white plastic with a mulch laying

bed press. Transparent plastic for solarization also was applied with the mulch laying bed

press, and was painted white after 45 days of solarization.

The soil temperatures in bare soil and in the solarization treatments were

monitored at depths of 5 and 20 cm with thermal couple sensors (Omnidata International,

Logan, UT). Ambient air temperature and daily precipitation totals were obtained from

an in situ weather station. Irrigation was provided through drip tubing A double-wall

single drip tube (Chapin Livinwall, Watertown, NY) with emitters spaced 30 cm apart

and a flow rate of 1 .9 liters per minute/30 m of row was placed under the plastic mulch

before planting. Postplant liquid fertilizer, 12.3-0-1 1.6 (N-P-K), was applied through

drip irrigation in 10 equal applications at weekly intervals at 1 1.2 kg/ha Tomato

seedlings, cv. Solar Set, were transplanted into beds with a plant spacing of 0.5 m on 14

August. Tomato fruits were harvested twice, on 2 and 13 November, and weighed after

each harvest.

Soil samples for final populations of the nematode were collected after the second

harvest, 91 days after planting the seedlings. For estimation of the final population of the

nematode, cores from the root zone of each of six plants per plot were bulked. The soil cores were 2.5 cm in diameter and were taken 20 cm deep. Second-stage juveniles were 3 extracted from 100-cm soil subsamples by centrifugal flotation (Jenkins, 1964). Six root

systems were removed from each plot, and plants were rated for root galling on a scale of

0 to 10, in which 0 = 0 galls, 1 = 10% of roots with galls, 2 = 20% of roots with galls, 3 =

30% of roots with galls, and so on until 10 = 100% of roots with galls (Barker et al.,

1986). Means of treatments were subjected to analyses of variance and compared using

Duncan's multiple-range test using SAS software (SAS Institute, Cary, NC). Contrasts

and factorial analysis were used to compare treatment differences using the 'proc glm'

procedure in the SAS software.

Evaluation of the effects of soil solarization, organic amendment, and chloropicrin

on P. penetrans in the greenhouse . The experiment was conducted in a greenhouse at the

University of Florida campus, Gainesville, Florida. Soil from the first 20-cm depth of

each of the 60 plots treated in the field experiment with methyl bromide, chloropicrin,

solarization, cabbage amendment, or the untreated control was collected and stored in

plastic bags in the shade. Three hundred and fifty cubic centimeters of soil from each

plot were spread on a paper plate and allowed to dry for 1 5 days on benches in the

greenhouse without ventilation. After this period, the dry soil was placed in sterilized,

10-cm-diameter clay pots and transferred to a bench in a room at 24 °C. The moisture of

the soil in the pots was adjusted to field capacity, and the pots were left for 3 days at

room temperature (approximately 24 °C). After this period, 2,000 1-to 4-day-old juveniles of M. arenaria were added in 10 ml of water to five holes, 5 cm deep, in the

soil of each pot. After 5 days, one 15-day-old seedling of the tomato cv. Solar Set was

transplanted into each pot. Treatments were arranged in a randomized, complete block design, with six replications per treatment. The plants were fertilized once a week with

18-18-21 (N-P-K) and micronutrients (Miracle-Gro for tomato; Stern's Miracle-Gro

Products, Port Washington, NY) at 0.05 g/pot, and insects were controlled as needed

using recommended insecticides.

3 Second-stage juveniles were extracted from 100-cm of soil by centrifugal

flotation (Jenkins, 1964). Plants were rated for root galling on a scale of 0 to 10 as

described above (Barker et ah, 1986), and eggs were extracted with 0.5% NaOCl.

Twenty females of M. arenaria were picked from each root system, and each of them was

crushed separately in 2.5 jo.1 of lactophenol plus 1% methyl blue (v/w) (Sigma Chemical

Co., St. Louis, MO 63 1 78) under a coverslip. The presence of P. penetrans and its

developmental stage were observed under an inverted microscope at a magnification.of

x400. The experiment was repeated once with a 15-day interval between inoculations with nematodes. Means of treatments were subjected to analyses of variance and compared using Duncan's multiple-range test in SAS software (SAS Institute, Cary, NC).

Contrasts and factorial analysis were used to compare treatment differences using the

'proc glm' procedure in the SAS software.

Evaluation of the effect of the exposure of soil with Pasteuria penetrans endospores, with and without organic amendment, to 50 °C . The effect of the exposure of soil with endospores of P. penetrans, with and without organic amendment, to 50 °C was determined in water-bath experiments. Field soil infested with P. penetrans was collected from a site suppressive to M. arenaria and was used in the experiment and in the following experiment to determine the thermal inactivation point of P. penetrans. 63

Two kilograms of soil were spread on paper plates and allowed to dry for 15 days on

benches in the greenhouse without ventilation to kill residual nematodes, and 2 kg were

submitted to two, 30-minute cycles on consecutive days in an autoclave at 120 °C. One

kilogram of soil from each treatment was mixed with 2.5 g of dry, chopped cabbage

(0.25% v:v). The experiment was a 2 x 2 x 2 factorial in which the treatments, with five

replicates per treatment, consisted of air-dried soil, autoclaved soil, air-dried soil plus

cabbage, and autoclaved soil plus cabbage incubated at 20 or 50 °C. Fifty cubic

centimeters of soil from each treatment with or without organic amendment were placed

into a pre-washed, 50-ml, polystyrene centrifuge tube (Blue Max MPS conical tubes,

Becton Dickinson and Company, NJ). The tubes were closed and maintained in a growth

chamber constantly at 20 °C, or in a water-bath tank adjusted to a daily cycle of 5 hours at

50 °C followed by 19 hours at room temperature (23 °C). The latter was an attempt to

simulate the high temperature attained in soil under solarization conditions in the field.

After 10 days of incubation, the tubes were opened and taken to the greenhouse, where they received 10 ml of water every day for a 15-day period, for the completion of the cabbage decomposition and release of toxic volatiles. After this period, 1,000 juveniles

of M. arenaria were added to each tube, and the tubes were placed in an incubator at 28

°C for 4 days. After incubation, one 10-day-old tomato seedling was planted in each tube. The tubes were distributed randomly in a growth chamber at 28 °C with a photoperiod of 13 hours. Thirty five days after planting, the shoots of the plants were cut at the soil surface level, and the soil with the root system and nematodes from each tube was poured into a bucket. The root system was washed to remove the soil and the 64

juveniles were extracted from the soil using centrifugal flotation (Jenkins, 1964); juveniles were stored in water at 5 °C until assessment. The washed roots had the excess

water removed and were weighed. The gall index was determined using a scale of 0 to

10, as described above (Barker et ah, 1986). The eggs were collected after dissolving the

gelatinous matrices containing eggs with NaOCl. Each root system was placed into a 50-

ml centrifuge tube with 30 ml of an aqueous solution of 0.5% NaOCl. The tubes were

closed and vigorously shaken for 30 seconds. The egg suspension was poured onto a

sieve with 25-um-pore openings (500-mesh), rinsed with tap water, and collected in another centrifuge tube. The process was repeated twice, using tap water instead of

NaOCl solution. Each root was cut into pieces 3 to 5 cm long, and the pieces were placed into a centrifuge tube with 30 ml of an 8:1 (v/v) aqueous solution of commercial Cytolase

PCL5 (Genecor International, Rolling Meadows, IL). The tubes were placed in an automatic shaker (Eberback Coorporation, Ann Arbor, MI) for 24 hours at room temperature (approximately 24 °C). Softened roots were placed on a sieve with 600-um- pore openings (30-mesh) over a sieve with 180-|im-pore openings (80-mesh) and sprayed with a vigorous stream of tap water. The dislodged females and root debris were transferred to a beaker by washing the sieve from the back with a jet of water. The females were separated from the root debris with a Pasteur pipette under a stereomicroscope, and examined for P. penetrans infection with an inverted compound microscope. Number of eggs per root, gall index, and percentage of infected females were recorded. The numbers of attached endospores were counted on 20 randomly selected juveniles per replicate using a Nikon inverted microscope at a magnification of x400. The data were subjected to regression analyses using SAS software (SAS Institute,

Cary, NC).

Determination of the thermal inactivation point of P. penetrans endospores in

vitro . Fifty cubic centimeters of field soil infested with P. penetrans were placed into a

pre-washed, 50-ml, polystyrene centrifuge tube (Blue Max MPS conical tubes, Becton

Dickinson and Company, NJ). The experiment had five treatments (incubation at 30, 40,

50, 60, or 70 °C) and five replicates per treatment. Five water-bath tanks were adjusted to

a daily cycle of 5 hours at 30, 40, 50, 60, or 70 °C, followed by 19 hours at room

temperature (23 °C); and five tubes were maintained for each temperature cycle for 10

days. After this period, 1 ,000 juveniles of M. arenaria were added per tube, and the

tubes were placed in an incubator at 28 °C for 4 days. After incubation, one 10-day-old

tomato seedling cv. Rutgers was planted in each tube. The tubes were distributed

randomly in a growth chamber at 28 °C with a photoperiod of 13 hours. Thirty five days

after planting, the shoots of the plants were cut at the soil surface level, and the root

system of each plant was washed in a bucket to remove the soil. Juveniles were extracted from the soil by centrifugal flotation (Jenkins, 1964), and stored in water at 5 °C until assessment. The roots had the excess water removed and were weighed. The gall index, number of eggs per root system, percentage of infected females, and number of attached endospores per juvenile were determined as described above. Data were subjected to regression analyses using SAS software (SAS Institute, Cary, NC). 66

Results

Bioassav for determining the density of endospores of P. penetrans in field soil .

Numbers of endospores attached to juveniles increased in proportion to the concentration

of endospores in artificially infested soil (Fig. 3.1). The numbers of endospores attached per juvenile in the soils that received 10,000, 100,000, and 1,000,000 endospores/g of

autoclaved soil were 1.1, 4.1, and 21.2, respectively.

The average number of endospores attached per juvenile in 40 juveniles examined

(20 juveniles per each of two replications) in the untreated field soil was 2.0. The endospore density in the field soil, estimated by entering 2.0 in the regression equation,

4 was 3.0 x 10 endospores/g of soil (Fig. 3.1).

Evaluation of the effects of soil solarization, organic amendment, and chloropicrin on root knot of tomato, caused by M. arenaria, in soil naturally infested with P.

penetrans in the field . A higher difference between day and night temperatures occurred in solarized soil at the 5-cm depth (approximately 25 °C) than at the 20-cm depth

(approximately 10 °C) (Figure 3.2). Solarized soil at 5-cm deep, with or without organic

amendment, had an average maximum daily temperature of 54 °C, and non-solarized soil had an average maximum daily temperature of 42 °C (Fig. 3. 3 A). At the depth of 20 cm, the average maximum daily temperature was 41 °C in solarized soil and 36 °C in non- solarized soil (Fig. 3.3B).

~ Solarization and organic amendment appeared to increase Mg and N03 compared to untreated soil (Table 3.1). Potassium appeared to be higher in amended soils 67

0 2 4 6 8 10 12 14 16 18 20 Endospores/J2

Fig. 3.1. Regression of the number of endospores of Pasteuria penetrans attached to second-stage juveniles (J2) of Meloidogyne arenaria race 1 to density of endospores in

soil. the The value for the number of endospores/g of field soil (

69

Solarization Sol.+Cabbage Nonsolarized

8 10 12 14 16 18 20 22 24 26 28 30

Days of solarization

62

Solarization 58 B Sol.+Cabbage

54 Nonsolarized

U 50

10 12 14 16 18 20 22 24 26 28 30

Days of solarization

Fig 3.3. Maximum daily temperatures of the soil under transparent plastic

(solarization), soil amended with cabbage under transparent plastic (sol. + cabbage), and soil under opaque-white plastic (nonsolarized) at 5-cm deep (graph A), and at 20-cm deep (graph B). *

70

Table 3.1. Soil analysis from the solarization experiment.

Treatment5' z PH Ca Mg K P NH/ N03 OM

Untreated control 5.1 223 10.5 45.3 56.2 3.5 9.1 1.0 Organic amendment (OA) 5.3 225 14.3 52.9 57.6 3.7 14.1 1.0 Solarization (PE) 5.2 204 14.1 43.0 57.3 3.5 14.1 0.9 OA + PE 5.0 198 14.2 53.5 59.0 4.2 22.2 1.0 Solarization (GI) 5.2 224 14.8 44.8 57.9 3.5 17.6 0.8 OA + GI 5.5 274 20.2 62.3 69.8 4.0 20.2 0.8

The chemical elements were reported as mg/kg of soil. y OA = Organic amendment (chopped cabbage) was incorporated into soil to provide approximately 0.25% of soil weight in the upper 20 cm; PE = solarization under clear, gas-permeable polyethylene plastic film; GI = solarization under clear, gas-impermeable polyethylene plastic film. z OM = Organic matter 71

than in nonamended soils. The interaction of amendment and solarization treatments

seems to have resulted in greater levels of P and nitrogen forms than all other treatments.

Galling was reduced by the application of methyl bromide or chloropicrin in the

soil, when compared to untreated controls and organic amendment treatment in

bothsolarized and non-solarized treatments (P < 0.05) (Table 3.2). No differences in

galling were detected in soils with or without cabbage amendment under either solarized

or non-solarized treatments. There were no differences among treatments in the number

ofjuveniles per 100 cm of soil. The number of endospores attached per juvenile in the

solarized plots treated with methyl bromide plus 33% chloropicrin was lower than that in

non-solarized plots treated with methyl bromide plus 33% chloropicrin and solarized or

non-solarized treatments with chloropicrin alone; no differences in attachment were

observed among other treatments.

Numbers of fruit and weight of fruit were higher in methyl bromide and

chloropicrin treatments than in untreated, solarized, or non-solarized controls. The numbers of fruit and weight of fruit in plots with cabbage amendment were not different from the controls, in both solarized and non-solarized soils. In the factorial analysis of variance, no significant effects were observed among treatments in the number of

* 3 juveniles per 100 cm of soil or in number of endospores attached per juvenile; ANOVA effects were highly significant (P > 0.001) when treatments with chemicals (methyl bromide plus 33% chloropicrin, chloropicrin alone, and cabbage volatiles) were compared to the untreated controls for root galling, number of fruit, and weight of fruit

(Table 3.2). Contrasts were not observed between cabbage under gas-permeable and gas- Table 3.2. Effects of solarization, organic amendment, methyl bromide and

chloropicrin on root knot of tomato, caused by Meloidogyne arenaria, in soil naturally

infested with Pasteuria penetrans in the field.

^ V f~^L oil in/lav oaii index Number of J2 / Number of Number or Weight (kg)

1 I L all 11 till 100 cm of soil endospores/ tomato fruits/ of tomato TO JZ plot rruits/plot Non-solarized w 1 - Methyl bromide 0.09 b 5.80 a 45.00 a 277 ab 46.30 ab 2 - Chloropicrin 0.59 b 13.80 a 41.80 a 293 a 48.80 ab 0*") *h ( Winn 1 /> o m An,-] rv> a r% t 0 o j urganic amenument 18.00 a 7.00 ab 245 be 41.70 be

"1 /i ^> £f\ ( Art _ 1 t - unireaieu control z.Dj a 342.50 a 4.00 ab 218 c 35.90 c

x Solarized (PE) 5 - Methyl bromide U.2U D 1.50 a 0.00 b 299 a 50.10 a 6 - Chloropicrin U.04 D 75.20 a 31.30 a 270 ab 48.00 ab 7 - Organic amendment 3.83 a 106.30 a 14.90 ab 233 c 42.00 be 8 - Untreated solarized 4.25 a 70.70 a 4.80 ab 230 c 38.30 c

Solarized (GI)

"7/1 OA rt, C /IA «U y UIllICtUEU bUlallZCU z.y / a fH.zy) a j.4U ab 225 c 38.40 c iu - vjrgamc amenurncni j.uo a 42.20 a 20.80 ab 243 be 41.80 be

Factorial effects Solari7ation z XTO NS rNo in a NS Chemicals *** NS NS *** *** Solarization x Chemicals NS NS NS NS NS

Contrasts 7x 10 NS NS NS NS NS 8x9 NS NS NS NS NS 7+8 x 9+10 NS NS NS NS NS 1+2 x 5+6 NS NS NS NS NS

"Gall index was based on a scale of 0 to 10, in which 0 = 0 galls, 1 = 10 % of roots with galls, 2 = 20 % of roots with galls, 3 = 30 % of roots with galls, and so on until 10 = 100 % of roots with galls. J2 = Second-stage juveniles. w Values are means of six replicates; means within columns and within experiments followed by the same letter do not differ at P < 0.05 according to Duncan's multiple-range 3 test (data for gall index, number of J2/100 cm of soil, and number of endospores/J2 were transformed to log 10 (x + 1) for analysis). solarization under clear, gas-permeable polyethylene (PE) plastic film. y solarization under clear, gas-impermeable (GI) polyethylene plastic film. z * ** *** represent < < 5 ? p o 05> P o oi, and P < 0.001, respectively, and NS = no significant differences at P < 0.05 according to the non-orthogonal contrast test. 73

impermeable plastic, control soil under gas-permeable and gas-impermeable plastic,

solarization using gas-permeable and gas-impermeable plastic, or treatments of soil with

methyl bromide plus 33% chloropicrin or chloropicrin alone without solarization or

undersolarization {P > 0.05. ). The absence of nematodes or low numbers of them in

some replicates, while not in others, generated great variation in the final population of

the nematodes in the plots among replicates of treatments (Figure 3.4).

Evaluation of the effects of soil solarization. organic amendment, and chloropicrin

on P. penetrans in the greenhouse . After treatment of soil in the field and subsequent

inoculation with M. arenaria, galling was lower in the non-solarized, untreated control

than in non-solarized soils fumigated with methyl bromide and chloropicrin (P < 0.05) in

the first trial (Table 3.3). Galling was also lower in the non-solarized soil amended with

cabbage when compared to non-solarized soils treated with methyl bromide or

chloropicrin in the second trial (Table 3.4). The gall index was similar for all treatments

in solarized soils, in both trials (Tables 3.3, 3.4). Factorial analysis showed that soils

exposed to methyl bromide or chloropicrin resulted in higher galling when compared to

untreated soils in both tests (P < 0.05).

There were no differences in the number of eggs per root system among all

treatments under solarization in both tests, but in non-solarized soils more eggs were produced in the methyl bromide and chloropicrin treatments than in soil amended with cabbage (Tables 3.3, 3.4). Solarization and chemicals were not significant factors in the first trial (P > 0.05), but solarization was significant (P < 0.05) and chemicals were significant (P < 0.01) in the second trial (Tables 3.3, 3.4); no interaction between : « 1 < 1 «

Initial Population of Final Population of Meloidogyne arenaria Meloidogyne arenaria

in the Field in the Field

i

700 700

< » 600 600

500 500

o o < "E < 1 400 u 400 5 o o o

J> "3 1 300 300 1 1 S — > > 3 3

• 200 200

100 100 —

« 4 4 I i II

i • t 1 >— t—41 11-1 1— »— Ui l-l 1 23456789 10 123456789 10 Treatments Treatments

Fig. 3.4. Initial and final population densities of Meloidogyne arenaria race 1 per

100 cm of field soil. Treatments included 1) methyl bromide plus 33% chloropicrin; 2) chloropicrin alone; 3) organic amendment (chopped cabbage incorporated into soil to provide approximately 0.25% in the upper 20 cm); 4) Untreated control; 5) solarization with clear, gas-permeable polyethylene plastic film (PE) with methyl bromide plus 33% chloropicrin; 6) PE plus chloropicrin alone; 7) PE plus organic amendment; 8) PE; 9) solarization with gas-impermeable plastic film (GI); and 10) GI plus organic amendment. 75

Table 3.3. Effects of field treatments on infectivity of Pasteuria penetrans to subsequently introduced second-stage juveniles (J2) of Meloidogyne arenaria race 1, grown on tomato under greenhouse conditions.

Number of Root Number of Number of %of Treatment 3 Gall index" eggs / root weight J2/ 100 cm endospores / females

system (g) of soil J2 with P. penetrans Non-solarized t v w v v w r\ r\ /- \ V.W v 1 - MB + chloropicrin y.\J\) a 2750 a 3.40 be 1600 a 0.06 b 0.00 d 2 - Chloropicrin 9.00 a 2810 a 2.15 c 1790 a 0.09 ab 0.00 d 3 - Organic amendment 8.33 ab loot/ 0 1.77 c 740 b 0.48 a 25.80 b

4 - Untreated control 1 ASfl ah 7.83 b 2.28 c 440 b 0.10 ab 34.20 a

x Solarized (PE) 5 - MB + chloropicrin 9.33 a 2180 ah 4.35 ab 1060 a 0.08 ab 0.00 d 6 - Chloropicrin 8.83 ab 2590 a 3.45 be 1670 a 0.11 ab 0.00 d 7 - Organic amendment 8.17 2670 a ab 5.42 a 1180 a 0.46 ab 9.20 c o - unircaicu soianzea 5.83 ab 2200 a 2.83 be 940 ab 0.10 ab 13.30 c

Solarized (GI) y o.8 7fl/U ah 3D 2580 a 3.53 be 1220 a 0.30 ab 10.80 c 10 - Organic amenHmpnt X 17 ah 2860 a 4.59 ab 1230 ab 0.08 ab 5.80 cd

Factorial effects z Solarization \IC NS ** NS * * * Chemicals * NS NS * NS *** Solarization x Chemicals NS NS * NS NS **

Contrasts 7x 10 NS NS NS NS NS NS 8x9 NS NS NS NS NS NS 7+8x9+10 NS NS NS NS NS NS 1+2 x 5+6 NS NS NS NS NS NS

1 Methyl bromide plus 33% chloropicrin. u Gall index was based on a scale = of 0 to 10, in which 0 0 galls, 1 = 10 % of roots with galls, = 2 20 % of roots with galls, 3 = 30 % of roots with galls, and so on until 10 = 100 % of roots with galls. v Values are means of six replicates; means within columns followed by the same letter do not differ at P < 0.05 according to Duncan's w multiple-range test. Actual means are shown, but values were transformed using log 10 (x + 1) before comparison with Duncan's multiple-range test. x Solarization under clear, gas-permeable polyethylene (PE) plastic film. polarization under clear, gas-impermeable (GI) polyethylene plastic Z film. *> **. *** represent P < 0.05, P < 0.01, and P < 0.001, respectively, and NS - no significant differences at P < 0.05 according to factorial and non-orthogonal contrast tests. 76

solarization and chemicals was observed in either trial. Based on contrast analysis, egg

production was lower in fumigated soil that was solarized than in fumigated, non-

solarized soil in the second trial. Treatment with cabbage under transparent, gas-

permeable plastic resulted in higher root weights than in all treatments except fumigation

with methyl bromide in non-solarized soil in the second trial and amendment with

cabbage under transparent, gas-impermeable plastic, in both trials (P > 0.05) (Tables 3.3,

3.4). Factorial analysis indicated that both chemicals and solarization increased the

weight of root systems in the second trial, but chemicals did not have a significant effect

on root weight in the first trial (P > 0.05); a significant interaction between chemicals and

solarization occurred in the first trial but not in the second trial. Contrast analysis

indicated that root weights were greater in chemical treatments supplemented with

solarization than in fumigated, non-solarized treatments in the second trial (P > 0.01).

Lower 3 numbers ofjuveniles per 100 cm of soil were detected in non-solarized,

untreated and cabbage-amended treatments than in non-solarized soils treated with

methyl bromide or chloropicrin in both trials (Tables 3.3,3.4). Contrast analysis showed a reduction in juvenile numbers when soil was amended with cabbage and solarized under gas-impermeable membrane as compared to cabbage-amended soil under gas- permeable membrane in the second trial, and fewer juveniles in fumigated, solarized treatments in the second trial (P > 0.05).

Number of endospores attached per juvenile was higher in non-solarized soil amended with cabbage than in nonsolarized, methyl-bromide treated soil in both tests, and higher than amended soil under solarization with either plastic film in the second trial '

77

Table 3.4. Effects of field treatments on infectivity of Pasteuria penetrans to subsequently introduced second-stage juveniles (J2) of Meloidogyne arenaria race 1, grown on tomato under greenhouse conditions, in the second test.

Number of Root Number of IN UIIlUCl VI % of U 3 /""oil \nAav pnHnQnnr^Q / fpmalf**; with Treatment Lraii index eggs / root weight J2/100cm IVlllUl^iJ Willi cvctpm (g) of soil J2 P. penetrans Non-solarized v w v v w v w v ' v w w A 11 ok 1 oor\ ~k 9.00 a 5320 a 4.JJ ab 1 OoU 3D 0.24 b 0.00 d 1 - MB + chloropicrin

2 - Chloropicrin 9.50 a 4575 ab 3.18 be 1670 abc 0.36 ab 0.00 d

3 - Organic amendment 7.00 b 1840 c 3.32 be 400 d 0.89 a 25.00 a

4 - Untreated control 8.00 a 2497 abc 2.12 c 560 d 0.46 ab 26.70 a

X Solarized (PE) 0.01 d 5 - MB + chloropicrin 8.00 ab 3785 ab 6.05 a 1430 be 0.69 ab ft ftl r*A 6 - Chloropicrin 8.33 ab 2105 be 3.23 be 1070 cd U.ol ao u.uz ca

1 QftC L~ 1 0 80 he 7 - Organic amendment 7.50 ab 18U5 DC 6.87 a 2240 a yj. i j u

8 - Untreated solarized 8.50 a 2525 abc 2.93 be 1070 cd 0.31 ab 20.00 ab

Solarized (GI) y

^ 1 C\f\ U. ~ ft ~)1 K 9 - Untreated solarized 6.80 ab 2100 be 2.93 be 1570 abc V.Zj D 1 J.6U aD c\ in ok 10 - Organic amendment 7.80 ab 3280 abc 4.73 ab 1350 be u.j / ao ca

Factorial effects Z ** Solarization NS * NS NS NS * *** •* *** Chemicals ** NS *** Solarization x Chemicals NS NS NS NS NS

Contrasts ** 7x 10 NS NS NS NS NS 8x9 NS NS NS NS NS NS 7+8x9+10 NS NS NS NS NS NS 1+2x5+6 * * NS * NS NS

1 Methyl bromide plus 33% chloropicrin. u Gall index was based on a scale of 0 to 10, in which 0 = 0 galls, 1 = 10 % of roots with galls, 2 = 20 % of roots with galls, 3 = 30 % of roots with galls, and so on until 10 = 100 % of roots with galls. v Values are means of six replicates; means within columns followed by the same

letter do not differ at P < 0.05 according to Duncan's multiple-range test. w Actual means are shown, but values were transformed using log 10 (x + 1) before comparison with Duncan's multiple-range test. x Solarization under clear, gas-permeable polyethylene (PE) plastic film. y Solarization under clear, gas-impermeable (GI) polyethylene plastic film. z *, **, *** represent P < 0.05, P < 0.01, and P < 0.001, respectively, and NS = no significant differences at P < 0.05 according to factorial and non-orthogonal

contrast tests. 78

(P > 0.05) (Tables 3.3,3-4). Differences were not significant among other treatments in

both tests.

Few or no endospores of P. penetrans were produced in females formed in tomato

roots in soil fumigated with methyl bromide or chloropicrin in either test (Tables 3.3,

3.4). These treatments resulted in lower percentages of females with endospores than

other treatments in both trials, except for soil amended with cabbage and solarized under

gas-impermeable plastic in both trials. Cabbage plus solarization, using either type of

plastic, resulted in lower percentages of females with endospores when compared with

the untreated control or cabbage in non-solarized soils in both trials. Overall fewer

females had endospores in solarized treatments than in non-solarized treatments in the

first trial (P > 0.001), but not in the second trial. Chemicals reduced the percentages of

females with endospores in both trials (P > 0.001); an interaction between solarization

and chemicals occurred in the first trial (P > 0.01).

Evaluation of the effect of the exposure of soil with Pasteuria penetrans

endospores . with and without organic amendment, to 50 °C . More eggs and juveniles were produced in soil autoclaved and exposed to either 20 or 50 °C than

in any other treatments (P > 0.05) (Tables 3.5 and 3.6). Egg and juvenile production was greatly reduced when autoclaved soil was amended with cabbage at both temperatures. In both trials, the numbers of eggs and juveniles produced in untreated soil at 20 °C were not different than the numbers in untreated soil exposed to 50 °C or than the numbers in untreated soil amended with cabbage and « '

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incubated at 20 °C. Incubating untreated soil with cabbage at 50 °C decreased the

production ofjuveniles in both tests.

Overall, amendment with cabbage reduced egg and juvenile production,

autoclaving soil increased production, and the interaction of cabbage amendment and

autoclaving was significant in both trials (P > 0.01). The interaction of temperature,

autoclaving, and cabbage amendment was significant for juvenile production in both tests

(P > 0.05).

The number of endospores attached per second-stage juvenile in untreated soil did

not differ from that in soil amended with cabbage and exposed to 20 °C, but was lower

than that in soil with cabbage at 50 °C in both trials. The number of endospore per

juvenile in untreated soil at 20 °C was not different from that in soil at 50 °C in both

trials. Autoclaving soil prevented endospore attachment in soil incubated either at 20 °C

or 50 °C in both trials. The disease of nematode females by P. penetrans was evaluated

differentially, according to the developmental stage of the bacterium, as percentage of

females with mature endospores or the percentage of females with bacterial vegetative

structures. Presence of either stage confirmed the presence of the bacterium in the

assessment of the overall percentage of females infected by P. penetrans. In the first

trial, there was no difference between the percentage of females with endospores in untreated soil and in untreated soil amended with cabbage and incubated at 20 °C; in the

second trial, amending soil with cabbage resulted in a higher percentage of females with endospores at 20 °C (P > 0.05) (Tables 3.5,3.6). At 50 °C, the percentage of females with

endospores was lower in the soil with cabbage than in the untreated soil in both trials. In the first trial, there was no difference in percentage of females with endospores between

soil with cabbage incubated at 50 °C and autoclaving soil with or without cabbage prior

to incubation at 50 °C. Temperature, autoclaving, cabbage, and interactions of these

factors were significant in both trials (P > 0.001).

A higher percentage of vegetative structures was produced in females in soil

amended with cabbage and incubated at 50 °C than in all other treatments in the second

test; in the first test there were no differences in amended and non-amended untreated

soils incubated at 50 °C (P > 0.05) (Tables 3.5,3.6). Vegetative structures of P.

penetrans were not produced in females in autoclaved soil, but more females contained

vegetative structures in soil incubated at 50 °C than in soil incubated at 20 °C.

Temperature, autoclaving, and the interaction of these two factors were significant in both

trials, but cabbage and its interaction with the other two factors were significant only in

the second trial (P < 0.05).

Neither vegetative structures nor mature endospores were formed in females of

M. arenaria in autoclaved soils (Tables 3.5,3.6). No difference was detected in the

presence of females with P. penetrans in untreated soils incubated at 20 or 50 °C in the

first trial, but in the second trial incubation of untreated soil at 50 °C resulted in a

reduction of females with the bacterium when compared with soil incubated at 20 °C (P

> 0.05). In soil amended with cabbage, incubation at 50 °C resulted in a reduction of

females with the bacterium when compared with incubation at 20 °C. Percentage of

females with P. penetrans did not differ in untreated soil and soil amended with cabbage

when incubated at 20 °C in the first trial, but more females contained the bacterium in 83

amended soil than in untreated soil at the same temperature in the second trial. When

incubated at 50 °C, untreated soil resulted in a higher percentage of females with P.

penetrans than in amended soil in both trials. Temperature, autoclaving, cabbage, and

interactions among the three factors were significant in both tests for the development of

P. penetrans in nematode females (P < 0.01).

Determination of the thermal inactivation point of P. penetrans endospores in

vitro . Numbers of eggs and juveniles increased drastically in soil incubated at

temperatures higher than 50 °C previous to nematode introduction (Fig. 3.5) and were

positively correlated with increasing temperature, in both tests (Tables 3.7, 3.8). The

number of endospores attached per juvenile decreased greatly in soil treated at

temperatures higher than 50 °C (Fig. 3.5). The percentages of infected females and of

females with mature endospores, in both experiments, decreased with temperatures above

40 °C (Figure 3.6). The presence of vegetative structures of P. penetrans in nematode

females increased pronouncedly when field soil was previously incubated at increasing

temperatures above 40 °C in both tests (Fig. 3.6). Endospores were not produced after

soil treatment at 70 °C in either test (Fig. 3.6). Endospore attachment, percentage of

infected females, and percentage of females with mature endospores were all negatively

correlated with temperature, and percentage of females with bacterial vegetative

structures was positively correlated with temperature (Tables 3.7, 3.8). 84

18000

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Fig. 3.5. Effect of exposure of Pasteuria penetrans endospores in field soil at different temperatures for 1 0 days on the biocontrol of Meloidogyne arenaria; Number of eggs of M. arenaria per root system of tomato (A), number of second-stage juveniles 3 per 100 cm of soil (B), and number of endospores of Pasteuria penetrans attached per second-stage juvenile of M. arenaria (C). 85

100 Vegetative structures Endospores 80

E 60

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30 40 50 60 70

100

80

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Fig. 3.6. Effect of exposure of Pasteuria penetrans endospores in field soil at different temperatures for 10 days on the biocontrol of Meloidogyne arenaria. Percentage of females of M. arenaria filled with endospores or vegetative structures of P. penetrans in the first test (A) and in the second test (B). i i

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Discussion

Bioassay for determining the density of endospores of P. penetrans in field soil .

Upon contact, the endospores of P. penetrans attach to the cuticle of their appropriate

nematode hosts. Endospore attachment may lead to infection by the bacterium and

reduction of nematode reproduction, but at high population densities in soil P. penetrans

may also diminish the ability of the encumbered juvenile to penetrate the roots. Thus, it

is important to determine the density of P. penetrans endospores in soil before

conducting experiments in the field, greenhouse and laboratory. Since no culture medium

has been developed for in vitro cultivation of P. penetrans and it is not possible to

quantify the bacterium density in soil by serial dilution, bioassays provide the best current

method of quantifying P. penetrans in the soil.

The present bioassay is an indirect, but relatively simple, method of estimating

soil density of the bacterial endospores, in which the attachment of endospores to

juveniles of M. arenaria in field soil is correlated with the attachment in soil to which

known concentrations of the bacterium have been added. In this bioassay, considerable

variation in attachment was observed among the 20 juveniles assessed from each

replicate. This supports the findings of Oostendorp et al. (1990) that tests relying on the

movement ofjuveniles through soil for attachment result in variation of attachment,

especially if the endospores are introduced into the soil. Although variation in

attachment occurred in the replicates, it was lower among means of replicates in the same

treatment. These means were used for the calculation of the regression curve. The field

4 density of 3.0 x 10 endospores/g of soil was calculated by entering the average of the 89

observed attachment in the regression equation. Techniques that do not depend on the

movement of the nematode in the soil may reduce the variation in attachment and are

desirable for bioassays.

Evaluation of the effects of soil solarization, organic amendment, and chloropicrin

on root knot of tomato, caused by M. arenaria, in soil naturally infested with P.

penetrans in the field . Methyl bromide was an effective biocide that provided better

nematode control than other treatments. The higher endospore attachment in methyl

bromide-treated plots also may have contributed to the nematode control in non-solarized

plots treated with the fumigant. Attachment in solarized plots treated with methyl

bromide could not be evaluated accurately because of the lack of nematodes for

assessment, which resulted in a low value for this treatment. The lack of statistical

differences in numbers ofjuveniles among treatments is a result of the high variation of

number ofjuveniles in replicates of treatments (Figure 3.2); the uneven distribution of the

initial population of the nematode in the field is probably the cause of the variation. The

higher yield of tomato fruits in fumigated plots resulted from the control of nematode

pathogens and possibly because of a growth promoting effect inherent to the use of

methyl bromide or chloropicrin (Rovira and Ridge, 1979). Differences in the effects of

solarization and cabbage amendment on P. penetrans could not be determined due to the

unequal nematode population and also to the effects of temperature and volatiles on the

nematodes.

Evaluation of the effects of soil solarization, organic amendment, and chloropicrin

on P. penetrans in the greenhouse . Increase of galling after exposure of soil infested with 90

P. penetrans to chemicals (methyl bromide, chloropicrin and volatiles from cabbage

decomposition) indicates that this bacterium is detrimentally affected by certain classes of chemicals. The lower number of eggs in non-solarized soil amended with cabbage in

comparison to amended soil after solarization with both kinds of plastic in the first test and with gas-impermeable plastic in the second test may be explained by the biocontrol of the nematodes by P. penetrans. Reduction in nematode egg production occured when

P. penetrans endospores were not inhibited by toxic volatiles, high temperatures, or the combination of both. The overall increase in the number ofjuveniles in fumigated or

solarized soil supports this hypothesis. The temperatures recorded under solarization

were demonstrated to be deleterious to P. penetrans attachment in Chapter 2, and the effect of temperature on the development of the bacterium will be discussed later in this chapter. Inactivation of P. penetrans by chloropicrin was reported by Nishizawa (1986).

According to Chen et al. (1991); fumigation with chloropicrin or methyl bromide reduces

bacterial populations in soil. Effects of cabbage volatiles on P. penetrans have not been demonstrated and deserve further investigation.

The increase in root weights of tomato plants grown in the greenhouse in soil that was previously fumigated with methyl bromide and solarized or amended with cabbage

and solarized in the field suggests that changes in soil microflora or in soil fertility caused by these treatments lasted more than one crop season. Soil disinfestation enhances plant

growth by altering chemical and biological properties of the soil (Chen et al. 1991). The

~, major mechanisms for chemical changes proposed are an increase in NH4 and N03

+ _ changes in NH4 /N03 ratio, increase in soluble organic matter (mostly fulvic acid), and increases in micronutrient availability. This study supports the hypothesis of increased

fertility after solarization. Solarization and organic amendment appeared to increased Mg

~ and N03 in soil; amending soils with cabbage also appeared to increased K levels. The

interaction of soil amendment with solarization appeared to result in increases of P and

nitrogen forms, but no increase of organic matter was registered in solarized soils, even

after introduction of cabbage. The low C/N ratio of cabbage leaves favored its rapid

mineralization, especially under solarization temperatures. Solarization temperatures

increase populations of plant growth-promoting rhizobacteria (PGPR) (Gamliel et al.,

1989), which also may act in biocontrol of root-knot nematode. Increase in soil fertility,

a possible soil colonization with PGPR, and thermal and chemical inactivation of P. penetrans may have interacted in the increase of root weight in this experiment and

increase of fruit number and fruit weight in the field experiment. The absence of P. penetrans in M. arenaria females from roots grown in soils previously fumigated and the

low numbers of females from amended and solarized soils support the hypothesis that

some chemicals are deleterious to P. penetrans.

Differences in maximum temperatures of solarization and in amplitude of temperature variation between day and night at the two depths suggest that endospores

located 5 cm deep would have suffered more stress than endospores located 20 cm or

deeper in the soil. The low number ofjuveniles in untreated soil solarized with gas- permeable plastic in the first test may be a result of the collection of soil in a layer deeper than soil used in the second test; thus endospores may have been exposed to lower temperatures and remained viable in higher numbers. Determination of the inactivation temperature of P. penetrans is important to the understanding of the role of solarization

on the life cycle of this bacterium.

Evaluation of the effect of exposure of soil with Pasteuria penetrans endospores.

with and without organic amendment, to 50 °C . The higher numbers of eggs and juveniles produced in autoclaved soils in comparison to those in untreated soils after

incubation at either temperature, in both tests, are the result of the inactivation of P.

penetrans endospores by two, 30-minute cycles on consecutive days in an autoclave at

120 °C. The lack of development of P. penetrans in nematode females in autoclaved

soils supports this premise. Reductions of endospore attachment, percentage of nematode

females infected by P. penetrans, and percentage of females with mature endospores

were observed in soil with cabbage after incubation at 50 °C, when compared to

treatments with nonamended soil incubated at this temperature and amended or non-

amended soil incubated at 20 °C, in both the first and second trials. These reductions

may also have been caused by toxic volatiles produced from the cabbage decomposition

or by the increase of temperature of the soil resulting from increased microbial activity

during decomposition of organic matter. Reductions in eggs and juveniles in autoclaved

soil with cabbage compared to autoclaved soil without cabbage in both tests at 50 °C

indicate that residues from cabbage decomposition, which are toxic to nematodes, were

still present in soil at the time of nematode introduction, and that the nematodes were

negatively affected by these compounds. It is also possible that plant-growth promoting

bacteria (PGPR) colonized soil after it was autoclaved at a higher rate when cabbage was

present than when it was omitted; these bacteria then could have acted as biocontrol agents of M. arenaria. A few pseudomonads, possibly introduced with the cabbage into

the autoclaved soil, could have gained an advantage in the temporary "biological

vacuum" by their ability to utilize the relatively large amounts of readily available

organic matter from the decomposing cabbage or from the nutrients released by the death

of much of the soil biomass (Rovira and Ridge, 1979). Brito-Alvarez et al. (1995) found

that compost treatments significantly improved tomato growth, and increased populations

of rhizosphere bacteria exhibiting antagonism towards various fungal plant pathogens.

Plant growth-promoting rhizobacteria have been reported as the cause of reduction of

populations of plant-parasitic nematodes and of the reduction of infection of host plants

by nematodes (Becker et al., 1988; Kluepfel et al., 1993; Oostendorp and Sikora, 1989;

Zavaleta-Mejia and VanGundy, 1982).

The higher percentage of M. arenaria females with vegetative colonies of P. penetrans in soil with cabbage incubated at 50 °C than at 20 °C or in the nonamended

controls at either temperature supports the hypothesis that more toxic volatiles are

released during decomposition of cabbage at 50 °C than at 20 °C. Thus, increased levels

of volatiles or an increase in temperature of soil resulting from increased microbial

activity during decomposition of organic matter, or a combination of the two factors,

appear to be deleterious to the development of P. penetrans in the nematode female and

may have been responsible for reducing the production of mature endospores.

Determination of the thermal inactivation point of P. penetrans endospores in

vitro . The reduction of attachment of P. penetrans endospores after incubation at a daily

cycle of 5 hours at 50 °C or more over 10 days supported the conclusion in Chapter 2 that 94

attachment of endospores to juveniles decreased with increasing temperature and that

little attachment occurred at temperatures above 50 °C. According to Davies and Danks

(1993), N-acetylglucosamine moieties linked to proteins or peptidoglycans, which are extentions of the cell wall of P. penetrans, are responsible for the adhesion of the bacterial endospore to the nematode cuticle. Decreased attachment resulting from

incubation of endospores at high temperatures suggests that N-acetylglucosamine or similar carbohydrates were solubilized or denatured. Proteins and peptidoglycans, or the bounds that link N-acetylglucosamine to them, also may have been harmed by increasing temperature.

Not only attachment, but also the percentage of infected females and percentage of females with mature endospores decreased after incubation at a daily cycle of 5 hours at 50 °C or more over 10 days. These decreases and the increased percentage of females with vegetative structures of P. penetrans that occurred with increasing temperatures

demonstrated that portions of the bacterial life cycle were retarded or terminated by long term exposure to temperatures higher than 50 °C for 10 days. Pasteuria penetrans has been related to other endospore forming bacteria, such as Bacillus spp. which can withstand 100 °C for many minutes (Stelow, 1992), due to similarities in morphological

and biochemical features (Stirling, 1984; Stirling et al., 1990). These similarities and the attachment of P. penetrans endospores to nematode cuticle after short term treatment of

endospores with high temperatures (Stirling et al., 1986) resulted in a belief that this

bacterium is resistant to high temperatures. However, Williams et al. (1989) determined

heating endospores for as little as 5 minutes reduced 98% of the infection of nematode females by P. penetrans. The relatively low resistance of P. penetrans to heat also was

reported by Dutky and Sayre (1978), who observed that infection by P. penetrans was

prevented by incubating endospores at 80 °C for 30 min. According to Mallidis and

Scholefield (1987), there is a correlation between spore heat resistance and dipicolinic

acid content. This chemical constitutes 5-15% of the dry weight of true endospores, but

endospores of P. penetrans have a much lower concentration (0.96%); this difference

may explain why P. penetrans endospores are less resistant to heat than other endospore-

forming bacteria (Williams et al., 1989).

In conclusion, differences in the effects of solarization and cabbage amendment

on P. penetrans under field conditions could not be determined because of the unequal

nematode population and also because of the limiting effects of temperature and volatiles

on the nematodes. The introduction of untreated juveniles, under greenhouse conditions,

into soil treated in the field allowed the detection of deleterious effects of cabbage

volatiles and solarization temperatures on the development of P. penetrans present in the

first 20-cm depth of field soil. At deeper layers in the soil, moderate increases in

temperature by solarization may increase both the rate of development of females of

Meloidogyne spp. and the production of P. penetrans endospores (Walker and Wachtel,

1988). The negative effects of high temperatures on the development of P. penetrans

were confirmed by another greenhouse experiment in which portions of the bacterial life

cycle were retarded or terminated by long term exposure to temperatures higher than 50

°C for 10 days. In another greenhouse experiment, deleterious effects of volatiles

released during cabbage decomposition on P. penetrans were observed in soil infested with the bacterium and amended with cabbage, and incubated at 50 °C, when compared to infested treatments with nonamended soil incubated at this temperature and infested, amended or non-amended soil incubated at 20 °C 4

CHAPTER THE EFFECTS OF FUMIGANT NEMATICIDES ON PASTEURIA PENETRANS AND

ITS INFECTIVITY TO MELOIDOGYNE ARENARIA RACE 1 IN TOMATO

Introduction

Nematicide treatment is considered one of the most reliable means of controlling a

wide variety of nematodes. Chemicals are used mainly to control nematode populations

in soil before planting annual crops. Application methods have been developed for

applying about 25 highly effective nematicides, which were used on approximately

700,000 hectares in the United States (Johnson and Fassuliotis, 1984). The two broad types of nematicides for control of nematodes on vegetable crops are soil fumigants and

non-fumigant nematicides. Soil fumigants are liquids that are injected below the soil

surface and volatilize to kill nematodes. Vapors from fumigants diffuse through the soil,

dissolve in soil water, and penetrate the cuticle of the nematodes. Fumigation is the most

effective method of chemical plant disease control, and fumigation with methyl bromide remains the only generally accepted method of eradicating soilborne pathogens and

weeds in many countries. It is used as a pre-plant fumigant in greenhouse soils and in field situations on high value cash crops (Minerbi, 1992). In four of the most important tomato growing regions in the world, Florida, California, Italy, and Israel, 80% of the fresh tomato crop is grown on soil fumigated with methyl bromide to control nematodes,

Fusarium and Verticillium wilts, and corky root (Crop Protection Coalition, 1995).

97 The elimination of soil fumigation with methyl bromide for tomato production by

the year 2001, due to its influence in the depletion of the ozone layer, will result in an

estimated 20% decrease in yield (Crop Protection Coalition, 1995). Research is being

conducted by governmental and academic institutions, as well as by the private sector, to

ensure that alternative materials and methods will be proven viable and available to the

agricultural community before methyl bromide is phased out. The potential alternatives

for methyl bromide's preplant uses can be divided into chemical and nonchemical. The

chemical alternatives, according to the United States General Accounting Office (1995),

include 1 ,3-dichloropropene, dazomet, metham soudium, sodium tetrathiocarbonate,

formalin or formaldehyde, chloropicrin, and nonfumigant nematicides. The nonchemical

alternatives include the use of steam, soil solarization, organic amendments, crop

rotation, disease-resistant cultivars, and biological control.

Pasteuria penetrans is an obligate parasite of nematodes that has significant potential

as a control agent (Davies et al., 1991). The use of P. penetrans as a biological control

agent in combination with other management practices, especially nematicides, is of great

interest to nematologists. Stirling (1984) claimed that infection of M. javanica by P. penetrans after treatment in vitro was not affected by the nematicides 1,3-D or DBCP.

However, his conclusion must be qualified because the author did not compare the

percentage of females infected with P. penetrans in fumigated soils with the percentage

in non-fumigated soil. Brown and Nordmeyer (1985) demonstrated a synergistic

reduction of root galling by M. javanica with carbofuran or aldicarb combined with P.

penetrans. A possible explanation for the synergism is that low concentrations of 99

carbamate nematicides, rather than killing the nematodes, stimulate the movement and orientation of the nematodes toward host roots; thus, the probability of nematode contact with bacterial endospores is increased. Brown and Nordmeyer (1985) did not report on

the reproduction of the bacterium within nematodes in the nematicide treated soils, but it

is expected that it was reduced compared with that obtained in a non-treated soil, since the galling was greatly reduced in the presence of the nematicides. Walker and Wachtel

(1988) observed a reduction in parasitism of M. javanica juveniles by P. penetrans following the use of nonfumigant nematicides. Organophosphate and carbamate

nematicides at high concentrations decreased the mobility of nematodes, and the most likely explanation of the reduction in infection was a decreased probability of contact between nematodes and passive endospores of the parasite. To date, no detailed study on the direct effects of nematicides on P. penetrans has been conducted. Thus, the objective of this work was to investigate the effects of fumigant nematicide alternatives to methyl

bromide on P. penetrans and its infectivity to M. arenaria race 1 in tomato.

Material and Methods

Field Experiment 1 . The experiment was conducted in a field at the University of

Florida Agronomy Farm, Alachua County, Florida. The treatments were arranged in a randomized, complete block design, with six replications per treatment. The plots were 2

m wide and 12 m long, and the bed width was 1 m. The treatments included: 1) methyl bromide plus 33% chloropicrin at 392 kg/ha, 2) chloropicrin at 375 kg/ha, 3) metham sodium at 936 liters/ha, 4) 1,3-dichloropropene (1,3-D) plus 16.5% chloropicrin at 327 .

100

liters/ha, 5) 1,3-D plus 16.5% chloropicrin at 203 liters/ha, 6) 1,3-D plus 25% chloropicrin at 224 liters/ha, 7) 1,3-D plus 35% chloropicrin at 259 liters/ha„ and 8) the untreated control.

A field suppressive to M. arenaria was deep-plowed and cultivated before the

treatment applications. Preplant fertilizer, 13-1.8-1 1 (N-P-K), was applied broadcast over

the area at 450 kg/ha. Soil from each plot was sampled for the initial population density

(Pi) of M. arenaria, before the treatment applications in March, by removing and combining 12 soil cores (2.5 cm in diameter and 20 cm deep) from each plot. The fumigants were applied on preformed beds with a closed-system, mulch-laying injection rig (Kennco, Ruskin, Florida). Nitrogen gas was used to pressurize the fumigant cylinders. The bed press had three chisels spaced 30 cm apart, and were adjusted to

deliver the fumigant at 20 cm deep beneath the final bed surface. The mulch (1 .25-mm- thick, black polyethylene plastic,) was applied simultaneously when the fumigants were applied. Tomato seedlings, cv. Agriset 761 (Speedling, Ruskin, Florida), were transplanted to beds, with a plant spacing of 0.5 m, 3 weeks after the treatments.

Irrigation was provided through drip tubing. A double-wall single drip tube (Chapin

Livinwall, Watertown, NY) with emitters spaced 30 cm apart and a flow rate of 1 .9 liters per minute/30 m of row was placed under the plastic mulch before planting. Postplant

liquid fertilizer, 12.3-0-1 1.6 (N-P-K), was applied through drip irrigation in 10 equal

applications at weekly intervals at 1 1 .2 kg/ha.

Twelve soil cores (2.5 cm in diameter and 20 cm deep) were collected and bulked

after the final harvest to determine final population densities of M. arenaria race 1 5

101

3 Second-stage juveniles were extracted from 100-cm subsamples of soil by centrifugal flotation (Jenkins, 1964). Six root systems were removed from each plot, and plants were

rated for root galling on a scale of 0 to 1 0, in which 0 = 0 galls, 1 = 1 0% of roots with galls, 2 = 20% of roots with galls, 3 = 30% of roots with galls, and so on until 10 = 100% of roots with galls (Barker et ah, 1986). Tomato yields were recorded. Data were subjected to analyses of variance and Duncan's multiple range test using SAS software

(SAS Institute, Cary, NC).

Greenhouse Experiment 1 . The experiment was conducted in the greenhouse at the University of Florida campus. Nematodes used in this experiment were grown on

tomato plants in the greenhouse as described in Chapter 2. The treatments were arranged in a randomized, complete block design, with six replications per treatment. Soil from

each of the 48 plots used in field experiment 1 was collected 1 5 days after the application of the chemicals and stored in a plastic bag in the shade. Three hundred and fifty cubic

centimeters of soil from each bag were spread on a paper plate and allowed to dry for 1 days on benches in the greenhouse without ventilation. After this period, the dry soil was placed in sterilized, 10-cm-diameter clay pots on a bench in a room at 24 °C. The moisture of the soil in the pots was adjusted to field capacity, determined by the point at which the added water stopped dripping from the hole in the bottom of the pot. After 3 days, 2,400 1-to 4-day-old juveniles of M. arenaria were added in 10 ml of water to five holes, 5 cm deep, in the soil of each pot, and incubated for 4 days. One 10-day-old seedling of dwarf cherry tomato, cv. Florida Petite (Florida Foundation Seed Producers,

Greenwood, FL) was transplanted into each pot. The plants were fertilized once a week 102

with 18-18-21 (N-P-K) and micronutrients (Miracle-Gro for tomato; Stern's Miracle-Gro

Products, Port Washington, NY) at 0.05 g/pot, and insects were controlled as needed using recommended insecticides.

3 Second-stage juveniles were extracted from 100 cm of soil by centrifugal

flotation (Jenkins, 1 964) and counted with the use of an inverted microscope. The number of P. penetrans endospores attached per juvenile in each sample was determined as the average attachment on 20 observed juveniles. Eggs were extracted and counted,

and plants were weighed and rated for root galling on a scale of 0 to 1 0, as described

above (Barker et al., 1986). Twenty females of M. arenaria were picked from each root system, and each of them was crushed separately in 2.5 ul of lactophenol plus 1% methyl

blue (v/w) (Sigma Chemical, St. Louis, MO) under a coverslip and examined microscopically to determine the percentage of females infected by P. penetrans. Data were subjected to analyses of variance and Duncan's multiple range test using SAS software (SAS Institute, Cary, NC).

Greenhouse Experiment 2 . The experiment was conducted in the greenhouse at the University of Florida campus. Nematodes used in this experiment were grown on tomato plants in the greenhouse as described in the Chapter 2. The treatments were arranged in a randomized, complete block design, with six replications per treatment.

Soil from each plot of the untreated control, methyl bromide and chloropicrin treatments

from field experiment 1 was collected and stored in a plastic bag in the shade. Three hundred and fifty cubic centimeters of soil from each bag were spread on a paper plate in

a thin layer and allowed to dry for 15 days in a greenhouse without ventilation at 30-40 103

°C to kill the nematodes. After this period, the dry soil from each plot was placed in six, sterilized, 10-cm-diameter clay pots per treatment and maintained on a bench in a room at

24 °C; non-dried soil from each treatment also was placed in six, sterilized, 10-cm- diameter clay pots per treatment and transferred to the same room at 24 °C. The moisture of the soil in the pots was adjusted to field capacity. After 3 days, 2,000 1-to 4-day-old juveniles of M. arenaria were added in 10 ml of water to five holes, 5 cm deep, in the

soil of each pot, and incubated for 4 days. One 10-day-old seedling of dwarf cherry tomato cv. Florida Petite (Florida Foundation Seed Producers, Greenwood, FL) was

transplanted into each pot. The plants were fertilized once a week with 1 8-18-21 (N-P-K) and micronutrients (Miracle-Gro for tomato; Stern's Miracle-Gro Products, Port

Washington, NY) at 0.05 g/pot, and insects were controlled as needed using recommended insecticides. The experiment was repeated once with a 15 -day interval

between inoculation of plants with nematodes in the first and second trial.

At harvest, the shoots of the plants were cut at the soil surface level, and the contents of each pot were stored in an individual plastic bag in a cold room at 5 °C until they were

processed. The root system of each plant was washed to remove the soil, the excess water was removed, and the roots were weighed. The gall index was determined using a scale of 0 to 10 scale, as described above (Barker et ah, 1986). The eggs were collected after dissolving the gelatinous matrices containing eggs with 0.5% NaOCl. Each root system was placed into a 50-ml centrifuge tube with 30 ml of an aqueous solution of

0.5% NaOCl. The tubes were closed and vigorously shaken for 30 seconds. The egg suspension was poured onto a sieve with 25-um-pore openings (500-mesh), rinsed with 104

tap water, and collected in another centrifuge tube. The process was repeated twice, using tap water instead of NaOCl solution. The roots were cut into pieces 3 to 5 cm long and placed into a centrifuge tube with 30 ml of an 8:1 (v/v) aqueous solution of commercial Cytolase PCL5 (Genecor International, Rolling Meadows, IL). The tubes were placed in a automatic shaker (Eberback Coorporation, Ann Arbor, MI) for 24 hours at room temperature (approximately 24 °C). Softened roots were placed on a sieve with

600-um-pore openings (30-mesh) over a sieve with 1 80-um-pore openings (80-mesh) and sprayed with a vigorous stream of tap water. The dislodged females and root debris were transferred to a beaker by washing the sieve from the back with a jet of water. The females were separated from the root debris with a Pasteur pipette under a stereomicroscope and examined for P. penetrans infection with an inverted compound microscope. Numbers of eggs per root, gall index, and percentage of infected females were recorded. The second-stage juveniles were extracted from soil using centrifugal flotation (Jenkins, 1964). The number of attached endospores was counted on each of 20

randomly selected juveniles per replicate using an inverted microscope at a magnification

of x400. Data were subjected to analyses of variance and Duncan's multiple range test using SAS software (SAS Institute, Cary, NC).

Field Experiment 2 . A second field experiment was conducted in 1 996 to

evaluate the suppressiveness of P. penetrans to M. arenaria race 1 in the same field site used in experiment 1. The plots were 2 m wide and 12 m long, and the bed width was 1.2 m. The treatments included: 1) methyl bromide plus 33% chloropicrin at 392.3 kg/ha, 2) chloropicrin alone at 375.5 kg/ha, and 3) the untreated control. The treatments, with six

replications, were arranged in a randomized, complete block design.

The field was deep-plowed and cultivated on 27 February. Fertilizer, 10-10-10

(N-P-K), was applied broadcast over the area used for raised beds at 784.6 kg/ha and

incorporated 10 cm into the soil with a rotatil. After the fertilization, the soil was

sampled for the initial population density of M. arenaria by removing and combining 12

soil cores (2.5 cm in diameter and 20 cm deep) from each of the 1 8 plots. On 29

February, the nematicides were applied on preformed beds with a closed-system, mulch-

laying injection rig (Kennco, Ruskin, Florida). Nitrogen gas was used to pressurize the

fumigant cylinders. The bed press had three chisels spaced 30 cm apart, and were

adjusted to deliver the fumigant at 20 cm deep beneath the final bed surface. The mulch

(1.25 mm thick, black polyethylene plastic) was applied simultaneously when the

fumigants were applied. Irrigation was provided through drip tubing. A double-wall,

single drip tube (Chapin Livinwall, Watertown, NY) with emitters spaced 30 cm apart

and a flow rate of 1 .9 liters per minute/30 m of row was placed under the plastic mulch

before planting. Postplant liquid fertilizer, 12.3-0-1 1.6 (N-P-K), was applied through

drip irrigation in 10 equal applications at weekly intervals at 1 1.2 kg/ha. Two cultivars of

tobacco were used in the experiment. The cultivar Coker 371 -Gold was planted on the

north side of the beds and the cultivar K-326 on the south side of the beds. Neither of the cultivars are resistant to M. arenaria.

For estimation of the final nematode population densities, samples from the root zone of each of six plants per plot were collecteded on 17 July. The soil cores were 2.5 106

cm in diameter and 20 cm deep. Second-stage juveniles were extracted from 100-cm3 subsamples of soil by centrifugal flotation (Jenkins, 1964).

The root systems of 10 tobacco plants from each replication of each treatment, five from each cultivar, were rated for root galling on 17 July on a scale of 0 to 10 (Barker

et ah, 1986). Females of M. arenaria were picked from root systems that had galls and observed under a microscope for the presence of attached P. penetrans endospores.

Results

3 Field Experiment 1 . Sixty second-stage juveniles/ 100 cm of soil, in average, were present in each field plot by the time of the harvest. Ninety six percent of the juveniles extracted from the field plots had endospores of P. penetrans attached on their cuticles, at an average of 34 ± 25 endospores/juvenile. No differences in number of endospores attached per juvenile and in gall index occurred among any of the treatments

3 (P > 0.05) (Table 4.1). The number ofjuveniles per 100 cm of soil was lower in plots fumigated with methyl bromide plus 33% chloropicrin than in plots treated with 1,3-D plus 33% chloropicrin, but no differences were observed among other treatments.

Number of fruit and weight of fruit were lower in soil treated with chloropicrin alone than

in the treatment with 1,3-D plus 16.5% chloropicrin at 203 liters/ha; no differences in number of fruit or weight of fruit were observed among any other treatments. Greenhouse Experiment 1 . Higher gall indices were observed in plants growing

in soil from plots treated with methyl bromide and chloropicrin than from those treated with metham sodium or the untreated control (P > 0.05) (Table 4.2). The number of

3 juveniles per 100 cm of soil was lower in the untreated control than in the other treatments, except that the number was not different from that in soil treated with metham sodium. Inversely, the percentage of nematode females with P. penetrans endospores was higher in the untreated control and in the soil treated with metham sodium than in the other treatments. No differences in number ofjuveniles per 100 cm of soil or in percentage of female nematodes with P. penetrans endospores were observed among the other treatments. No differences in number of eggs per root system, root weight, or number of endospores attached per second-stage juvenile were observed among any of the treatments.

Greenhouse Experiment 2 . No differences in endospore attachment among

treatments occurred in the first test, but attachment was lower in chloropicrin treatment

than in the untreated control in the second test (P > 0.05) (Table 4.3). The percentage of

females with P. penetrans endospores was higher in the untreated soil than in fumigation

treatments in both dried and non-dried soils, and in both tests. No endospores were observed in females in fumigated soil. The number of eggs produced in the root systems was higher in fumigated soils than in the untreated control when soil had been dried in

test 1 and in both dried and non-dried soil in test 2. There was no difference in the

number of eggs produced in the methyl bromide and chloropicrin treatments in the first test, but more eggs were produced in chloropicrin treated soils than in methyl bromide 108

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3 treated soils in the second test. Number ofjuveniles per 100 cm of soil was higher in chloropicrin treated soils in both dried and non-dried soils in both tests. In both tests, no differences in number ofjuveniles were observed between treatments with methyl bromide and the untreated control when soil was not dried, but more juveniles were produced in methyl bromide treated soil than in the untreated control when soil was dried.

No differences were observed in the gall indices among treatments in the first trial, but they were lower in untreated soil than in soil fumigated with methyl bromide or

chloropicrin in the second trial. Root weight was lower in the untreated control than in the fumigation treatments in dried soils in both tests and in non-dried soil in the second

test.

Field Experiment 2 . Juveniles were present in only six of the 1 8 beds sampled at

population densities of 1, 1, 2, 6, 9, and 21 juveniles/100 cm of soil. Pasteuria penetrans endospores were attached on most of the juveniles, and the attachment rate

varied with locality sampled from 1 endospore per juvenile in the plot with 6

3 juveniles/100 cm of soil to 20 endospores per juvenile in the plot with 9 juveniles/100

cm of soil. By the end of the tobacco season, juveniles were found in only four of the 18 beds with tobacco. Three of the beds were untreated controls and the numbers of

juveniles/100 cm of soil in them were 1, 14, and 25. An average of 12 endospores were attached to each juvenile in the control plots. One methyl bromide treated plot had a

nematode population density of 1 8 juveniles/1 00 cm of soil, and the average attachment was 7 endospores/juvenile. 112

The average of the gall indices determined for 1 0 root systems of tobacco per plot was only 0.2 in the untreated controls, 0.02 in the methyl bromide treated plots and 0.05 in the chloropicrin treatment. Few galls were produced in either cultivar of tobacco.

Females were extracted from the few galled roots encountered in the field and assessed for the presence of P. penetrans. Twelve females were extracted from roots from each of two untreated control plots. The percentages of females with endospores from these plots were 58% and 75%. Twenty females were extracted from one methyl bromide treated plot and only one female had endospores (5%). Sixteen females were extracted from a chloropicrin treated plot and none of them contained endospores of P. penetrans. The low numbers ofjuveniles, galls and females did not allow the performance of statistical analysis.

Discussion

Field Experiment 1 . Although nematodes were present in considerable numbers in most of the plots at the end of the tomato season, except in methyl bromide treated plots, few galls developed in the tomato roots. High numbers of endospores attached to

juveniles in all treatments may have reduced the penetration ofjuveniles into the tomato

roots. Reductions in plant infectivity have been observed when nematodes had 1 5 to 20 endospores attached to their cuticles (Brown and Smart, 1985), and, most of the juveniles extracted from field soil in this study each had more than 30 endospores attached to their cuticles. Chemical treatments did not affect the endospore attachment to the juveniles under field conditions. Effects of chemicals on the development of the bacterium could 113

not be evaluated because few females completed their life cycles, and because effects of the chemicals on the nematode host could have changed indirectly the bacterial

development. These results indicate that the field site used in this study may have been

suppressive to M. arenaria race 1 , and that P. penetrans probably played an important role in the nematode suppressiveness.

Greenhouse Experiment 1 . Introducing untreated, second-stage juveniles into

field soil with a natural population of P. penetrans that had been treated under field conditions and maintained in the greenhouse allowed the evaluation of the effects of nematicides on endospores of P. penetrans without interference of the effects of nematicides on the nematode hosts. Higher gall indices in methyl bromide 67-33 and chloropicrin treatments compared to untreated soil and metham sodium treated soil indicated that methyl bromide and chloropicrin reduced attachment of the endospores to the introduced juveniles. Thus, more juveniles with fewer endospores attached would have been able to penetrate the tomato roots in these treatments than in the untreated control, which had the highest levels of attached endospores. Although there were no differences in the number of eggs produced among any of the treatments, fewer juveniles were detected in the untreated control than in any of the nematicide treatments, except the metham sodium treatment. Percentages of females with bacterial endospores were higher

in the untreated control and in the metham sodium treated soil than in soil treated with other nematicides. Disease in M. arenaria females caused by P. penetrans was inversely related with the gall index and number ofjuveniles in the soil in this study, which indicates that P. penetrans suppresses the nematode and that chemical nematicides, 114

except for methan sodium, are detrimental to the development of P. penetrans in the nematode host.

Greenhouse Experiment 2 . Attachment of P. penetrans endospores to juveniles was not affected by fumigation with methyl bromide or chloropicrin, except in the non-

dried soil in test 2, in which attachment in the chloropicrin treatment was lower than in

untreated soil. Attachment was highly variable among juveniles, and differences may have resulted from experimental error. Although reductions in attachment were not constant, an effect of methyl bromide and chloropicrin on the endospore production in

females was detected in both dried and non-dried soil. These results support the findings by Nishizawa (1986), that chloropicrin had a bactericidal effect on P. nishizawae and reduced the percentage of cysts of soybean cyst nematode filled with P. nishizawae from

82% to 5% under field conditions. The results also support the conclusion in Chapter 3

that P. penetrans is detrimentally affected by some chemicals.

The low number of eggs in dried soil from the untreated control in test 1 and in both dried and non-dried soils in test 2 may be explained by the inhibition or impediment of reproduction of the nematode when colonized by P. penetrans, which supports earlier studies by Mankau (1980) and Minton and Sayre (1989). The fact that fewer juveniles were produced in untreated soil than in soil fumigated with methyl bromide or chloropicrin when the soil was dried, but that there was no difference between the untreated control and the methyl bromide treatment in non-dried soil, may have been due to the selective toxicity of chloropicrin in the interruption of P. penetrans development.

The mixture methyl bromide with 33% chloropicrin had less effect on P. penetrans than 115

chloropicrin alone. According to Chen et al. (1991), fumigation with chloropicrin or

methyl bromide reduces bacterial populations in soil. While methyl bromide affects

Gram-positive and Gram-negative bacteria, chloropicrin primarily affects Gram-positive

bacteria, such as P. penetrans. Methyl bromide, at commercial dosages, is a relatively

weak bactericide. Its bactericidal effect may be enhanced by the addition of 33% chloropicrin, as indicated in this study.

Pasteuria penetrans was more effective in controlling root-knot nematode after the fumigated soil was air-dried. This may have resulted form the disruption of bacterial sporangia with drying, which may increase the attachment of endospores to the nematode.

With bacterial efficacy enhanced by soil drying, differences between untreated endospores and endospores fumigated with methyl bromide or chloropicrin became more evident.

The lower root galling in the untreated control than in fumigation treatments in the second test indicate that fumigation may reduce galling in soils with high densities of

P. penetrans, but may augment it in soil with low densities of P. penetrans (Chapter 3).

In the later case, the increase of PGPR populations after fumigation may have a stronger

effect than P. penetrans in reducing galling (Chapter 3).

Field Experiment 2 . The low density of the final population of M. arenaria in the field, even after one season of a susceptible crop, and the extremely low gall indices suggest that the soil had become suppressive to the nematode. The presence of endospores of P. penetrans attached to juveniles before and after the crop season indicates that this bacterium may be the cause of the nematode supressiveness. The 116

presence of endospores attached to juveniles in plots fumigated with methyl bromide indicates that this chemical does not prevent bacterial attachment, but the low percentage of females filled with endospores in this treatment indicates that methyl bromide may be reducing bacterial development in the nematodes. This reduction and the lack of bacterial

reproduction in chloropicrin treated soil supports observations in the greenhouse

experiments that chloropicrin and methyl bromide have a detrimental effect on the life cycle of P. penetrans.

The nematicides evaluated in this study had differential effects on P. penetrans.

Chloropicrin alone or in combination with methyl bromide was highly detrimental to P.

penetrans because it inhibited endospore formation. Formulations of 1,3 -D and chloropicrin had moderate effects on the bacterium. The chloropicrin present in these formulations may have been responsible for the bactericide effect on P. penetrans.

Metham sodium was the only nematicide harmless to the bacterium and was also the only

chemical not containing chloropicrin. Further studies are necessary to determine if

chloropicrin is the most important detrimental fumigant to P. penetrans. CHAPTER 5 SUPPRESSION OF MELOIDOGYNE ARENARIA BY PASTEURIA PENETRANS IN THE FIELD

Introduction

Soil suppressiveness occurs when soilborne disease is absent, or is not severe, in the presence of the pathogen (Hornsby, 1983). Naturally occurring soil microorganisms are responsible for numerous examples of biological control (Baker and Cook, 1974).

There are many reports of the occurrence of parasites and predators of nematodes in cultivated soils, of which fungi and the bacterium Pasteuria penetrans (Thorne) Sayre and Starr are the most important (Stirling, 1988).

It has been difficult to determine the extent of the role played by P. penetrans in the suppression of nematode deseases because the bacterium has not been cultured or critically differentiated in soil populations. Stirling (1984) devised a pot experiment with soil from a vineyard in South Australia to determine if P. penetrans was the causal agent

of the reduction of root-knot nematodes in the field. Field soil was autoclaved or treated with a nematicide and then infested with nematodes. Autoclaving resulted in the inactivation of P. penetrans and in a higher multiplication of the nematodes than in

untreated soil. Chen (1994) determined that microwaving soil for 4 minutes/kg of soil eliminated most soilborne fungi but not soilborne bacteria; this technique may be useful

to elucidate the biological nature of suppressiveness in field soil. To determine the role

117 118

of antagonists in typical field situations, methods of quantifying levels of parasitism are needed and must be developed (Stirling, 1988).

Pasteuria penetrans has been observed in nematode suppressive soils (Bird and

Brisbane, 1988; Chen et al., 1995, Dickson et aL, 1994; Minton and Sayre, 1989;

Weibelzahl-Fulton et al., 1996) and has suppressed nematodes in greenhouse and

microplot experiments (Brown et al., 1985; Daviesetal., 1991;De Leij etal., 1992;

Minton and Baujard, 1990; Oostendorp et al., 1991 ; Rodriguez-Kabanaet al., 1986;

Stirling, 1984). After an isolate of P. penetrans acquired from a peanut {Arachis hypogeae L.) farm in Williston, Florida was introduced into soil at the Green Acres

Research Farm of the University of Florida, Alachua County in the summer of 1987 for microplot studies, soil that was conducive to root-knot became suppressive to the disease

within 3 years (Oostendorp et al. 1991). Meloidogyne javanica and M. incognita race 1 also were suppressed in a tobacco field located 300 m distant from the 1991 introduction area, and P. penetrans was determined to be the primary cause of the nematode suppression (Chen, 1994; Weibelzahl-Fulton et al, 1996).

Race 1 of M. arenaria caused yield losses in peanut (Arachis hypogeae L.) in a

nematicide trial conducted in 1 994 in a site 1 00 m distant from the microplot area used by

Oostendorp et al. (1991), but a few endospores were observed attached to second-stage juveniles collected from the field site at the end of the season (D. W. Dickson, personal

communication). A tomato (Lycopersicon esculentum L.) cultivar, Agriset 761, which is

not resistant to M. arenaria race 1, was planted in the same area in 1995, in an experiment to evaluate fumigant nematicides as alternatives to methyl bromide for 119

management of root-knot nematodes. The low indices of root galling and the high number of endospores attached to juveniles by the end of the crop season suggested that

P. penetrans could be causing soil supressiveness. Thus, soil from this site provides a source of nematode-suppressive soil for elucidation of the role of P. penetrans in disease suppression. The objective of this study was to evaluate the suppressiveness of P. penetrans to M. arenaria in field soil under greenhouse conditions.

Material and Methods

Determination of P. penetrans density in the infested soil . The density of the bacterial endospores was estimated with a bioassay in which the attachment of the endospores to juveniles of M. arenaria in field soil was compared with the attachment in

soil to which known concentrations of the bacterium had been added. Two kilograms of

infested soil were placed in a plastic bag and shaken in circular movements for approximately 5 minutes to homogenize the endospore distribution. One kilogram of this soil was autoclaved twice for 30 minutes at 120 °C on consecutive days to kill nematodes and P. penetrans endospores. Both autoclaved and nonautoclaved soils were allowed to dry for 15 days in layers of 0.5 cm on paper trays placed on benches in a greenhouse with no ventilation at temperatures varying from 30 to 40 °C; thus, both soils were equally dry and did not contain nematodes. One gram of soil and 200 ul of distilled water were placed in each of five, 1.5-ml microcentrifuge tubes (Conical-bottom tubes, Fisher

Scientific, Pittsburgh, PA), which had been washed with detergent to prevent juveniles

and endospores from adhering to the tube walls. Tubes with the sterilized soil received P. 120

penetrans endospores in 500 ul of water at concentrations that resulted in densities of 5 x

4 5 6 10 , 5 x 10 , or 5 x 10 endospores/g of soil. The endospores were incubated in the soil at room temperature (approximately 24 °C) for 3 days. The experiment consisted of four

treatments (the soils infested at three different concentrations and the non-autoclaved,

naturally infested soil) and five replicates per treatment. One hundred and eighty, 1- to 3- day-old juveniles of M. arenaria were introduced to each centrifuge tube in 300 ul of water. The tubes were placed on an automatic shaker (Eberback Coorporation, Ann

Arbor, MI) for 24 hours. Juveniles were extracted using Cobb's decanting and sieving method (Hooper, 1986). Each tube was shaken, and after the sand was allowed to settle for a few seconds, the nematode suspension was poured onto a sieve with 25-um-pore openings (500-mesh). The process was repeated twice for each tube, and the nematodes

caught on the sieve were rinsed with tap water and collected in a 5. 5 -cm-diameter petri dish. Twenty juveniles per replication were randomly chosen for assessment of the number of P. penetrans endospores attached per juvenile. The endospore density in the field was determined by comparison with soil in which known concentrations of the bacterium were added. The data were subjected to regression analyses with SAS software (SAS Institute, Cary, NC).

Suppressiveness Test . An experiment was conducted in the greenhouse to evaluate the role of P. penetrans in the suppressiveness of soil to M. arenaria by P.

penetrans. The origin of the nematodes is the same as described in the Chapter 2. The field site, from which samples of soil infested with P. penetrans endospores were collected, was the same field used in the study on the effects of chemicals on P. penetrans 121

(Chapter 4). Soil samples were combined and used in the following four treatments: 1) untreated control, 2) air dried soil, 3) soil microwaved for 4 minutes/kg of soil (Ferris,

1984) at full power (625 W; 2,450 MHz), and 4) soil subjected to two, 30-minute cycles on consecutive days in an autoclave at 120 °C. The experiment was repeated once. The

soil for the second treatment, air-dried soil, was spread in thin layers on paper plates in

the greenhouse and allowed to dry at 30 °C to 40 °C for 1 5 days to kill the nematodes.

Each replicate in the microwave treatment consisted of one plastic bag containing 0.5 kg

of soil infested with P. penetrans endospores. In the first trial, the six replicates were placed side by side in the microwave oven and the time of heating was calculated according to the hypothesis that the effect of microwave treatment on soil

microorganisms is proportional to treatment time divided by soil weight (Bollen, 1969).

Since six replicates of 0.5 kg each were to be treated simultaneously and each kilogram of

soil should have been treated for 4 minutes, the time was adjusted for 12 minutes (6 x 0.5

x 4). After observing the deleterious effect on P. penetrans in the first trial, the

methodology was changed in the second trial, and each 0.5-kg sample of soil was microwaved individually for 4 minutes. The soil moisture in both trials was

approximately 9%. Soil from all treatments was placed in 1 0-cm-diameter clay pots. The treatments were arranged in a completely randomized block design, and each treatment had six replications. The experiment was repeated once with a 15-day interval between

inoculation of plants with nematodes in the first and second trial. Five thousand eggs of

M. arenaria were introduced into each pot, and one, 15-day-old tomato seedling cv.

Rutgers was planted in each pot. Both trials were run for 45 days. The shoots of the 122

plants were cut at the soil surface level and the contents of each pot were stored in a individual plastic bag in a cold room at 5 °C until they were processed. The root system

of each plant was washed to remove the soil, the excess water was removed, and the roots

were weighed. The gall index were determined using a scale of 1 to 10 (Barker et al.,

1986), in which 0 = 0 galls, 1 = about 10% of roots with galls, 2 = 20% of roots with galls, 3 = 30% of roots with galls, and so on until 10 = 100% of roots with galls. The eggs were collected after dissolving the gelatinous matrices containing eggs with 0.5%

NaOCl (Hussey and Barker, 1973). Each root system was placed into a 50-ml centrifuge tube with 30 ml of an aqueous solution of 0.5% NaOCl. The tubes were closed and vigorously shaken for 30 seconds. The egg suspension was poured onto a sieve with 25- um-pore openings (500-mesh), rinsed with tap water, and collected in another centrifuge tube. The process was repeated twice, using tap water instead of NaCIO solution. The roots were cut into pieces 3 to 5 cm long and placed into a centrifuge tube with 30 ml of an 8:1 (v/v) aqueous solution of commercial Cytolase PCL5 (Genecor International,

Rolling Meadows, IL). The tubes were placed on an automatic shaker (Eberback

Coorporation, Ann Arbor, MI) for 24 hours at room temperature (approximately 24 °C).

Softened roots were placed in a sieve with 600-um-pore openings (30-mesh) over a sieve with 180-um-pore openings (80-mesh) and sprayed with a vigorous stream of tap water.

The dislodged females and root debris were transferred to a beaker by washing the sieve from the back with a jet of water. The females were separated from the root debris with a

Pasteur pipette under a stereo microscope and examined for P. penetrans infection with an inverted compound microscope. Number of eggs per root, gall index, and percentage 123

of infected females were recorded. The second-stage juveniles were extracted from the soil by centrifugal flotation (Jenkins, 1964). The number of attached endospores was counted on each of 20 randomly selected juveniles per replicate using an inverted microscope at a magnification of x400. The data were subjected to analyses of variance and Duncan's multiple-range test using SAS software (SAS Institute, Cary, NC).

Results

Determination of P. penetrans density in infested soil . Numbers of endospores attached to juveniles increased in proportion to the concentration of endospores in

artificially infested soil (Fig. 5.1). The numbers of endospores attached per juvenile and their standard deviations in the soils that received 10,000, 100,000, and 1,000,000 endospores/g of sterilized soil were 1.4 ± 2.0, 15.9 ± 8.3, and 100.40 ± 44.8, respectively.

The average number of endospores attached per juvenile in 100 juveniles examined (20 juveniles per each of five replications) in the untreated field soil was 18.1

± 8.7. The endospore density in the field soil, estimated by entering 18.1 in the

5 regression equation, was 1.27 x 10 endospores/g of soil (Fig. 5.1).

Suppressiveness Test . The number of endospores attached per juvenile was lower

in the autoclaved soil than in untreated soil or air-dried soil (P < 0.05) in both trials

(Table 5.1). The number of endospores attached per juvenile in soil treated by

microwaving for 12 minutes in the first trial was lower than that in untreated and air-dried soil; when soil was treated for 4 minutes, attachment was reduced from that in untreated

soil, but was greater than attachment in autoclaved soil. Similar responses to soil 124

0 10 20 30 40 50 60 70 80 90 100 Endospores/J2

Fig. 5.1. Regression of the number of endospores of Pasteuria penetrans attached to second-stage juveniles (J2) of Meloidogyne arenaria race 1 to density of endospores in the soil; the value for the number of endospores/g of field soil (())), 126,962, was obtained by entering the number of endospores/J2 (18.1) observed in the bioassay into the regression equation. i

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treatments were observed for endospore production in female nematodes. No reproduction of P. penetrans occurred after soil was autoclaved. The extended

microwaving of soil in the first trial reduced the reproduction of P. penetrans compared

with untreated soil and air-dried soil, but in the second trial endospore production was as

high in soil treated with 4 minutes of microwaving as in the untreated control.

Autoclaved soil resulted in the egg production 122 and 64 times higher than in the

air-dried soil in the first and second trials, respectively; and 18 and 13 times higher than

in the non-treated soil in the first and second trials, respectively (Table 5.1.). The number

3 of second-stage juveniles per 100 cm of autoclaved soil was significantly higher than in

soil from the other three treatments (P < 0.05), which, in turn, were not different in juvenile numbers among themselves.

The gall index for tomato roots was higher for autoclaved soil than for untreated or air-dried soil in both tests (P < 0.05). The index was lower in the second trial in soil microwaved for 4 minutes than in the autoclaved soil; however, the extended

microwaving in the first trial resulted in similar indices for microwaved and autoclaved

soil. Root weights were not different among treatments in both the first and second trials.

Discussion

Determination of P. penetrans density in the infested soil . When field soil

infested with P. penetrans is used in studies on the biology of the bacterium, it is

important to quantify its endospore density. Since no culture medium has been developed for the cultivation of P. penetrans in vitro, serial dilution methods with 127

infested soil cannot be used, and estimations of the density of the bacterial endospores are limited to bioassays. Bioassays have been used by several authors to study attachment of

P. penetrans to various nematodes (Dutky and Sayre, 1978; Freitas et al., 1995; Hatz and

Dickson, 1992; Oostendorp et al., 1990; Singh and Dhawan, 1990; Stirling, 1981; Stirling

et al. 1990). As an example, Stirling and White (1982) used a bioassay to determine the presence of P. penetrans in soil under agitation. They added juveniles in water to field

soil and extracted them after 24 hours of agitation that induced attachment. Their method

detected the presence of P. penetrans in soil, but did not determine the endospore density

in the soil.

A bioassay to determine soil endospore density, devised by Freitas et al. (1995)

and used in the Chapter 3, compared the attachment of endospores to juveniles of M. arenaria in field soil with the attachment in soil in which known concentrations of the

bacterium had been added. According to Oostendorp et al. (1990), factors such as temperature, soil moisture, and soil texture are likely to influence endospore attachment to nematodes. Studies relying on the movement ofjuveniles in soil for attachment result in variation of attachment, especially if the endospores are introduced into the soil

(Oostendorp et al., 1990; personal observation).

The present methodology proved to be an efficient and rapid way to determine

endospore density in field soil, and presented less variation in attachment than the earlier assays described by Freitas et al. (1995) and used in Chapter 3. A higher homogeneity was observed in attachment because the attachment did not depend on the movement of

the juveniles in the soil, which is affected by temperature and moisture. In the new 128

bioassay, clumps of endospores in soil are dispersed by agitation in water, and the

chances of all juveniles to come into contact with endospores are basically the same and do not depend on movement of the juveniles.

Another benefit of this method is that small amounts of soil are required, which

results in lower amounts of endospores necessary to run the bioassay. There is a requirement of large quantities of endospores in other bioassays that use 30 to 50 g of soil

3 6 per dish with five to six replications and concentrations of 10 to 10 endospores/g of soil to obtain a regression curve. Such high endospore concentrations are usually required

5 because suppressive soils often have endospore densities higher than 10 endospores/g of

soil (Chen et al., 1996). In contrast, since endospores have to be collected from infected females from fields to be assayed and because attachment of some isolates of P.

penetrans is specific (Channer and Gowen, 1992; Davies et al., 1988; Sayre et al., 1991;

Stirling, 1985), the present bioassay may be used with the low number of endospores that may be obtained from a few females extracted from plants grown in the field, or from

females produced in a few pots in the greenhouse, as devised by Stirling and Wachtel

(1980).

If reduced soil volume is used in the bioassay, it is important to determine if the

sample is representative of the field soil. A systematic field sampling and a

comprehensive mixture of the samples are required to obtain the 1 g of soil used for each replicate of the assay. In this study, the attachment on nematodes of the same replicate and among replicates was generally consistent and compared favorably to the previous

method. If proper care is taken during soil sampling and mixing of the samples, the test 129

is a good indicator of the average endospore density in the field. High densities, such as

5 the level of 1 .27 x 10 endospores of P. penetrans determined in the field soil, are desirable for studies, such as those on environmental impacts on P. penetrans, because the differences in attachment may become more extreme, and because low densities of endospores may not be detected in soils by attachment bioassays after endospore numbers are greatly reduced by the treatments.

Suppressiveness Test . The results confirm that the soil from the field used in the

nematicide experiment (Chapter 4) is suppressive to M. arenaria race 1 and that P.

penetrans is the causal agent of the suppressiveness. The reduced attachment in

autoclaved soil, when compared with untreated and air-dried soil, supports the findings in

Chapter 2, and those of Bird et al. (1989), Dutky and Sayre (1978), and Stirling (1984); only very high temperatures are deleterious to endospores and their attachment to the cuticle of the nematode. The attachment was evaluated on the juveniles of the second generation of the nematode, 45 days after the introduction of the nematode inoculum, and was not different between untreated and air-dried soils. However, the greater

reproduction of the bacterium in the nematode females in air-dried soil in the first trial,

although not validated in the second trial, suggests that drying may have influenced the

endospores in the soil. Desiccation may have resulted in the disruption of the sporangium

membrane and subsequently in higher infection after attachment (Stirling et al., 1986).

High temperatures prevent P. penetrans from infecting host nematodes, as demonstrated

in Chapter 3 and by Dutky and Sayre (1978) and Williams et al. (1989). The low concentration of dipicolinic acid and calcium in P. penetrans endospores, compared to 130

other endospore-forming bacteria, may explain why this bacterium is less heat-resistant

(Williams et al., 1989).

The 92% to 99% reduction of egg production in the untreated and air-dried soils

compared with the autoclaved soil was the major mechanism of the suppressiveness of M.

arenaria by P. penetrans. Bird (1986), Mankau (1980), Minton and Sayre (1989), and

Sayre and Wergin (1977) also reported that infection by P. penetrans reduced or impaired

egg production. The reduction of egg production also may be a result of the decrease of

the number ofjuveniles that penetrate the roots in soil with high endospore density

(Brown and Smart, 1985; Davies et al., 1988; Stirling, 1984; and Stirling et al., 1990).

Reductions in infectivity have been observed when nematodes had 1 5 to 20 endospores

attached to their cuticles (Brown and Smart, 1985), and 96% of the juveniles extracted

from soil from the same field in the previous study (Chapter 4) each had an average of 34

endospores attached to their cuticles. The reduced penetration by juveniles also results in

less root galling. The lower gall index in untreated and dry soils compared to autoclaved

soil in the present study supports earlier reports, in which greenhouse tomato inoculated

with M. incognita had fewer galls on roots of plants growing in soil infested with P. penetrans than in soil free of the bacterium (Mankau, 1980; Mankau and Prasad, 1972).

The reduction of second-stage juveniles that occurred in this study was a consequence of

the reduction of eggs.

The lack of differences in root weight among treatments probably resulted from

an increase of weight due to the high number of galls, especially in autoclaved soil, which compensated for the reduction in root growth caused by the nematode. Weekly 131

fertilization, good irrigation and temperatures optimal for the tomato plants to tolerate nematode infection resulted in minimal plant damage. Since the goal of the experiment

was to study the effect of the bacterium on nematode reproduction, it was important to maintain the nematode plant host under optimum health conditions. Under field conditions, however, plants are subjected to more stresses and have less tolerance to nematode infection.

Differences between microwaved soil in the first and second trials resulted from differences in methodology. When the six bags of soil were arranged in the microwave

oven in the first trial, they were placed side-by-side in such a way that one bag did not

block exposure of the other bags. Thus, the soil in the first trial was exposed to the same

radiation of the soil as in the second trial, except it was treated 8 minutes longer.

Although the temperatures of the soil were not recorded after the microwave treatment,

the longer period of treatment of endospores in soil in the first trial most likely resulted in an exposure of them to temperatures that denatured the proteins responsible for

attachment. The inactivation of P. penetrans in the soil in the first trial does not follow

Bollen's (1969) hypothesis that the effects of microwaves on microorganisms are

proportional to the ratio of treatment time over soil weight, but it does support Ferriss

(1984), who concluded that a decrease in the ratio of time per unit weight of soil required to achieve a given effect on soil microorganisms decreased with the increase of soil weight. Increased temperatures or time of exposure to high, but non-lethal temperatures,

reduce the capability of endospores to attach to juveniles (Chapter 2). Because of the

impact of the higher temperatures attained with extended microwaving in the first trial, no 132

differences, except in the numbers ofjuveniles in soil, were observed in response to

microwaving or autoclaving. In the second trial with reduced microwave treatment, attachment and reproduction of P. penetrans were greater after microwaving than after autoclaving, and egg production, juvenile numbers and gall index were reduced. Thus, the extended time of microwaving was obviously detrimental to the bacterium and should

be avoided when treating soil to eliminate other antagonists of nematodes and retain P. penetrans.

Fungi are an important segment of the microbial biomass in soils. Many fungal species parasitize nematodes and have been the subjects of numerous studies. Barron

(1977) and Stirling (1991) summarized investigations on the many nematophagous fungi.

Over 140 fungal species that attack nematodes have been identified. Microwaving soil for 4 minutes/kg of soil eliminates most soilborne fungi but not soilborne bacteria (Chen,

1994). Since there were no differences in number of eggs and number ofjuveniles produced between untreated soil and soil treated by microwaving for 4 minutes/kg of soil, this technique was useful in determining that fungi did not play a significant role in the

suppressiveness of the field soil. Hence, females, eggs and juveniles collected from the field were free of nematophagous fungi.

The determination of specificity and adaptability of isolates of P. penetrans to specific nematodes will be important for the establishment of fields suppressive to root- knot nematodes and the diseases they cause. Subsequent effectiveness will depend on the survival and dissemination of selected isolates. Currently, the attachment of endospores

of P. penetrans to different nematode species is considered inadequate for 133

characterization of bacterial isolates because variability in host specificity occurs with field populations of P. penetrans that are genetically heterogeneous and are influenced by nematode hosts selected for endospore production (Channer and Gowen, 1992; Davies

and Danks, 1993; Davies et al, 1992; and Davies et al, 1994). Currently, it is not

possible to determine if the bacterial isolate infesting the field in the present study is

indiginous or was spread from the infested site used by Oostendorp et al. (1991) or from

the field studied by Weibelzahl-Fulton et al. (1996). The development of molecular biological techniques in the near future for genetic characterization of P. penetrans isolates may clarify dissemination factors, distribution, and the population dynamics of this bacterium.

Concluding, P. penetrans greatly suppressed Meloidogyne arenaria in tomato under field and greenhouse conditions, and the density of the bacterium in this soil was

5 determined to be 1.27 x 10 endospores/g of soil. CHAPTER 6 SUMMARY AND CONCLUSIONS

Plant-parasitic nematodes cause losses exceeding 100 billion dollars annually

(Statistics Division of the Economical and Social Policy Division, FAO, 1985). Most of the losses are caused by Meloidogyne spp., which are considered to be the most important

species of plant-parasitic nematodes worldwide (Sasser and Freckman, 1 987).

Nematodes cause yield losses of 24 to 38% in tomato in the tropics (Sasser, 1979b).

Nematicide treatment is considered one of the most reliable means of controlling a wide

variety of nematodes, and fumigation is the most effective method of chemical control.

Fumigation with methyl bromide remains the only generally accepted method of

eradicating pathogens in many countries. Because the phaseout of methyl bromide by the

year 2001, due to its deleterious effects on the ozone layer, is projected to result in an estimated 20% yield decrease in tomato production (Crop Protection Coalition, 1995),

combinations of treatments may become increasingly important as pesticide usage is

reduced. Thus, management of nematodes with biological control organisms, soil solarization, organic amendments, environmentally safer nematicides, or the integration

of these methods is of great interest for agriculture.

Pasteuria penetrans is a Gram-positive bacterium that prevents Meloidogyne spp. from reproducing and diminishes their ability to penetrate roots. In this study, P.

penetrans greatly suppressed Meloidogyne arenaria race 1 in tomato under field and

134 135

greenhouse conditions. This microorganism is considered to be one of the most

promising biocontrol agents of nematodes because it is resistant to desiccation and relatively resistance to heat. Good nematode control has been reported with combinations of P. penetrans and solarization or nematicides (Brown and Nordmeyer,

1985; Tzortzakakis and Gowen, 1993; Walker and Wachtel, 1988). Solarization alone decreases population densities of many genera of plant-parasitic nematodes. The addition

of organic matter into soil under solarization provides levels of disinfestation greater than with either method alone, apparently by releasing toxic volatile compounds and

stimulating antagonistic micro-organisms (Stapleton et al., 1993; Stirling, 1991); however no studies had been conducted on the effects of these volatiles on P. penetrans.

In this study, the effects of temperature, solarization, cabbage amendment, and

fumigant nematicides on P. penetrans and its development in M. arenaria in tomato were

evaluated under field, greenhouse, and controlled environment conditions. Overall, the results of this work indicate that solarization may be used in combination with P. penetrans for management of root knot of tomato, but the use of cabbage amendments under solarization or fumigation with chloropicrin should be avoided because they inhibit endospore production.

As a preliminary study to a field solarization test, the effects of temperature on the

development of P. penetrans in Meloidogyne arenaria race 1 were investigated.

Exposing second-stage juveniles of M. arenaria to approximately 30 °C increased their receptivity to endospore attachment when compared to treating juveniles at 25 °C and 35

°C. Changes in the nematode surface coat may have been altered at extreme temperatures and, thus, may have been responsible for changes in endospore attachment. Incubating P.

penetrans endospores in soil at 30 °C to 70 °C for 5 hours a day over 10 days resulted in reductions of endospore attachment to nematodes and subsequent reproduction of the bacterium in the nematode as temperatures of incubation increased to 50 °C and higher.

Highest attachment occurred when nematode juveniles were incubated in soil infested with endospores and maintained at 20 °C to 30 °C for 4 days. Heating juveniles to

sublethal temperatures (35 °C to 40 °C) decreased endospore attachment. In another

study, incubating endospores in soil with cabbage amendment at 50 °C for 10 days

reduced subsequent bacterial endospore formation in female nematodes when compared

to incubation of endospores in soil with cabbage at 20 °C or with endospores in non-

amended soil at 50 °C. Volatiles released from cabbage decomposition may have been

toxic to the bacterium.

Differences in the effects of solarization and cabbage amendment on P. penetrans

under field conditions were not determined due to the unequal nematode population and

also because of the limiting effects of temperature and volatiles on the nematodes. The

introduction of untreated juveniles, under greenhouse conditions, into soil treated in the

field allowed the detection of deleterious effects of cabbage volatiles and solarization

temperatures on the development of P. penetrans present in the first 20-cm depth of soil

in the field. At deeper layers in the soil, moderate increases in temperature may increase

both the rate of development of females of Meloidogyne spp. and the production of P. penetrans endospores (Walker and Wachtel, 1988). The negative effects of high

temperatures on the development of P. penetrans were confirmed in a greenhouse 137

experiment in which portions of the bacterial life cycle were retarded or terminated by

long term exposure to temperatures higher than 50 °C for 10 days. In another greenhouse

experiment, deleterious effects of volatiles released during cabbage decomposition on P. penetrans were observed in soil infested with the bacterium, amended with cabbage, and

incubated at 50 °C, when compared to treatments with nonamended, infested soil

incubated at this temperature and amended or nonamended, infested soil incubated at 20

°C.

Effects of fumigant nematicide alternatives to methyl bromide on P. penetrans

and its infectivity to M. arenaria race 1 in tomato roots were studied under field and

greenhouse conditions. Treatments included methyl bromide plus 33% chloropicrin;

chloropicrin alone; metham sodium; 1,3-dichloropropene plus 16.5% chloropicrin; 1,3-

dichloropropene plus 25% chloropicrin; 1,3-dichloropropene plus 35% chloropicrin; and

the untreated control. The effects of nematicides on the nematodes did not allow the

elucidation of the effects of chemicals on P. penetrans endospores. Endospore

s population density in the field soil was determined to be 1.27 x 10 endospores/g of soil,

and, consequently, the high level of endospore attachment to second-stage juveniles

reduced the penetration ofjuveniles into the tomato roots under field conditions. Soil

from field plots treated with chemicals was taken to the greenhouse and untreated juveniles were introduced to evaluate the development of P. penetrans. Data from the greenhouse experiments indicated that chloropicrin alone or in combination with methyl bromide was highly detrimental to P. penetrans because they inhibited endospore formation. Formulations of 1,3-dichloropropene plus 16.5% chloropicrin; 1,3- dichloropropene plus 25% chloropicrin; and 1,3-dichloropropene plus 35% chloropicrin had moderate effects on the bacterium. The chloropicrin present in these formulations may have been responsible for the bactericide effect on P. penetrans. Metham sodium was the only nematicide harmless to the bacterium and was also the only chemical not

containing chloropicrin. Further studies are necessary to determine if chloropicrin is the chemical detrimental to P. penetrans.

Overall results of this work indicate that solarization may be used in combination with P. penetrans, but the use of chloropicrin alone or in combination with methyl bromide should be avoided in integrated management of nematodes with P. penetrans. LITERATURE CITED

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BIOGRAPHICAL SKETCH

Leandro Grassi de Freitas was born on October 6, 1963, at Sao Paulo city, Sao Paulo,

Brazil. In 1983 he entered the University Federal de Vicosa, Minas Greais, and graduated in

January 1987, receiving an Engineer degree in Agronomy.

In January 1987, he entered the Graduate School in the Department of Plant Pathology of

the University Federal de Vicosa. The title of his thesis was "Biological Control of Meloidogyne

javcmica by the fungi Paecilomyces lilacinus and Cylindrocarpon destructans" . He was awarded

a Master of Science degree in January 1991

In January 1991, Leandro started his doctoral studies in the Department of Plant

Pathology in the University of Florida under supervision of Dr. D. J. Mitchell and Dr. D. W.

Dickson.

156 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.

David D. MhVhell, Chair Professor of Plant Pathology

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

Donald W. Dickson, Cochair 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.

lobert McSorley 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. 1_ Raghavan Charudattan Professor of Plant Pathology

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

Donald L. Hopkins Professor of Plant Pathology This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy.

May, 1997 XcuA Y . J^/w DeanTCollege of Agriculture

Dean, Graduate School