Title: Pathogenicity of Bacillus penetrans to Meloidogyne incognita
CITATION THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00102800/00001
 Material Information
Title: Pathogenicity of Bacillus penetrans to Meloidogyne incognita
Physical Description: Book
Language: English
Creator: Brown, Stephen Michael, 1946-
Copyright Date: 1983
 Record Information
Bibliographic ID: UF00102800
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: ltuf - ACG6852
oclc - 10955886

Full Text













PATHOGENICITY OF Bacillus penetrans
TO Meloidogyne incognita




BY


STEPHEN MICHAEL BROWN


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



UNIVERSITY OF FLORIDA


1983






























to



Marie, Lora and Johanna














ACKNOWLEDGEMENTS


I express my sincere gratitude to my major professor, G. C. Smart,

Jr., for guidance, encouragement and continued interest throughout the

course of this study. I am grateful to D. W. Dickson, R. P. Esser,

D. J. Mitchell and E. P. Previc for serving on my committee and for con-

structive review of my dissertation. I am most grateful to D. J. Mitchell

for his friendship, for his innovative plant pathology course and for his

considerable encouragement throughout my studies, and to J. L. Kepner for

allowing me to attend his multivariate, nonparametric and regression

courses in statistics, for use of an unpublished statistical test and

for many helpful comments on the manuscript. I thank D. Nordmeyer for

many suggestions about techniques, Nguyen Ba Khuong for Figure 3.1, P. C.

O'Brien for help in initiating the field experiments, C. Baeza Arag6n

for collaboration on the sampling of nematodes in soil and E. B. Whitty

for providing tobacco seedlings and agronomic advice.

This study was supported in part by a research assistantship from

the Department of Entomology and Nematology and a United Brands student

loan and research grant.















TABLE OF CONTENTS


ACKNOWLEDGEMENTS. ..


Page

. . . . . . . . . . . . iii


LIST OF TABLES . . . .

LIST OF FIGURES . . . . .

ABSTRACT . . . . . .

INTRODUCTION . . . . .

CHAPTER 1: INCREASED CROP YIELDS
OF Bacillus penetrans TO PLOTS
Meloidogyne incognita . . .

Introduction . . . .

Materials and Methods . .

Results . . . . .

Discussion . . . . .

Summary . . . .

CHAPTER 2: INCREASED CROP YIELDS
OF Bacillus Denetrans TO PLOTS
AND Meloidogyne incognita . .

Introduction . . . .

Materials and Methods. . .
Field Experiments . .


FOLLOWING APPLICATION
INFESTED WITH













FOLLOWING APPLICATION
INFESTED WITH B. penetrans




. . . . . I .


. . . . . . . . . . 22


Determination of a Dosage-Response Function
for the Inoculum of Bacillus penetrans. .
Attachment of Spores to Meloidogyne javanica,
M. incognita and M. arenaria . . .

Results . . . . . . . . . .
Field Experiments . . . . . . .
Determination of a Dosage-Response Function
for the Inoculum . . . . . .


. . . 24

. . . . 25

. . . . 27
. . . . 27


. . . 29


viii

xi

1



6

6

7

12

18

20












Attachment of Spores to Meloidogyne javanica,
M. incognita and M. arenaria . . .

Discussion . . . . . . . . . .

Summary . . . . . . . . . .

CHAPTER 3: EFFECT OF Bacillus penetrans ON MOVEMENT OF
AND ROOT PENETRATION BY Meloidogyne incognita . .

Introduction . . . . . . . . . .

Materials and Methods . . . . . . .

Root Penetration by Meloidogyne incognita
Infected with Bacillus penetrans . . .

Movement of Meloidogyne incognita Infected
with Bacillus penetrans . . . . .

Results . . . . . . . . . .

Discussion . . . . . . . . . .

Summary . . . . . . . . . .

DISCUSSION AND CONCLUSIONS . . . . . . .

LITERATURE CITED . . . . . . . . .

BIOGRAPHICAL SKETCH . . . . . . . . .


Page


. . . . 29

. . . . 32

. . . . 34


. . . . 35

. . . . 35

. . . . 35


. . . . 35


. . . . 37

. . . . 40

. . . . 43

. . . . 47

. . . . 48

. . . . 52

55














LIST OF TABLES

Table Page

1.1 Yields of tobacco, soybean and winter vetch planted
in sequence in noninfested soil and soil infested
with Meloidogyne incognita alone and M. incognita
plus Bacillus penetrans. . . . . . . . . ... 13

1.2 Meloidogyne incognita in soil and roots 0, 59 and 91
days after planting tobacco. . . . . . . . ... 15

1.3 Meloidogyne incognita in soil 0, 72 and 107 days
after planting soybean . . . . . . . .... . 16

1.4 Meloidogyne incognita in soil and roots 71 and 103
days after planting winter vetch . . . . . .... 17

2.1 Yields of tobacco and soybean planted in sequence in
noninfested soil and soil infested with Meloidogyne
incognita alone and M. incognita plus Bacillus penetrans . 28

2.2 Meloidogyne incognita in soil and roots 0, 60 and 77
days after planting tobacco. . . . . . . . ... 30

2.3 Meloidogyne incognita in soil and roots 39 and 118 days
after planting soybean . . . . . . . .... . 31

3.1 The effect of number of juveniles and number of juveniles
with spores of Bacillus penetrans on root penetration
by Meloidogyne incognita . . . . . . . .... 38

3.2 The effect of number of juveniles and number of juveniles
with spores of Bacillus penetrans on the movement of
Meloidogyne incognita. . . . . . . . .... 39

3.3 Number of galls on tomato roots 7 days after inoculation
with 50 juveniles of Meloidogyne incognita noninfected
(control) and infected with Bacillus penetrans . . ... 42










Table Page

3.4 Numbersof Meloidogyne incognita in greenhouse tomato
roots 14 or 30 days after inoculation with juveniles
not infected (control) and infected with Bacillus
penetrans. . . . . . . . . ... ....... 44

3.5 Proportions of juveniles of Meloidogyne incognita
not infected (control) and infected with Bacillus
penetrans that moved more than 5.25 cm toward a
tomato plant . . . . . . . . .. .. .. . .45














LIST OF FIGURES

Figure Page

2.1 Dosage-response function for inoculum of Bacillus
Denetrans added to field plots on 26 March 1983. . . .. 26

3.1 Spores of Bacillus penetrans attached to the cuticle
of a second-stage juvenile of Meloidogyne incognita. .... .41


viii














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


PATHOGENICITY OF Bacillus penetrans
TO Meloidogyne incognita


By

STEPHEN MICHAEL BROWN

April 1983

Chairman: G. C. Smart, Jr.
Major Department: Entomology and Nematology


The pathogenicity of Bacillus penetrans to Meloidogyne incognita was

tested in field plots bordered by concrete, and the mode of action of B.

penetrans on the nematode was investigated under controlled conditions.

Bacillus penetrans was added to the soil of plots into which tobacco,

soybeans, winter vetch, tobacco and soybeans were planted in sequence

during the 1981 and 1982 growing seasons. Tobacco (1981 and 1982), winter

vetch and soybeans (1982) showed trends of increasing yields with decreas-

ing pathogenicity of M. incognita. There was insufficient evidence to

declare a similar trend for soybeans during 1981. Yields from plots

infested with M. incognita and treated with B. penetrans were 23% greater

for tobacco (1981), 38% greater for winter vetch, 24% greater for tobacco

(1982) and 35% greater for soybeans (1982) than those from plots infested

with M. incognita alone.










In a laboratory experiment, juveniles of M. incognita infected with

B. penetrans caused fewer galls (a total of 10) to form on the roots of

tomato plants than did noninfected juveniles (a total of 61). Bacillus

penetrans reduced the number of nematodes in tomato roots in two green-

house experiments, and in a third experiment there was insufficient evi-

dence to declare a lower number of nematodes in tomato roots inoculated

with juveniles infected with B. penetrans than in roots inoculated with

noninfected juveniles. Bacillus penetrans reduced the percentage of

juveniles moving toward tomato plants in only one of four laboratory

experiments.

Spores from the population of B. penetrans used in this study

attached to M. javanica, M. incognita and M. arenaria. A greater pro-

portion of juveniles of M. javanica were infected than those of either

M. incognita or M. arenaria, and the corresponding proportions for M.

incognita and M. arenaria were not found to be different.














INTRODUCTION


Cobb (1906) was the first to report distinctively shaped spores

parasitizing nematodes. Numerous reports of nematodes with this parasite

occurred thereafter. It is probable that all of these reported parasites

are the same organism or closely related organisms. However, none of the

authors who assigned a taxonomic position or proposed a life cycle for

the organism obtained it in pure culture.

Thorne (1940) observed spores within the pseudocoelom and attached

to the cuticle of a lesion nematode, Pratylenchus pratensis (de Man 1880)

Filipjev 1936. On the basis of that observation only, he named the organ-

ism Duboscqia penetrans Thorne (Microsporidia, Nosematidae).

After observing smears and sections of parasitized nematodes with an

electron microscope, Mankau (1975) renamed the organism Bacillus penetrans

(Thorne) Mankau. He based the name change on an absence of both organelles

and a membrane-surrounded nucleus, the presence of a typical bacterial cell

wall and plasma membrane, and the presence of spores morphologically identi-

cal to spores of typical Bacillus spp. although shaped differently. The

spores were refractive, a characteristic shared by the milky disease organ-

isms B. popilliae and B. lentimorbus. Mankau (1975) also considered the

disease in nematodes caused by B. penetrans as identical etiologically to the

milky disease of scarabaeid grubs caused by B. popilliae and B. lentimorbus.

Unfortunately, none of the evidence given by Mankau (1975) is diagnos-

tic for the genus Bacillus. No members of the family Bacillaceae produce






2


filamentous vegetative cells, and although the cells examined were prokary-

ote, they were not rod-shaped or spherical, and thus do not fit in Bacil-

laceae as presently defined. Bacterial cell walls and plasma membranes

cannot be distinguished morphologically from corresponding eukaryotic

structures. Prokaryotic cell walls are chemically distinctive from those

of any eukaryotic cell, but Mankau (1975) did not report a chemical analysis

of the cell wall of this organism. Moreover,a reliable chemical analysis

can be carried out only from a pure culture, which has not been achieved

for this organism.

Refractive endospores are not unique to Bacillus spp. Refractiveness

is a characteristic of all endospores, which are defined as spores formed

endogenously within a sporangiumpossessing a multilayered coat, a cortex

and a contained cytoplasm or core that is capable of resisting high temper-

atures and exposure to radiation (Cross 1970). In addition to Bacillaceae,

endospores have been shown to be formed by some Actinomycetes (Cross et al.

1968, Dorokhova et al. 1968).

The similarities between the nematode disease and the milky disease

of insects lie only in that the pathogens are bacteria and that multiplica-

tion occurs in a clear body fluid. Spores of B. penetrans attach to the

cuticle of nematodes when nematodes contact them in soil. After attachment,

a spore germinates and a germ tube penetrates the body wall (Allen 1957,

Mankau et al. 1976, Sayre and Wergin 1977). Insect larvae ingest B. popil-

liae spores that then germinate in the midgut, producing motile rods.

Splittstoesser et al. (1975) proposed that the motile rods penetrate the

midgut wall, although vegetative cell contact with the midgut epithelium

has not been observed (Splittstoesser et al. 1978). They suggested that

penetration may occur by phagocytosis of the midgut cells similar to the









invasion of honey bee larvae by B. larvae (Davidson 1973). Splittstoesser

et al. (1978) and Kawanishi et al. (1978) documented the interaction of

insect midgut cells and B. popilliae, although penetration of the basement

membrane and entry into the hemocoel was not observed.

Sayre and Wergin (1977) suggested that-B. penetrans might be included

among genera of the order Actinomycetales, Actinomycetes--Part 17 of Bergey's

Manual (1974). They studied parasitized specimens of a root-knot nematode,

Meloidogyne incognita (Kofoid and White 1919) Chitwood 1949, (smears and

sections of adult females and whole juveniles), but again the parasite was

not studied from a pure culture. They gave three reasons for a relationship

to Actinomycetales: an absence of both organelles and a membrane-surrounded

nucleus; the presence of branching filamentous mycelia, which are less than

1 m in diameter; and a double-track cell wall .with hairlike projections

similar to that of several species of Actinomyces described by Slack and

Gerencser (1975). Sayre and Wergin (1977) concluded that a precise classi-

fication could not be made until the organism had been cultivated in vitro.

They called the organism a bacterial spore parasite of nematodes (BSPN).

Although the absence of organelles and of a membrane-surrounded nucleus

and the presence of vegetative cells with narrow branching mycelia suggest

a bacterium related to Actinomycetes, a double-track cell wall with

fimbriae is not diagnostic for Actinomycetales (Bergey's Manual 1974).

No existing evidence precludes B. penetrans from the order Actinomycetales.

Although the classification of this organism is in question, it will be

called Bacillus penetrans in this study.

The experimental evidence that supports the pathogenicity of B. pene-

trans to nematodes has been derived from greenhouse and laboratory studies.









Prasad (1971) reported that infected juveniles of M. javanica (Treub 1885)

Chitwood 1949 produced fewer galls on tomato roots (Lycopersicon esculentum

Miller) than did noninfected juveniles under greenhouse conditions. Tomato

plants inoculated with M. incognita had more leaves and fewer root galls

when grown under greenhouse conditions in soil containing B. penetrans

than when grown in steamed soil without B. penetrans (Prasad 1971). Green-

house snap beans (Phaseolus vulgaris L.) inoculated with Pratylenchus

scribneri Steiner 1943 had fewer nematodes after 55 days when grown in

soil containing B. penetrans than when grown in soil without B. penetrans.

The total number of P. scribneri collected from soil and roots was 1220

from the treatment with B. oenetrans compared with 3034 from the treatment

without B. penetrans. However, the population size of P. scribneri from

soil containing B. penetrans may have been underestimated, since the

Baermann funnel technique was used to extract the nematodes from the soil

and roots. Using a modified Baermann funnel technique (a petri plate re-

placed the funnel), Prasad (1971) demonstrated that the number of M. javanica

extracted from soil containing B. penetrans was less than that from steamed

soil. He collected 279 of 2000 M. javanica juveniles from soil containing

B. penetrans compared with 1719 of 2000 from steamed soil. If B. penetrans

has a similar effect on P. scribneri, the population size of P. scribneri

in soil infested with B. penetrans may have been underestimated.

Worldwide, the root-knot nematodes, Meloidogyne spp., are the most

economically important group of plant-oarasitic nematodes (Franklin 1979,

Lamberti 1979, Sasser 1979). They are geographically widespread, have a

wide host range and cause crop damage both alone and together with other

plant pathogens (Sasser 1980). Although the genus contains more than 40









species, M. incognita is the most important species throughout the world

(Sasser 1980).

If the pathogenicity of M. incognita to plants could be hindered by

B. penetrans over several growing seasons, B. penetrans might be suitable

to include as a component of a biological control program where a combina-

tion of pathogens, resistant varieties and crop rotations are used to re-

duce damage to crops caused by M. incognita.

The purpose of this study was to determine the practicality of using

B. penetrans against M. incognita under field conditions and to determine

aspects of the mode of action of B. penetrans. In addition, M. javanica

and M. arenaria (Neal 1889) Chitwood 1949 as well as M. incognita were

exposed to B. penetrans to determine if the three species would become

infected.














CHAPTER 1
INCREASED CROP YIELDS FOLLOWING APPLICATION OF
Bacillus penetrans TO PLOTS INFESTED
WITH Meloidogyne incognita



Introduction


Prasad (1971) reported that greenhouse tomatoes inoculated with

Meloidogyne incognita (Kofoid and White) Chitwood had improved growth and

that greenhouse snap beans had fewer nematodes 55 days after inoculation

with Pratylenchus scribneri Steiner when grown in soil containing B. pene-

trans than when grown in soil without the antagonist. Should this bacter-

ium prove to be pathogenic to plant-parasitic nematodes under field condi-

tions, damage to plants could be reduced by applying spores of B. penetrans

to soil.

Several advantages of this bacterium for control of nematodes are

known. Bacterial spores are resistant to heat, desiccation and many chemi-

cals. Although information about predation is scant (Alexander 1977),

spores may have few antagonists. Four populations of B. penetrans have

shown host specificity (Prasad 1971, Dutky 1978). Therefore, it may be

possible to select isolates pathogenic to plant parasites leaving saproph-

agous and predatory nematodes unaffected.

Disadvantages that may limit the use of this bacterium are that its

spores are nonmotile and apparently attach to the cuticle of nematodes only

when nematodes contact them in soil. Of most importance, B. penetrans has

not been grown in vitro. The fact that it now can be grown only in a









nematode host hinders experimentation and may prevent commercial produc-

tion of inoculum.

The purpose of this chapter was to determine if the addition of spores

of B. penetrans to soil hindered the parasitism of M. incognita to three

crops planted in sequence.



Materials and Methods


In the first year of this experiment, a population of M. incognita

was established in eight of 12 plots located in Gainesville, Florida, while a

population of B. penetrans was produced in a greenhouse. The following

year B. penetrans was added to four of the plots infested with M. incognita

and three crops were planted in sequence: tobacco (Nicotiana tabacum L.

'NC 2326'), soybeans (Glycine max (L.) Merrill 'Hood') and winter vetch

(Vicia villosa Roth).

In spring 1980, twelve 2.4-m x 0.9-m concrete-bordered plots contain-

ing Arredondo fine sand (92.9% sand, 4.9% silt and 2.2% clay) to a depth

of 60 cm were treated with methyl bromide (53 g/m2) under plastic. One

month after fumigation, okra (Hibiscus esculentus L. 'Clemson Spineless')

was planted in all plots. The plots were grouped into four blocks, and

three treatments were assigned randomly within the blocks in a randomized,

complete-block design. The treatments were 1. M. incognita alone, 2.

M. incognita plus B. penetrans and 3. neither M. incognita nor B. penetrans.

Forty-five days after planting okra, the soil of treatments 1 and 2 were

infested with eggs of M. incognita (300,000/plot). The eggs were obtained

by shaking tomato roots (cultivar Rutgers) infected with M. incognita in

a 0.5% sodium hypochlorite solution for 30 sec (Hussey and Barker 1973).









In late fall, okra was removed from the plots and sweet blue lupine (Lupinus

angustifolius L. 'Frost') was planted. In early spring 1981, the lupine

was turned into the soil. Okra and lupine were grown to distribute the

population of M. incognita as might be found in agricultural soil.

Concurrent with the establishing of M. incognita in the field plots, B.

penetrans was produced in M. incognita. Juveniles of Meloidogyne spp.

parasitized by B. penetrans were found in association with Japanese privet

(Ligustrum japonicum Thunberg) from MacClenny, Florida. The isolated ju-

veniles were suspended in water and injected into the soil of potted green-

house tomato plants (cultivar Rutgers) that then were grown for approxi-

mately three months. The tomato roots either were cut into pieces and

mixed with the soil or comminuted in a blender, filtered through a coarse

screen and the filtrate poured into the soil. In both cases the soil was

air-dried for one month. The dried soil was diluted (1 to 1) with fumigated

potting soil (90.6% sand, 3.9% silt and 5.5% clay), planted with tomato

seedlings and reinfested with healthy juveniles of M. incognita.

From spring 1981 to spring 1982, three crops (tobacco, soybean, winter

vetch) were planted in sequence to test the hypotheses that the yields of

the crops increase as the pathogenicity of M. incognita decreases. With

the model for a randomized, complete-block design, X.. = V + i + T. + Ei..

where u represents a location parameter, Bi represents the effect of the

i block and T. represents the effect of the j treatment and is subject

to the constraint Z.j. = 0; the error terms (Eij's) are mutually indepen-

dent random variables from some continuous population that is symmetric

about zero; the null hypothesis of no treatment effect, Ho: T1 = T2 = T3,

was tested against the ordered alternative research hypothesis, Hi: Ti

T2 < T3, where at least one of these inequalities is strict. Here, T1 is









the effect of the presence of nematodes on yield, T2 is the effect of the

presence of both nematodes and bacteria on yield, and T3 is the effect of

having no nematodes or bacteria on yield. Because the assumption of

normally distributed error terms seemed questionable, a nonparametric

test specifically designed to detect these ordered alternatives was used.

The composite Wilcoxon test, proposed by Kepner and Robinson (1982) was

selected for use in this research. A significant omnibus test for ordered

alternatives was followed with point estimates of the differences between

treatment effects based on one-sample median estimators (Hollander and

Wolfe 1973). Had the omnibus test not been significant, estimates of treat-

ment effects would not have been made. It was decided that pairwise com-

parisons to determine which treatment effects were strictly ordered should

not be conducted because of the small sample sizes.

Samples of nematodes from soil and roots (except for soybeans) were

taken during the growing of the three crops. The effect on the nematode

population of adding B. penetrans to soil was tested with the Wilcoxon

signed rank test (Hollander and Wolfe 1973). The proportions of parasitized

nematodes in the samples taken at harvest were compared by normal approxi-

mation. Also biological assays on the soil samples at harvests were

carried out to provide additional estimates of the proportion of parasitized

nematodes in the population. The independence of the proportion of para-

sitized juveniles and crop type was investigated with the log likelihood

ratio G statistic of Sokal and Rohlf (1981).

On 10 April 1981 soil samples were taken from all plots to determine

the nematode densities, B. penetrans was added to the soil of treatment 2

and tobacco was planted in all plots. Six soil cores (2.5-cm diam x 25-cm

deep) were collected randomly 15 cm from the planting sites of tobacco plants.









The soil was mixed and nematodes were extracted from the soil by a proce-

dure adapted from Jenkins (1964). A 250-cm3, random subsample of soil was

rinsed into a container and mixed with a stream of water to a volume of

approximately 500 ml. One minute later the water was decanted onto stacked

sieves of 75-vm and 25-vm openings (200 and 500 mesh). Mixing and decant-

ing were repeated three times. The residue from the 25-pm sieve was washed

into a 50-ml centrifuge tube and spun in a centrifuge for 5 min at a

relative centrifugal force of 1071 g. The supernatant was discarded, and

the pellet was mixed in a sugar solution (454 g/l) and spun for 2 min

at the same force. The supernatant was decanted onto a 25-vm sieve, and

the residue from the sieve was collected and diluted to 10 ml. The nema-

todes from a l-ml sample were counted in a Peters counting slide. When

nematodes were too numerous to count, a greater dilution was used, and the

number of nematodes was converted to nematodes per 25 cm3 of soil. Bacil-

lus penetrans was added to the soil of treatment 2 by placing approximately

1200 cm3 of potting soil infested with B. penetrans into six 10-cm diam x

15-cm deep holes into which tobacco was transplanted. Fumigated potting

soil was placed similarly into the soil of the other two treatments, and

six tobacco seedlings were transplanted into each of those plots.

During the growing of tobacco, samples of nematodes in soil and roots

were taken at 59 and 91 days (final harvest) after transplanting. The

nematodes in soil were estimated as described previously, and roots from

the randomly selected 250-cm3 subsamples of soil were stained with acid

fuchsin in lactophenol to aid in counting the nematodes (Southey 1970).

After the nematodes were counted, the roots were oven-dried, and the number

of nematodes was converted to nematodes per gram of dry roots. Tobacco

leaves were harvested six times at 7-day intervals beginning 56 days after

transplanting. Leaves were dried at 65 C for 7 days before weighing.









Biological assays of soil were taken at the harvest of each crop to

determine whether the proportions of parasitized juveniles followed the

same trend as those obtained from field sampling. A 500-cm3, random sample

of soil taken from the soil collected at harvest from each plot of the

treatment with nematodes and bacteria was air-dried for approximately 2

months in the laboratory. Three 30-cm3, random subsamples of dried soil

from each plot were placed in 30-ml plastic cups, moistened with 10 ml of

distilled water and incubated for 3 days at 28 C. Fumigated potting soil

was used as a control. Then 1000 juveniles of M. incognita in water were

injected into each cup and incubated for 3 days at 28 C. The juveniles

were extracted from the soil by the method described previously, and a ran-

dom sample of 50 juveniles was examined (at 200X) for parasitism by B. pene-

trans. Moistening the soil for 3 days before adding nematodes was selected

following a previous experiment comparing moistening the soil for 3 days

with no prior moistening before adding nematodes to soil. The proportions

of parasitized nematodes from these treatments were compared by normal ap-

proximation.

After the last tobacco harvest, the control plots were treated with

methyl bromide (210 g/m2) to kill the contaminating nematodes. On 27 July

1981, 11 days after the application of methyl bromide, soybeans were planted

in the plots in two 2.4-m rows spaced 40 cm apart. Seven days later the

seedlings were thinned to a 5-cm to 7.5-cm spacing. Soil samples were

taken and processed for nematodes at 0, 72 and 107 days (harvest) after

planting. The number of soil cores was increased to 15 at the 107-day

sample. After harvest and before weighing, seeds were allowed to reach a

mean moisture content of 9.46% (s = 0.19, with all observations 2s of

the mean).









On 9 December 1981, 28 days after the soybeans had been harvested,

winter vetch was planted. The final sample of nematodes in soil from soy-

beans was used as a preplant sample for winter vetch. Soil samples from

winter vetch were collected randomly 71 and 103 days (harvest) after plant-

ing. At harvest, roots from a random sample of five plants from each plot

were collected and cut into small pieces. A 2-a subsample of roots was

selected randomly and stained with acid fuchsin in lactophenol to aid in

counting nematodes. The winter vetch forage was dried at 65 C for 7 days

before weighing.


Results


The test of Keener and Robinson (1982) indicated a trend of increas-

ing yields with decreasing pathogenicity of the M. incognita population for

both tobacco and winter vetch (Table 1.1). The attained significance level

of the tests, which is the probability of observing a value of the statistic

at least as extreme as the value observed, was P = .012 for tobacco and

P = .008 for winter vetch. The same test provided insufficient evidence to

enable one to conclude a trend of increasing yield with decreasing patho-

genicity of the nematode population for soybeans (P = .23). The dry leaf

weight of tobacco (based on one sample median estimators) from plots in-

fested with nematodes and bacteria minus that from plots infested with

nematodes alone was 150 g, and between the control minus the treatment with

nematodes alone was 653 g. The increase in yield for lots infested with

nematodes and bacteria over those infested with nematodes alone was 23% of

the increase in yield observed for the control plots over the plots infested

with nematodes alone. The dry forage weight of winter vetch from plots

infested with nematodes and bacteria minus that from plots infested with

nematodes alone was 122 g, and between the control minus the treatment with



















Table 1.1 Yields of tobacco, soybean and winter vetch planted in sequence in noninfested
soil and soil infested with Meloidogyne incognita alone and M. incognita plus
Bacillus penetrans.


Weight (g)


Treatment


Control


Nematodes +
Bacteria

Nematodes


Tobacco leaf (dry)

976 1076 1253 965*

592 460 1011 334

375 618 368 256


Soybean seed


808 619 606


784 760 729 772

512 495 668 809


Vetch forage (dry)


736 632

373 426


792 580

576 805


227 311 484 440


Values are from four blocks of a randomized,complete-block design.









nematodes alone was 324 g. The increase in yield for plots infested with

nematodes and bacteria over those infested with nematodes alone was 38% of

the increase in yield observed for the control plots over the plots infested

with nematodes alone.

The Wilcoxon signed rank test indicated fewer nematodes in the plots

with nematodes and bacteria than in the lots with nematodes alone at the

59-day sample of tobacco soil (P = .062), and more nematodes in the plots

with nematodes and bacteria than in the plots with nematodes alone at the

91-day sample of soil and tobacco roots (P = .062) and at the 0-iay sample

of soybean soil (P = .062) (Tables 1.2 and 1.3). In the remaining samples

of soil and roots, there was insufficient evidence to enable one to declare

that the numbers of nematodes in the plots with nematodes and bacteria and

with nematodes alone were different (Tables 1.2, 1.3, and 1.4).

The proportions of parasitized juveniles of M. incognita in the final

soil samples of each crop were tobacco, 40 of 1192 (3.4%); soybeans, 41 of

951 (4.3%); and winter vetch, 39 of 381 (10.2%). Winter vetch had a greater

proportion of nematodes with spores on the cuticle than either tobacco or

soybean, and the corresponding proportions for tobacco and soybean were

not found to be different. The significance level for this family of

comparisons (the probability of making at least one Type I error in the

three conclusions) was a = .03.

The proportions of parasitized juveniles of M. incognita in biological

assays of the soil collected at harvests were tobacco, 168 of 600; soybeans

155 of 600; and winter vetch, 358 of 600. The hypothesis that the true

proportions of parasitized nematodes in the soil from the three crops

are simultaneously equal was rejected (P < .001) by the log likelihood

ratio test. Follow-up pairwise comparisons among the proportions indicated












Table 1.2 Meloidogyne incognita in soil and roots 0, 59 and 91 days
after planting tobacco.


Treatment


Block Control

1 (0,-)*
2 (0,-)
3 (0,-)
4 (0,-)


Nematodes + Bacteria


(12,-)
(12,-)
(5,-)
(21,-)


Nematodes

(6,-)
(4,-)
(8,-)
(6,-)


(0,63)
(0,0)
(0,11)
(0,179)


(288,500)
(2,13)
(190,181)
(1680,338)


(1540,1579)
(790,3000)
(425,2538)
(730,5786)


(2930,3067)
(4370,2700)
(2400,734)
(2220,8300)


(1935,1659)
(1090,1567)
(1290,908)
(890,2025)


(1445,2234)
(1895,1645)
(1085,0)**
(1885,0)**


* 3
In the ordered pair (A,B), A is the number of juveniles/25 cm3 of soil,
and B is the number of nematodes/g of dry roots. A dash (-) for B
indicates that samples were not taken.
**
No roots were present.


Days
After
Planting



0












Table 1.3 Meloidogyne incognita in soil 0, 72 and 107 days after
planting soybean.


Days
After
Planting


Treatment


Block Control Nematodes + Bacteria


375
1175
185
305


Nematodes

135
715
135
295


419
197
105
146


338
190
248
175


*Values are the number of juveniles/25 cm3 of soil.
Values are the number of juveniles/25 cm of soil.











Table 1.4 -


Meloidogyne incognita in soil and roots 71 and 103 days
after planting winter vetch.


Treatment


Control Nematodes + Bacteria

(0,-)* (70,-)

(0,-) (49,-)

(0,-) (52,-)

(0,-) (38,-)


(79,152)

(98,357)

(36,160)

(168,108)


(0,0)

(0,0)

(0,0)

(0,0)


Nematodes

(49,-)

(41,-)

(174,-)

(64,-)


(48,318)

(61,171)

(68,409)

(64,280)


In the ordered pair (A,B), A is the number of juveniles/25 cm3 of
soil, and B is the number of nematodes/g of fresh roots. A dash (-)
for B indicates that samples were not taken.


Days
After
Planting


Block









that the proportion of nematodes with spores on the cuticle from soil

collected at harvest of winter vetch exceeded those from the preceding

crops, tobacco and soybean (P <.0001 for each test). No difference could

be claimed between the proportions for tobacco and soybean (P = .1977).

Spores were observed on 119 of 200 randomly selected M. incognita

juveniles in a biological assay of the same potting soil used to infest

the treatment with nematodes and bacteria before planting tobacco.

The proportions of infected nematodes from the experiment comparing

bacteria-infested soil moistened for 0 or 3 days before adding test nema-

todes were 59 of 200 for 0 days and 146 of 200 for 3 days. The hypothe-

sis that the proportions are equal was rejected by normal approximation

(P <.0001).

Samples of mature females from roots collected at harvest of tobacco

and winter vetch were examined for spores within the body. The proportions

of parasitized females were tobacco, 8 of 42. and winter vetch, 0 of

40. The number of parasitized females from roots of winter vetch was

lower than that from roots of tobacco (P = .0008).



Discussion


The increased yields of tobacco and winter vetch resulting from add-

ing B. penetrans to field soil containing an established population of

M. incognita demonstrated that B. penetrans reduced the pathogenicity of

the population of M. incognita. Prasad (1971) reported that greenhouse

tomatoes inoculated with M. incognita had more leaves when grown in soil

containing B. penetrans than when grown in steamed soil. The effect of

B. penetrans on M. incognita now can be extended to field conditions.

Fifteen days after planting soybean seed, leaf chlorosis caused by









Rhizoctonia sp. was observed on soybean plants only in the control plots.

Probably Rhizoctonia sp. was introduced into plots on soybean seed and

thrived in the fumigated soil of the control. The effect of the disease

on yield could not be determined, but the possibility of a suppressed

yield for the control should be considered when interpreting the results.

Prasad (1971) reported that greenhouse tomatoes inoculated with M.

incognita had fewer nematode galls on roots and that greenhouse snap beans

inoculated with P. scribneri had fewer nematodes after 55 days when grown

in soil containing B. Denetrans. However, the population of P. scribneri

from soil containing B. penetrans may have been underestimated, since the

Baermann funnel technique was used to extract the nematodes from the soil

and roots. Using a modified Baermann funnel technique (a petri plate re-

placed the funnel), Prasad (1971) demonstrated that the number of M.

javanica (Treub) Chitwood extracted from soil containing B. penetrans was

less than that from soil without the antagonist. He collected 279 of 2000

M. javanica juveniles from soil containing B. penetrans compared with 1719

of 2000 from steamed soil. If B. penetrans has a similar effect on P.

scribneri, the population of P. scribneri in soil infested with B. penetrans

may have been underestimated. I suspect that a low soil concentration of

B. penetrans causes a reduction in the rate of population growth of M.

incognita. If parasitized juveniles are prevented from feeding or if feed-

ing is hampered, the rate of nematode population growth is decreased. The

lower number of juveniles at the 59-day sample of tobacco soil in plots

treated with bacteria may be a result of a reduced rate of population growth.

The lower number of nematodes in soil and roots was a result of the reduced

carrying capacity of the tobacco plants. In two of the four plots containing









nematodes only, all of the plants were dead at the end of the experiment

(Table 1.2).

The proportion of M. incognita juveniles with spores on the cuticle

was greater in the final soil samples from winter vetch than in those from

tobacco or soybean. It is conjectured that the larger proportion of para-

sitized juveniles at the end of the cultivation of the three crops indi-

cated that the number of B. penetrans in the soil increased. Alexander

(1981) reviewed studies of the addition of bacteria to natural ecosystems

that suggested that for bacteria that persist in soil there is a char-

acteristic number that is maintained. Determining this characteristic

number for B. penetrans in agricultural soil, and in turn, the optimum

effect of that concentration of B. penetrans on a population of plant

parasitic nematodes would be an interesting continuation of the present

study. Such work would be aided if an artificial medium for growing B.

penetrans were developed. In addition, this would lead to a pure culture

of B. penetrans allowing the study of its biology and taxonomy.


Summary


The pathogenicity of Bacillus penetrans to Meloidogyne incognita

was tested under field conditions. Bacillus penetrans was added to

concrete-bordered plots into which tobacco, soybeans and winter vetch

were planted in sequence. Tobacco and winter vetch showed trends of

increasing yields with decreasing pathogenicity of M. incognita. There

was insufficient evidence to declare a similar trend for soybeans. Yields

from plots infested with M. incognita and treated with B. penetrans were

23% greater for tobacco and 38% greater for winter vetch than those from

plots infested with M. incognita alone. Soil samples taken midway through






21


the tobacco season revealed fewer second-stage-juvenile nematodes in plots

infested with B. penetrans than in the nematode-infested plots without B.

penetrans. The proportion of juvenile nematodes infected with B. pene-

trans was greater at the harvest of winter vetch than at the harvests

of tobacco or soybeans, while the proportion of mature females infected

with B. penetrans was less at the harvest of winter vetch than at the

harvest of tobacco.














CHAPTER 2
INCREASED CROP YIELDS FOLLOWING APPLICATION OF
Bacillus penetrans TO PLOTS INFESTED WITH
B. penetrans AND Meloidogyne incognita



Introduction


The purpose of this chapter was to determine whether adding inoculum

of Bacillus penetrans to plots infested during the previous year with

Meloidogyne incognita and B. penetrans would decrease further the effects

of M. incognita to two crops planted in sequence. In addition, M. javanica

and M. arenaria as well as M. incognita were exposed to B. penetrans to

determine if the three species would become infected.



Materials and Methods


Field Experiments

Tobacco (Nicotiana tabacum L. 'NC 2326') and then soybeans (Glycine

max (L.) Merrill 'Hood') were grown during the 1982 growing season in twelve

2.4-m x 0.9-m concrete-bordered plots containing Arredondo fine sand (92.8%

sand, 4.9% silt and 2.2% clay) to a depth of 60 cm in which three crops

had been grown in sequence from spring 1981 to spring 1982 (CHAPTER 1). The

plots were assigned randomly to the 12 block-by-treatment combinations in a

randomized, complete-block design involving four blocks and three treatments.

The treatments were 1. M. incognita alone, 2. M. incognita plus B. penetrans









and 3. neither M. incognita nor B. oenetrans. The procedures used to

infest the Plots initially with these organisms are described in CHAPTER

1. In spring 1982 additional inoculum of B. penetrans was added to the

clots containing M. incognita and B. penetrans. This inoculum was produced

by air-drying and grinding tomato root systems previously colonized by M.

incognita parasitized by B. penetrans (Stirling and Wachtel 1980). The

original source of B. nenetrans was MacClenny, Florida.

Tobacco and then soybeans were planted in spring and summer 1982 to

test the hypotheses that the yields of the two crops increase as the path-

ogenicity of M. incognita decreases. The composite Wilcoxon test (Kepner

and Robinson 1982) was used to test the null hypothesis of no treatment

effect, H : T1 = 2 = T3, against the ordered alternative research hypoth-

esis, HI: T1 < T< T3, where at least one of the inequalities is strict.

Here, r1 is the effect of the presence of nematodes on yield, T2 is the

effect of the presence of both nematodes and bacteria on yield, and 73 is

the effect of having no nematodes or bacteria on yield. A significant

omnibus test for ordered alternatives was followed with point estimates

of the differences between treatment effects based on one-sample median

estimators (Hollander and Wolfe 1973). If the omnibus test was not sig-

nificant, no point estimates were to be made. Samples of nematodes from

soil and roots were taken during the growing of the two crops. The effect

on the nematode population of adding B. oenetrans to soil was analyzed

with the Wilcoxon signed rank test (Hollander and Wolfe 1973). The pro-

portions of infected nematodes were compared by normal approximation.

On 26 March 1982, 150 g of inoculum of B. penetrans were incorporated

into the top 2.5 cm of soil in the plots containing M. incognita and B.

penetrans. Soil samples were taken from all plots to determine the









densities of nematodes (CHAPTER 1), and six tobacco seedlings were trans-

planted into each plot. During the growing of tobacco, samples of nema-

todes in soil were taken 60 and 77 days (final harvest) after transplanting,

and samples of nematodes in roots were taken 77 days after transplanting.

The nematodes in soil were estimated from 15 soil cores (2.5-cm diam x

25-cm deep) collected randomly 15 cm from the planting sites of tobacco.

The cores were mixed, and the nematodes were extracted as described pre-

viously (CHAPTER 1) from a random subsample of 250 cm3 of soil. The nema-

todes in roots were estimated from a random sample of 1 g of roots/plot.

The samples of roots were stained with acid fuchsin in lactophenol to aid

in counting the nematodes (Southey 1970). Tobacco leaves were harvested

four times at 7-day intervals beginning 56 days after transplanting.

Leaves were dried at 65 C for 7 days before weighing.

After the last tobacco harvest, the control plots were treated with

methyl bromide (210 g/m2) under plastic to kill the contaminating nematodes.

On 23 June 1982, 12 days after the final harvest of tobacco, soybeans were

planted in the plots in two 2.4-m rows spaced 40 cm apart. Seven days

later the seedlings were thinned to a 5-cm to 7.5-cm spacing. The final

sample of nematodes in soil for tobacco was used as a preplant sample for

soybeans. Soil samples were processed for nematodes at 39 and 118 days

(harvest) after planting. The nematodes in roots were estimated, as de-

scribed for tobacco, 39 days after planting. Soybean seed weight was

reported at 11% seed moisture.


Determination of a Dosage-Response Function for Inoculum of Bacillus penetrans

Biological assays of the inoculum were conducted to determine the

percentage of juveniles of M. incognita infected when varying amounts of









inoculum were used. Zero, 10, 100, and 1000mg of inoculum were added to 30-ml

plastic cups and mixed with 30 g of quartz sand; each amount of inoculum

was replicated five times. The mixture of inoculum and sand was moistened

with 10 ml of distilled water and incubated for 3 days at 28 C. Then

1000 juveniles of M. incognita in water were injected into each cup and

incubated for 3 days at 28 C. The juveniles were extracted from the cups

by modifications (CHAPTER 1) of the sieving and centrifugation procedure of

Jenkins (1964), and a random sample of 50 juveniles was examined (at 200X)

for attached spores of B. penetrans.

A third-order-polynomial regression model was fit to this data using

the Statistical Analysis System (SAS) to express the percentage of infected

nematodes as a cubic function of the base 10 log of 1 mg more than the

weight (in mg) of the inoculum (Fig. 2.1).A residual analysis then was

performed to determine which, if any, of the model assumptions failed to

be satisfied (Neter and Wasserman 1974). The observation y = 14 at log

(100 + 1) = 2 was determined to be an outlier by fitting a cubic regression

to the remaining n-1 observations and constructing a 95% prediction interval

for a new random response at log (100 + 1) = 2. Because the response y = 14

fell outside of this prediction interval, it was considered as not having

come from the same population as the other n-1 observations and was de-

leted from further study.


Attachment of Spores to Meloidogyne javanica, M. incognita and
M. arenaria

Biological assays were used to determine the relative attachment of

spores to three species of Meloidogyne: M. javanica, M. incognita and M.

arenaria. Potting soil (90.6% sand, 3.9% silt and 5.5% clay) infested

with B. penetrans was used in the assays using the plastic-cup procedure














90-


70*




50-




30-




10-


y:- 49 x + 91 x2- 24 x3
(0.7

1 2 3
INOCULUM LOG (mg + 1)


Figure 2.1 Dosage-response function for inoculum of Bacillus
penetrans added to field plots on 26 'larch 1983.
The observation y = 14 was considered an outlier
(P <.05).









previously described with four replications for each species. The inde-

Dendence of the percentage of infected juveniles and species was investi-

gated with the log likelihood ratio G statistic of Sokal and Rohlf (1981).



Results


Field Experiments

The test of Kepner and Robinson (1982) indicated a trend of increas-

ing yields with decreasing pathogenicity of M. incognita for both tobacco

and soybean (Table 2.1). The attained significance level of the tests was

P = .M04 for tobacco and P = .008 for soybeans (the attained significance

level of a test if the probability of observing a value of the statistic

at least as extreme as the value observed). The dry leaf weight of tobacco

from plots infested with nematodes and bacteria minus that from plots in-

fested with nematodes alone was 235 g, and between the control minus the

treatment with nematodes alone was 962 g. The increase in yield for plots

infested with nematodes and bacteria over those infested with nematodes

alone was 24% of the increase in yield observed for the control plots over

the plots infested with nematodes alone. The seed weight of soybeans from

plots infested with nematodes and bacteria minus that from plots infested

with nematodes alone was 149 a, and between the control minus the treatment

with nematodes alone was 431 g. The increase in yield for plots infested

with nematodes and bacteria over those infested with nematodes alone was 35%

of the increase in yield observed for the control plots over the plots in-

fested with nematodes alone.

The Wilcoxon signed rank test indicated fewer nematodes in the plots

with nematodes and bacteria than in the plots with nematodes alone at the
















Table 2.1 -


Yields of tobacco and soybean planted in sequence in non-
infested soil and soil infested with Meloidogyne incognita
alone and M. incognita plus Bacillus penetrans.


Weight (g)
Treatment Tobacco leaf (dry) Soybean seed

Control 968 1000 1062 1076* 1475 1365 1301 1412

Nematodes +
emates + 733 379 273 102 1165 1050 1194 1043
Bacteria

Nematodes 60 228 0 0 934 942 913 1107


Values are
Values are


from four blocks of a randomized, complete-block design.








118-day (harvest) sample of soybean soil (P = .062). In the remaining

samples of soil and roots, there was insufficient evidence to enable one

to declare that the numbers of nematodes in the plots with nematodes and

bacteria and with nematodes alone were different (Tables 2.2 and 2.3).

The proportion of infected juveniles in the soil sample taken from

the treatment with M. incognita plus B. penetrans before planting tobacco

was 39 of 381 (10.2%). The proportions of infected juveniles in the final

soil samples of each crop were tobacco, 309 of 1647 (18.8%) and soybeans,

192 of 901 (21.3%). Soybeans had a greater proportion of infected juveniles

than that of tobacco (P = .063). Samples of mature females from roots col-

lected at harvest of tobacco and 39 days after planting soybeans were ex-

amined (at 400X) for spores within the body. The proportions of females

with spores within the body were tobacco, 8 of 40, and soybeans, 3 of 40.

The proportion of females containing spores from roots of soybeans was

lower than that of tobacco (P = .0495).


Determination of a Dosage-Response Function for the Inoculum

A third-order-polynomial regression function for the percentage of

infected juveniles (y) versus the log of 1 mg more than the weight (in mg)

of the inoculum (x) is y = -49x + 91x 24x3 (Fig. 2.1).

Attachment of Spores to Meloidogyne javanica, M. incognita
and M. arenaria

Spores of population of B. penetrans attached to the cuticle of all

three nematode species. The proportions of infected juveniles of the three

species of Meloidogvne were M. javanica, 157 of 200; M. incognita, 95 of

200; and M. arenaria, 105 of 200. A greater proportion of juveniles of M.

javanica were infected than were those of either M. incognita or M. arenaria,

and the corresponding proportions for M. incognita and M. arenaria were not











Table 2.2 Meloidoqyne incognita in soil and roots 0, 60 and 77 days
after planting tobacco.


Days
After
Planting


Treatment


Block

1
2
3
4


Control Nematodes + Bacteria


(0,-)*
(0,-)
(0,-)
(0,-)


(63,-)
(1,-)
(0,-)
(0,-)


(252,63)
(0,2)
(12,5)
(0,0)


(79,-)
(98,-)
(36,-)
(168,-)


(16,-)
(95,-)
(94,-)
(207,-)


(881,115)
(203,228)
(358,172)
(205,254)


Nematodes

(48,-)
(61,-)
(68,-)
(64,-)


(69,-)
(314,-)
(155,-)
(143,-)


(167,163)
(389,92)
(57,0)**
(25,0)**


In the ordered pair (A,B), A is the number of juveniles/25 cm3 of
soil, and B is the number of nematodes/g of fresh roots. A dash
(-) for B indicates that samples were not taken.
**
No roots were present.











Table 2.3 Meloidogyne incognita in soil and roots 39 and 118 days
after planting soybean.


Treatment


Control

(0,0)*
(0,0)
(0,0)
(0,0)


(0,-)
(0,-)
(0,-)
(0,-)


Nematodes + Bacteria


(55,196)
(54,97)
(9,55)
(20,134)


(142,-)
(254,-)
(208,-)
(297,-)


Nematodes

(13,241)
(33,136)
(11,143)
(7,80)


(271,-)
(371,-)
(786,-)
(330,-)


* 3
In the ordered pair (A,B), A is the number of juveniles/25 cm of
soil, and B is the number of nematodes/g of fresh roots. A dash
(-) for B indicates that samples were not taken.


Days
After
Planting


Block


118









found to be different. The significance level for this family of compari-

sons (the probability of making at least one TyDe I error in the four con-

clusions) was a = .04. Nearly all infected M. javanica and the majority of

infected M. incognita had more than 15 attached spores; the infected M.

arenaria had fewer than seven attached spores, except for one specimen

that was heavily infected.



Discussion


The increased yields of tobacco and soybeans from field plots infested

with B. penetrans and M. incognita over those infested with M. incognita

alone agree with previous field experiments (CHAPTER 1) and provide further

supporting evidence that B. penetrans is pathogenic to M. incognita under

field conditions. The addition of B. penetrans at the beginning of this

experiment may have contributed to the increased incidence of infection of

juveniles in soil; however, this incidence of infection did not affect yield

appreciably when compared with those reported previously (CHAPTER 1).

A tentative conclusion that B. Denetrans reduces the population size

of M. incognita could be drawn from the samples of juveniles in soil at

the harvest of soybeans. However, the size of the population at a given

time is dependent on so many factors that a generalization is difficult

to make. For example, a high initial infestation of juveniles may damage

a host to such a degree that subsequent lack of food limits the increase

in nematode numbers. On the other hand, if juveniles are in some way

inhibited from penetrating roots or developing once within the roots,

damage to the host may be less and juveniles that do successfully complete









their life cycles may produce more offspring due to less competition. As

a result of this early protection of the host, the size of the population

may increase later in the season. Future research should emphasize studies

on the mode of action of B. penetrans to root-knot nematodes.

This population of B. penetrans attaches to M. javanica and M. arenaria

as well as M. incognita. Assuming that attachment of spores indicates a

host-pathogen relationship between the organisms, this population would be

promising for use against the three most important Meloidogyne spp. (M.

javanica, M. incognita, M. arenaria) in Florida (R. A. Dunn, personal

communication). Moreover, since this population of B. penetrans attached

in greater numbers to M. javanica than to M. incognita, it may be more

effective against M. javanica than M. incognita.

In the laboratory, a dosage-response experiment showed an increase

in infection with increasing inoculum until a maximum in infection was

reached. Then infection decreased as the amount of inoculum increased.

If a similar function describes this relationship in field soil, a maxi-

mum concentration of B. penetrans may not have been reached in the present

study because the infection of juveniles in soil increased during the

tobacco and soybean seasons. Even though the full potential of B. penetrans

in preventing the oarasitism of M. incognita may not have been reached,

the consistent benefit of B. penetrans in this and the previous year

(CHAPTER 1) indicates that B. penetrans may be a good component of con-

trol programs where integrated techniques and a combination of pathogens

are used to reduce M. incognita damage to crons.

For example, experiments using a combination of antagonists such as

B. penetrans, Dactylella oviparasitica Stirling and Mankau, Paecilomyces

lilacinus (Thom) Samson and Nematophthora gynoohila Kerry and Crump against









Meloidognye spp. might be practical. Dactylella oviparasitica is a para-

site of M. incognita eggs (Stirling and Mankau 1978). Paecilomyces

lilacinus is a parasite of M. incognita eggs and females (Jatala et al.

1980). Nematophthora gynophila is a parasite of Heterodera spp. females

(Kerry and Crump 1977) and has been found parasitizing M. acronea Coetzee

(Kerry and Mullen 1981). These organisms should be tested against the

economically important Meloidogyne spp. If B. penetrans and N. gynophila

could be produced on a large scale, extensive field testing could be ini-

tiated. Perhaps organisms already described should be evaluated alone

and in combination with other organisms, and the search for other pathogens

should be continued.



Summary


The pathogenicity of Bacillus penetrans to Meloidogyne incognita was

tested under field conditions. Bacillus oenetrans was added to concrete-

bordered plots infested with M. incognita and B. penetrans into which

tobacco and soybeans were planted in sequence. Both crops showed trends

of increased yields with decreasing pathogenicity of M. incognita. Yields

from plots infested with M. incognita and treated with B. penetrans were

24% greater for tobacco and 35% greater for soybeans than those from

plots infested with M. incognita alone. Soil samples taken at the harvest

of soybeans revealed fewer second-stage-juvenile nematodes in plots infested

with B. penetrans than the nematode-infested plots without B. penetrans.

Spores from this population of B. penetrans attached to M. javanica, M.

incognita and M. arenaria.













CHAPTER 3
EFFECT OF Bacillus penetrans ON MOVEMENT OF AND
ROOT PENETRATION BY Meloidogyne incognita



Introduction

Possible modes of action (impaired motility, reduced root penetration

and reduced natality) of Bacillus Denetrans to Meloidogyne spp. were sug-

gested in previous studies (Prasad 1971, CHAPTERS 1, 2). The purpose of

the studies reported in this chapter was to determine the effects of B.

penetrans on root penetration by and movement of M. incognita.



Materials and Methods

Root Penetration by Meloidogyne incognita
Infected with Bacillus penetrans

The effect of B. Denetrans on the penetration of M. incognita into

tomato roots (Lycopersicon esculentum Miller 'Rutgers') was investigated

using a modified penetration inhibition test (Bunt 1975). Ten cubic cen-

timeters of quartz sand (99.3% sand, 0.3% silt and 0.4% clay with a sand

particle size distribution of 22% 1-0.5 mm, 41.4% 0.5-0.25 mm, 35.4% 0.25-

0.1 mm and 0.5% 0.1-0.05 mm) and 4 ml of water were added to 25-ml glass

vials into which 50 juveniles of M. incognita in water were injected.

There were two treatments: juveniles infected with B. penetrans and

juveniles not infected (control). Each treatment was replicated 25 times.

The infected juveniles were obtained by adding healthy juveniles to

100 cm3 of B. penetrans-infested potting soil (90.6% sand, 3.9% silt and









5.5% clay) that had been moistened and incubated for 3 days at 28 C prior

to the addition of nematodes. Then the juveniles were extracted from

the soil by modifications (CHAPTER 1) of the sieving and centrifugation

method of Jenkins (1964). The juveniles used for the controls were

treated identically except that they were added to soil not infested with

B. penetrans. A random sample of 30 juveniles from each treatment was

examined (at 200X) to determine the number of attached spores before add-

ing juveniles to the vials. Twenty-five juveniles from the infested soil

had more than 20 spores attached to their cuticles. Of the remaining five

juveniles,onehad 13 spores,onehad 10 spores and three had one spore each. None

of the 30 juveniles from noninfested soil had spores on their cuticles.

Twenty-four hours after infesting the vials with M1. incognita, tomato

seedlings were planted in each vial. The vials were placed randomly in

an incubator at 28 C and a cycle of 13 hr light (260 Lux) and 11 hr dark.

After 7 days the galls on the roots were counted, and the effect on the

formation of galls by nematodes parasitized by B. penetrans was tested

with the Wilcoxon rank sum test (Hollander and Wolfe 1973).

Immediately after counting the galls the seedlings were transplanted

into 10-cm-diam clay pots containing potting soil and allowed to develop

further in a greenhouse. After 14 days in the greenhouse, the seedlings

were removed from the pots, and the root systems were stained with acid

fuchsin in lactophenol to aid in counting the nematodes (Southey 1970).

This procedure was undertaken to corroborate that the galls were formed

by nematodes in established feeding sites.

The effect of B. penetrans on root penetration by M. incognita was

investigated further under greenhouse conditions. Tomato plants were









inoculated with healthy or infected juveniles, which were obtained as

described previously. In three experiments, which were carried out for

either 14 or 30 days, either 35 or 325 juveniles were added to the six

pots of each treatment (Table 3.1).

At the end of the experiments, the plants were removed from the pots

and when 325 juveniles were used a 4-g random sample of roots was stained

with acid fuchsin in lactophenol. When 35 juveniles were used, the entire

root system was stained. The numbers of nematodes within the roots in the

two treatments were compared with the Wilcoxon rank sum test.


movement of Meloidogyne incognita Infected with Bacillus penetrans

The effect of B. Denetrans on the movement of M. incognita was inves-

tigated in polyvinyl chloride (PVC) tubes filled with 50 g of quartz sand

or nothing soil (Table 3.2). One-half inch PVC tubes (18-mm diam) were

cut and glued into 0.5-in PVC 90 elbows. In half of the tubes, polyester

screen with 13-pm openings was glued onto the ends of the tubes before

they were glued into the elbows. The tubes then were cut to fit flush

into a 0.5-in PVC coupling. The resulting tube had an internal diameter

of 18 mm and a length of 105 mm from center to center of the elbow open-

ings.

A tomato seedling was planted into the soil at the end of the tube

nearest the polyester screen. The screen restricted passage of roots and

nematode juveniles. The tubes were placed horizontally for 2, 3 or 6 days

in an incubator as described previously. Infected or noninfected juveniles

then were injected into the end of the tube farthest from the screen and

the tubes were incubated for 7 days. Each treatment was replicated four

times.












Table 3.1 -


The effect of number of juveniles and number of juveniles
with spores of Bacillus Denetrans on root penetration by
Meloidogyne incognita.


Duration
(days)


Exot.


Number of
juveniles/pot





325


Number of
spores/juvenile*

0
1
2
5
10
12
15
16
>20


Number of
juveniles

10
8
3
1
1
1
1
1
4


No spores were observed on juveniles from the control.












Table 3.2 -


The effect of number of juveniles and number of juveniles
with spores of Bacillus penetrans on the movement of
Meloidogyne incognita.


Soil
Expt. type


Days seedlings
present before
juveniles


Number of
juveniles/tube


Number of
spores/juvenile*


Number of
juveniles


1 sand


1500


2 sand


3 sand


Dotting


600


3
5
7
11
>20


0
1
2
3
6
9
10
15
>20


1500


3000


observed on juveniles from the control.


No snores were









The infected juveniles were obtained as described previously except

for the last experiment in which potting soil infested and not infested

with B. penetrans was used. In this experiment, noninfected-second-stage

juveniles not older than 2 days were added to the

tubes to determine whether the previous extraction process or quartz

sand were inhibiting the movement of juveniles.

After 7 days the juveniles in each half of the tube were extracted

from the soil, and the proportions of juveniles from each treatment moving

to the half of the tube with the tomato seedling were compared by normal

approximation. When sand was used in the tubes, the nematodes were ex-

tracted by mixing the sand with water and decanting into a Millipore

Swinnex-25 with a polyester screen of 10-nm openings. When 1500 juveniles

per tube were used, the screen was washed in 10 ml of water. When 600

juveniles per tube were used, the screen was washed in 5 ml of water.

In each case the nematodes in a 1-ml sample were counted in a Peters

counting slide. When potting soil was used in the tubes, the nematodes

were extracted by sieving and centrifugation.



Results

In the penetration experiment in the laboratory, the Wilcoxon rank

sum test indicated fewer galls on the roots of tomato plants inoculated

with second-stage juveniles of M. incognita infected with B. penetrans

(Fig. 3.1) than on tomato plants inoculated with noninfected juveniles

(Table 3.3). The attained significance level of the test was D <.001 (the

attained significance level of a test is the probability of observing a

value of the statistic at least as extreme as the value observed). The





41



































2 Imr




Figure 3.1 Spores of Bacillus penetrans attached to the cuticle of
a second-staqe juvenile of Meloidogyne incognita.
















Table 3.3 Number of galls on tomato roots 7 days after inoculation
with 50 juveniles of Meloidoqyne incognita noninfected
(control) and infected with Bacillus penetrans.


Control
Number of
qalls/plant


Frequency


Infected
Number of
galls/plant


Frequency

17

7









total number of galls formed on the roots after 7 days was 10 for the

infected nematodes and 61 for the control nematodes; after 14 days, the

number of infected nematodes in the roots was nine compared with 101

noninfected nematodes.

In two of three penetration experiments in the greenhouse, fewer

infected then noninfected nematodes developed in tomato roots (Experiment

1, P = .066; Experiment 3, P = .004) (Table 3.4). No difference could be

claimed between the number of infected and noninfected nematodes in tomato

roots in Exoeriment 2 (P = .197).

In the movement experiments, the proportion of infected juveniles

moving to the half of the tube with the tomato seedling was less than that

of the noninfected juveniles in Exoeriment 1 (P <.001) (Table 3.5). In

the remaining experiments there was insufficient evidence to enable one

to declare a smaller proportion of infected juveniles than noninfected

juveniles moving to the half of the tube containing the tomato seedling

(Experiment 2, P = .92; Exoeriment 3, P = .90; Exneriment 4, P = .40).

Of the samples of juveniles from the infected populations that were

recovered from the half of the tube containing a olant, two of 20 were in-

fected with B. nenetrans (one with 15 spores and the other with five spores).



Discussion


The decrease in root penetration of juveniles of M. incognita when

infected with B. Denetrans establishes one effect of B. nenetrans on nema-

todes (Tables 3.3, 3.,). Prasad (1971) reported that greenhouse tomatoes

inoculated with M. incognita had fewer galls on roots when grown in soil

containing B. Denetrans; juveniles of M. javanica that were heavily






44





Table 3.4 Numbersof h1eloidoqyne incognita in greenhouse tomato roots
14 or 30 days after inoculation with juveniles not infected
(control) and infected with Bacillus penetrans.


ExDt.


Control


Infected

8
24
32
32
38
53


Nematodes/4 g of roots.
Nematodes/root system.
Nematodes/root system.
















Table 3.5 Proportions of juveniles of Meloidogyne incognita not
infected (control) and infected with Bacillus penetrans
that moved more than 5.25 cm toward a tomato plant.


Exot.


Control


12/164


7/183


1/259


4/202


Infected*


1/164


14/201


3/173


2/120


*
Only two of the 20 juveniles that moved more than 5.25 cm toward a
tomato plant had snores attached to the cuticle;onehad 15 spores
and the other had five spores. Both juveniles were observed in
Experiment 2.









infected with B. penetrans formed four to six galls on tomato roots after 60

days in a greenhouse, while noninfected juveniles produced numerous galls.

In other treatments of Prasad's study, juveniles not heavily infected

(not quantified) produced a moderate number of galls on tomato roots.

This experiment suggests that the inhibition of root penetration is relat-

ed to the number of spores attached to a nematode.

The relationship of the number and degree of infected juveniles in

a population and the inhibition of root penetration needs further investi-

oation. Also, when infected juveniles establish feeding sites in root

tissue, the effects of B. oenetrans on developing nematodes (such as up-

take of nutrients, natality and longevity) need to be quantified.

Reduced movement toward plant roots by juveniles of M. incognita when

infected with B. Denetrans (Table 3.5) may explain in part the inhibition

of root penetration by M. incognita (Tables 3.3, 3.4). Prasad (1971)

demonstrated that the number of juveniles of M. javanica extracted from

soil containing B. penetrans was less than that from soil without the

bacterium. He collected 279 of 2000 M. javanica from Baermann funnels

containing soil infested with B. Denetrans compared with 1719 of 2000

from s o i l without the bacterium. One possible explanation of

these results is impaired motility of the juveniles after infection with

B. penetrans. Additional experiments should be carried out to determine

the causes of the reduced directed movement of nematodes infected with B.

penetrans. Two possible effects of B. penetrans on directed movement of

N. incognita might be impaired motility and disorientation.

The proportions of noninfected juveniles of 1. incognita that moved

5 to 10 cm toward tomato plants (Table 3.5) were smaller than those re-

ported in a previous study of horizontal movement of M. javanica.









Prot (1976) demonstrated that approximately 40% of juveniles of M. javanica

penetrated tomato roots in 5 days when placed 10 cm horizontally from them.

T he a u t h o r w a s unable to determine the reasons for the

poor movement of M. incognita in the present studies. Although movement

was poor in these experiments, most of the nematodes in both treatments

that moved toward the tomato plants were not infected. This observation

suggests that movement of infected juveniles is impaired.



Summary


Laboratory and greenhouse experiments were conducted to determine the

effect of B. penetrans on root penetration and movement of M. incognita.

In a laboratory experiment, juveniles of N. incognita infected with B.

penetrans caused fewer galls (a total of 10) to form on the roots of

tomato plants than did noninfected juveniles (a total of 61). In two

greenhouse experiments B. nenetrans reduced the number of nematodes in

tomato roots. In a third experiment there was insufficient evidence to

declare a lower number of nematodes in tomato roots inoculated with juve-

niles infected with B. penetrans than in roots inoculated with noninfected

juveniles. In one of four laboratory experiments B. penetrans reduced

the percentage of juveniles moving toward tomato plants from seven to one.

In the remaining three experiments there was insufficient evidence to

claim that B. penetrans reduced the directed movement of M. incognita.














DISCUSSION AND CONCLUSIONS


The increased crop yields in four of five experiments resulting from

adding B. penetrans to plots containing an established population of M.

incognita, demonstrated that B. penetrans reduced the pathogenicity of

the population of M. incognita. The effect of B. oenetrans on M. incognita

now can be extended from greenhouse conditions (Prasad 1971) to field

conditions.

The proportion of M. incognita juveniles with spores on the cuticle

increased through the field experiments. It is conjectured that this in-

crease in incidence of infection indicates that the number of B. penetrans

in the soil increased. The addition of B. Denetrans at the beginning of

the second year of experiments (CHAPTER 2) may have contributed to the

increased incidence of infection of juveniles in soil. Alexander (1981)

reviewed studies of the addition of bacteria to natural ecosystems that

suggested that for bacteria that persist in soil, there is a characteristic

number that is maintained. Determining this characteristic number for B.

penetrans in agricultural soil and, in turn, the optimum effect of that

concentration of B. oenetrans on a population of plant-parasitic nematodes

would be an interesting continuation of the present study.

A dosage-response experiment in the laboratory showed an increase in

infection of juveniles with increasing inoculum until a maximum in infec-

tion ,was reached. Then infection decreased as the amount of inoculum

increased. If a similar function describes this relationship in field

soil, a maximum concentration of B. penetrans may not have been reached









in the present study because of the continued increase in the incidence

of infection of juveniles during the experiments. Even though the full

potential of 3. penetrans in Dreventing the parasitism of M. incognita

may not have been reached, the consistent benefit of B. penetrans during

the two growing seasons indicates that B. penetrans may be a good compo-

nent in a control program where pathogens, resistant varieties, crop

rotations and other techniques are used to reduce M. incognita damage

to crops.

Experiments using combinations of antagonists such as B. penetrans,

Dactylella oviparasitica Stirling and Mankau, Paecilomyces lilacinus

(Thon) Samson and Nematophthora gynophila Kerry and Crump against

Mleloidogyne sop. should be undertaken. Dactylella oviparasitica is a

parasite of M. incognita eggs (Stirling and Mankau 1978). Paecilomyces

lilacinus is a parasite of M. incognita eggs and females (Jatala et al.

1980). Nematophthora gynophila is a parasite of Heterodera soD. females

(Kerry and Mullen 1981). These organisms need to be tested against the

economically important Meloidogyne sop., and efforts to culture B. pene-

trans and N. gynophila should be initiated. Perhaps organisms already

described should be reevaluated as well as continuing the search for

biological control organisms.

A tentative conclusion that B. penetrans reduces the population size

of M. incognita could be drawn from the samples of juveniles in soil at

the midseason of tobacco (1931) and at the end of the soybean season

(1992). Two of three greenhouse experiments also supported this conclu-

sion. However, the size of the population at a given time is dependent

on so many factors that a generalization is difficult to make. For example,

a high initial infestation of juveniles may damage a host to a degree that









subsequent lack of food limits the increase in nematode numbers. On the

other hand, if juveniles are in some way inhibited from penetrating roots

or developing once within the roots, damage to the host may be less and

juveniles that do successfully complete their life cycle may produce more

offspring due to less competition. As a result of this early protection

of the host, the size of the population may increase later in the season.

The decrease in root Denetration by juveniles of 1. incognita when

infected with B. penetrans establishes one effect of B. penetrans on nema-

todes (CHAPTER 3). These results aaree with Prasad (1971) who reported

that greenhouse tomatoes inoculated with M. incognita had fewer galls on

roots when grown in soil containing B. penetrans than when grown in soil

without B. penetrans and that juveniles of M. javanica that were heavily

infected with B. penetrans formed less than six galls on tomato roots after

60 days in a greenhouse, while noninfected juveniles produced numerous

galls. The relationship of the number and degree of infected juveniles

in a population and the inhibition of root penetration needs to be inves-

tigated. Also when infected juveniles establish feeding sites in root

tissue, the effects of B. penetrans on developing nematodes (such as up-

take of nutrients, natality and longevity) need to be quantified.

The reduced movement toward plant roots of juveniles of M. incognita

when infected with B. penetrans (CHAPTER 3) may explain in part the inhi-

bition of root penetration of M. incognita. The proportions of noninfected

juveniles of M. incognita that moved 5 to 10 cm toward tomato plants were

smaller than those reported in a previous study of horizontal movement

of M. javanica. Prot (1976) demonstrated that approximately 40% of the

juveniles of M. javanica penetrated tomato roots in 5 days when placed 10

cm horizontally from the tomato plants. I w a s unable to determine









the reasons for the ooor movement of [I. incognita in the present studies.

Prasad (1971) demonstrated that the number of M. javanica extracted from

soil containing B. penetrans was substantially less than that from soil

without B. penetrans. One possible explanation of these results is

impaired motility of the juveniles after infection with B. penetrans.

Additional experiments should be carried out to determine the importance

of reduced directed movement of nematodes infected with B. oenetrans.

The population of B. penetrans used in this study also attaches to

I. javanica and M. arenaria. Assuming that attachment of spores indicates

a host-pathogen relationship between the organisms, this population would

be promising for use against the three most important Meloidogyne spn.

(M. javanica, I. incognita, 1. arenaria) in Florida (R. A. Dunn, person-

al communication). Moreover, since this population of B. nenetrans

attached more readily to M. javanica than to M. incognita, it might be

possible to use it more effectively against '. javanica than M. incognita.














LITERATURE CITED


Alexander, M. 1977. Soil microbiology. 2nd ed. John Wiley & Sons,
New York. 467 o.

Alexander, M. 1981. Wihy microbial predators and parasites do not elim-
inate their prey and hosts. Ann. Rev. Microbiol. 35:113-133.

Allen, M. W. 1957. A new species of the genus Dolichodorus from Cali-
fornia (Nematoda: Tylenchida). Proc. Helminthol. Soc. Wash. 24:
95-98.

Bergey's Manual of Determinative Bacteriology. 1974. 8th ed. R. E.
Buchanan and N. E. Gibbons, eds. Williams & Wilkins Co., Baltimore.
1268 p.

Bunt, J. A. 1975. Effect and mode of action of some systemic nematicides.
Meded. Landbouwhogeschool, Wageningen. 75-10:1-127.

Cobb, N. A. 1906. Fungus maladies of the sugarcane, with notes on asso-
ciated insects and nematodes. Hawaiian Suqar Planters Assoc. Expt.
Sta. Div. Path. Physiol. Bull. 5:153-195.

Cross, T. 1970. The diversity of bacterial spores. J. Appl. Bacteriol.
33:95-102.

Cross, T., P. D. Walker, and G. W. Gould. 1968. Thermophilic Actino-
mycetes producing resistant endospores. Nature 220:352-354.

Davidson, E. W. 1973. Ultrastructure of American foulbrood disease
pathogenesis in larvae of the worker honey bee, Apis mellifera.
J. Invert. Pathol. 21:53-61.

Dorokhova, L. A., N. S. Agre, L. V. Kalakoutskii, and N. A. Krassilnikov.
1968. Fine structure of spores in a thermophilic actinomycete
Micromonospora vulgaris. J. Gen. Appl. Microbiol. 14:295.

Dutky, E. M. 1978. Some factors affecting infection of plant parasitic
nematodes by a bacterial spore parasite. M.S. Thesis. Univ. of
Maryland, College Park. 44 o.

Franklin, IM. T. 1979. Economic importance of Meloidoqvne in temperate
climates. Pages 331-339 in F. Lamberti and C. E. Taylor, eds.
Root-knot nematodes (Meloidogyne species) systematics, biology
and control. Academic Press, New York. 477 D.









Hollander, M., and D. A. 1olfe. 1973. Nlonoarametric statistical methods.
John Uiley & Sons, New York. 503 p.

Hussey, R. S., and K. R. Barker. 1973. A comparison of methods of col-
lecting inocula of Meloidogyne spp., including a new technique.
Plant Dis. Reptr. 57:1025-1028.

Jatala, P., R. Kaltenbach, M. Bocanael, A. J. Devaux, and R. Campos. 1980.
Field application of Paecilomyces lilacinus for controlling Meloidogyne
incognita on potatoes. J. Nematol. 12:226-227. (Abstr.)

Jenkins, W. R. 1964. A rapid centrifugal-flotation technique for separ-
ating nematodes from soil. Plant Dis. Reptr. 48:692.

Kawanishi, C. Y., C. M. Splittstoesser, and H. Tashiro. 1978. Infection
of the European chafer, Amnhimallon majalis, by Bacillus ponilliae:
Ultrastructure. J. Invert. Pathol. 31:91-102.

Kepner, J. L., and D. H. Robinson. 1982. A distribution-free rank test
for ordered alternatives in randomized complete block designs. Tech.
Rep. No. 176, Department of Statistics, Univ. of Florida.

Kerry, B. R., and D. H. Crumo. 1977. Observations on fungal parasites of
females and eggs of the cereal cyst-nematode, Heterodera avenae, and
other cyst nematodes. Nematologica 23:193-201.

Kerry, B. R., and L. A. Mullen. 1981. Fungal parasites of some plant
parasitic nematodes. Nematropica 11:187-189.

Lamberti, F. 1979. Economic importance of Meloidogyne spo. in subtropi-
cal and Mediterranean climates. Pages 341-357 in F. Lamberti and
C. E. Taylor, eds. Root-knot nematodes (Meloidogyne species) system-
atics, biology and control. Academic Press, New York. 477 p.

Mankau, R. 1975. Bacillus penetrans n. comb. causing a virulent disease
of plant-narasitic nematodes. J. Invert. Pathol. 71:333-339.

Mankau, R., J. L. Imbriani, and A. H. Bell. 1976. SEM observations on
nematode cuticle penetration by Bacillus penetrans. J. Nematol.
8:179-181.

Neter, J., and W. Wasserman. 1974. Applied linear statistical models.
Richard D. Irwin, Inc., Homewood, IL. 842 n.

Prasad, N. 1971. Studies on the biology, ultrastructure and effective-
ness of a sporozoan endoparasite of nematodes. Ph.D. Dissertation.
Univ. of California, Riverside. Available from: University rMicro-
films, Ann Arbor, MI; Publication No. 72-10,388. 108 D.

Prot, J. C. 1976. Amplitude et cinetique des migrations du nematode
Meloidoqyne iavanica sous l'influence d'un plant de tomate. Cah.
ORSTOM, Ser. Biol. 11:157-166.









Sasser, J. N. 1979. Economic importance of Meloidoqvne in tropical
countries. Pages 359-374 in F. Lamberti and C. E. Taylor, eds.
Root-knot nematodes (Meloidoqyne species) systematics, biology
and control. Academic Press, New York. 477 p.

Sasser, J. N. 1980. Root-knot nematodes: A global menace to crop pro-
duction. Plant Dis. 64:36-41.

Sayre, R. M., and W. P. Wergin. 1977. Bacterial parasite of a plant
nematode: Morphology and ultrastructure. J. Bacteriol. 129:1091-
1101.

Slack, J. M., and M. A. Gerencser. 1975. Actinomyces, filamentous
bacteria: Biology and pathogenicity. Burgess Publishing Co.,
Minneapolis. 158 p.

Sokal, R. R., and F. J. Rohlf. 1981. Biometry. 2nd ed. W. H. Freeman
& Co., San Francisco. 776 p.

Southey, J. F., ed. 1970. Laboratory methods for work with plant and
soil nematodes. Tech. Bull. 2. Ministry of Agriculture, Fisheries
and Food, London. 148 p.

Splittstoesser, C. M., C. Y. Kawanishi, and H. Tashiro. 1975. Germina-
tion and outgrowth of Bacillus Pooilliae in hemolymoh slide mounts.
J. Invert. Pathol. 25:371-374.

Splittstoesser, C. M., C. Y. Kawanishi, and H. Tashiro. 1978. Infection
of the European chafer, Amphimallon majalis, by Bacillus popilliae:
Light and electron observations. J. Invert. Pathol. 31:84-90.

Stirling, G. R., and R. Mankau. 1978. Parasitism of Meloidogyne eggs
by a new fungal parasite. J. Nematol. 10:236-240.

Stirlina, q. R., and ". F. Wachtel. 1980. Mass production of Bacillus
penetrans for the biological control of root-knot nematodes.
Nematologica 26:308-312.

Thorne, G. 1940. Duboscqia oenetrans, n. sp. (Sporozoa, Microsporidia,
Nosematidae), a parasite of the nematode Pratylenchus oratensis (de
Man) Filipjev. Proc. Helminthol. Soc. Wash. 7:51-53.














BIOGRAPHICAL SKETCH


Stephen Michael Brown was born in Glendale, California, in 1946.

He received a Bachelor of Science degree with distinction in zoology

and a Master of Arts dearee in bioloay from San Diego State University

with the first year of his graduate studies spent at the Free University

of Berlin, !West Germany.

After teaching for several years, he was hired by the United Fruit

Company to study the Dlant-oarasitic nematodes of banana in Central

America. This position brought about his matriculation at the Univer-

sity of Florida to pursue a Doctor of Philosophy degree in nematology.

Stephen is a member of the American Phytopatholoqical Society, the

American Statistical Association, the National Audubon Society, the

Organization of Tronical American Nematologists, the Sierra Club, Sigma

Xi, the Society of Nematologists and the Southeastern Biological Control

Working Group.














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




Grover C. Smart, Jr., Chairman
Professor of Entomology and Flematology





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.




Don W. Dickson
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.


--- _- ". --. -

Robert P. Esser
Assistant 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 Philosoohy.




David J. Mitchell
Professor of Plant Patholoqy


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 scooe and quality, as a dissertation for the degree of
Doctor of Philosophy.




Edward P. Previc
Associate Professor of M!icrobiology
and Cell Science





This dissertation was submitted to the Graduate Faculty of the College
of Agriculture and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.


April 1983


LDean, C


grilture
Agriclture


Dean for Graduate Studies and Research




University of Florida Home Page
© 2004 - 2010 University of Florida George A. Smathers Libraries.
All rights reserved.

Acceptable Use, Copyright, and Disclaimer Statement
Last updated October 10, 2010 - - mvs