Biological control of fusarium crown rot of tomato

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Biological control of fusarium crown rot of tomato
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Fusarium crown rot of tomato
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Marois, James J
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Tomato wilts   ( lcsh )
Tomatoes -- Diseases and pests   ( lcsh )
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Thesis--University of Florida.
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Includes bibliographical references (leaves 51-54).
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by James J. Marois.
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Typescript.
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Vita.

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University of Florida
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BIOLOGICAL CONTROL OF FUSARIUM CROWN ROT OF TOMATO


BY

JAMES J. MAROIS
















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


1980












ACKNOWLEDGMENTS


The author would like to thank David Mitchell, Eric Moore, Ronald

Sonoda, and James Thomas, for this work would not have been so enjoyable,

or even possible, without the patience and tolerance they have demon-

strated during the course of these studies.

The love, friendship, and expert advice provided by the author's

wife, Kathy, have made the difficult times pass with little notice, and

the good times treasured forever.

The author especially is grateful towards his parents, for the

guidance and support they have provided so consistently.













TABLE OF CONTENTS


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

ABSTRACT ........................................................ iv

SECTION I. EFFECTS OF FUMIGATION AND ANTAGONISTIC SOIL FUNGI ON THE
RELATIONSHIPS OF INOCULUM DENSITY TO INFECTION INCIDEUCE
AND DISEASE SEVERITY IN FUSARIUM CROWN ROT OF TOMATO

Introduction ....................................... 1

Materials and Methods ................................... 3

Results ........................................ ..... 6

Discussion .............................................. 11

SECTION II. EFFECT OF FUNGAL COMMUNITIES ON THE PATHOGENIC AND
SAPROPHYTIC ACTIVITIES OF FUSARIUM OXYSPORUM F. SP.
RADICIS-LYCOPERSICI

Introduction ....................................... ... 15

Materials and Methods ................................... 17

Results ................................................ 21

Discussion ...................................... ........ 32

SECTION III. BIOLOGICAL CONTROL OF FUSARIUM CROWN ROT OF TOMATO
UNDER FIELD CONDITIONS

Introduction ........................................ 38

Materials and Methods ................................ 39

Results .......................... ............. 41

Discussion ................................... .... 48

LITERATURE CITED ................................... ... 51

BIOGRAPHICAL SKETCH ...................................... .... 55

iii













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


BIOLOGICAL CONTROL OF FUSARIUM CROWN ROT OF TOMATO

by

James J. Marois

June, 1980

Chairman: David J. Mitchell
Major Department: Plant Pathology

Fusarium crown rot of tomato was controlled effectively with a

composite of several biological agents under growth-chamber, greenhouse,

and field conditions. The biological agents were selected for their

abilities to proliferate in freshly fumigated soil, to establish high

populations in the root zone of the host, and to interact with the

pathogen to reduce the incidence of infection or disease. The antago-

nists selected were three isolates of Trichoderma harzianum, one isolate

of Penicillium restrictum, and one isolate of Aspergillus ochraceus.

Chlamydospores of the causal agent, Fusarium oxysporum f. sp.

radicis-lycopersici, were formed under axenic conditions so that defined

concentrations of specific inocula could be added to freshly fumigated

soil. The relationship of inoculum density to incidence of infection

was determined under growth-chamber conditions. The inoculum concen-

trations of the pathogen at which 50% of the plants were infected were

300, 900, and 6500 chlamydospores per gram of soil which had been

iv







fumigated, not fumigated, or fumigated and infested with antagonists,

respectively. In greenhouse experiments the mean lesion length on

stems increased as the inoculum density was increased in fumigated soil;

lesion length, however, did not increase as the inoculum density was

increased in fumigated soil with antagonists added. In nonamended soils,

the disease incidence and mean lesion length were 70% and 2.22 cm,

respectively, at the highest inoculum density, 5 X 10 chlamydospores

per pot. At that inoculum density in antagonist amended soils, the

incidence of disease and mean lesion length were 44% and 0.96 cm,

respectively.

In field experiments, disease incidence increased as the inoculum

density of the pathogen was increased in soils that were fumigated but

not amended with the antagonists; disease incidence, however, did not

increase as the inoculum density was increased in soils that were

fumigated and amended with the antagonists. At 5000 chlamydospores of

the pathogen per plant, disease incidence at harvest was 7% in soils

amended with the antagonists and 37% in nonamended soils. The pathogen

population decreased from 600 to 200 propagules per gram of soil during

the growing season in the soils amended with the antagonists, but

increased from 1000 to over 5 X 104 propagules per gram of soil in non-

amended soils.

Control of disease was attributed to the abilities of the antago-

nists to inhibit the saprophytic activities of the pathogen. The

population of the pathogen was stable or decreased in fumigated soil

that had been amended with the antagonists, but the pathogen population

increased 20-fold in nonamended soils. Microbial interactions which

V







influenced succession during the early recolonization stages of treated

soils explain the observations that the activities of the pathogen were

more closely related to the total number of fungal propagules detected

in soils than to any single soilborne fungal species. Successful con-

trol of Fusarium crown rot of tomato with biological agents was depen-

dent upon the production practices, the biology of the pathogen, and the

methods used for selection and application of the antagonists.













SECTION I
EFFECTS OF FUMIGATION AND ANTAGONISTIC SOIL FUNGI ON THE
RELATIONSHIPS OF INOCULUM DENSITY TO INFECTION INCIDENCE
AND DISEASE SEVERITY IN FUSARIUM CROWN ROT OF TOMATO


Introduction

Fusarium crown rot of tomato (Lycopersicon esculentum Mill.),

caused by Fusarium oxysporum Schlecht f. sp. radicis-lycopersici Jarvis

and Shoemaker, is a disease which is severe when tomatoes are grown in

soil treated with biocides (14,15,28). This phenomenon fits Kreutzer's

(17) concept of disease trading in which dominant pathogens are con-

trolled by soil treatments but minor pathogens are elevated to major

importance because they can recolonize soil in which their competitors

and antagonists have been eliminated. The traditional methods of

applying fungicides directly to plants growing in previously treated

soil have proved ineffective in the control of Fusarium crown rot (27).

Rowe and Farley (27), however, controlled the disease with the applica-

tion of captafol to freshly steamed soil before planting. The success-

ful results were attributed to the selective action of the fungicide

which inhibited reinvasion by the pathogen but did not adversely affect

the recolonization of the soil by other airborne microorganisms.

Thompson (36), as early as 1929, realized that chemical agents

would be useful in disease control mainly where conditions are rela-

tively unfavorable for the pathogen and that biological agents would be

more important when the environment is conducive to activity of the

1






2
pathogen. In Florida, an environment conducive to the development of

Fusarium crown rot is established when plastic mulch is applied during

fumigation and maintained during the entire growing season. If antago-

nists of F. oxysporum f. sp. radicis-lycopersici could be introduced

under the plastic before the soil is recolonized by the pathogen, it

should be possible to reduce disease. Thus, the nature of the pathogen

and tomato production methods provide an excellent system for a quanti-

tative field study of biological control.

Before field studies are undertaken, however, the importance of

fumigation and recolonization of soil by antagonists can be evaluated

critically by quantitatively determining the relationships of the

pathogen and antagonists to incidences of infection and disease in

fumigated or nonfumigated soil under growth-chamber and greenhouse

conditions. The relationships of inoculum density to disease severity

in root rots caused by Fusarium spp. are well documented (1,7,11,31).

Baker (3) proposed that biological effects of antagonists on disease

could be quantified by the analyses of curves derived by plotting dis-

ease severity to inoculum density of the pathogen. Significantly

greater inoculum densities of the pathogen should be required to cause

a proportionate amount of disease when antagonists are present.

The quantification of inoculum required the development of a pro-

cedure in which defined levels of chlamydospores of the pathogen could

be established in freshly fumigated soil. The present procedure used

in soil density studies with Fusarium spp. involves placing plants in

soil with populations of the pathogen established by assaying artifi-

cially infested, aged soil with selective media and diluting the as-

sayed infested soil with noninfested soil (11). This procedure is not






3
applicable to a system which demands freshly treated soil because popu-

lations of many microorganisms can become established during the time

required for the aging of soil infested with the pathogen. An alterna-

tive to this method is to infest soil with chlamydospores produced under

axenic conditions and quantified by direct count; a subsequent estima-

tion of the population can be obtained by soil dilution plating.

The objectives of this study were: 1) to determine the relation-

ships of densities of chlamydospores of F. oxysporum f. sp. radicis-

lycopersici to the incidence of infection in fumigated and nonfumigated

soils, 2) to determine the effects that selected antagonists have on

the relationship of inoculum density to the incidence of infection, and

3) to determine the effects of antagonists on disease severity. The

techniques and procedures were developed so that they can be applied to

any disease which is severe after fumigation due to decreased competitor

populations and subsequently increased pathogen populations.


Materials and Methods

The isolate of F. oxysporum f. sp. radicis-lycopersici was obtained

from a diseased tomato plant collected in a south Florida field.

Cultures were stored in soil tubes according to the method of Toussoun

and Nelson (37).

Pompano fine sand was treated with methyl bromide-chloropicrin

(67/33% v/v) at the rate of 1 kg of fumigant to 50 kg of soil for 2 days

in a sealed container and then allowed to air in the greenhouse for 4

days.

Chlamydospores of the pathogen were used as inoculum to simulate

natural conditions in which chlamydospores of Fusarium spp. are the





4
major survival structure (37). For the production of chlamydospores,

macroconidia were washed from 2-wk-old cultures grown on potato dextrose

agar (Difco, Detroit, MI 48201) at 25 C under continuous fluorescent

light (3000 lux). The macroconidia formed intercalary chlamydospores

after 4 wk of incubation at 106 macroconidia per milliliter of auto-

claved deionized water at 28 C in the dark.

The potential antagonists were isolated from recolonized soils 1 wk

after fumigation. A soil dilution of 1 g of air-dried soil in 1 liter

of water was plated on potato dextrose agar which contained 1 ml of

Tergitol NPX (Sigma Chemical Co., St. Louis, MO 63178) and 50 mg of

chlortetracycline hydrochloride (Sigma Chemical Co., St. Louis, MO 63178)

per liter of medium (PDA-TC). Conidial suspensions of each isolate were

obtained by washing 2-wk-old cultures grown on potato dextrose agar at

25 C under 10 hr of fluorescent light (2000 lux) per day. Each suspen-

sion then was added to 1 kg of freshly fumigated soil (final water

concentration = 10% wt/wt). One-half kilogram of the infested soil then

was placed in a plastic container and stored at 25 C for 1 wk after

which the population density of each isolate was determined by dilution

plating on PDA-TC. The remaining soil that had been infested with an

individual isolate was placed in 100-ml polypropylene beakers at 80 g

of soil per beaker. Two germinated 'Bonnie Best' tomato seeds were

placed in each beaker, and the beakers were moved to growth chambers set

at 20 C and 12 hr of light (4000 lux) per day. After 2 wk the roots

were washed lightly and plated on PDA-TC. One week later the number of

colonies of each potential antagonist growing from the roots was used to

evaluate its ability to occupy the root environment. Those isolates






5
which increased populations rapidly in the freshly fumigated soil and

occupied the root environment then were tested for their potential to

increase the ratio of inoculum density to infection incidence in pre-

liminary growth-chamber experiments. Of the 26 isolates which fulfilled

the first two requirements, the five that were selected for the rest of

the tests included three isolates of Trichoderma harzianum Rafai, one

isolate of Penicillium restrictum Gilman and Abbott, and one isolate of

Aspergillus ochraceus Wilhelm.

Concentrations of conidia of the antagonists and chlamydospores of

F. oxysporum f. sp. radicis-lycopersici were determined by counting 40

fields of a standard hemocytometer, and the desired dilutions were added

to soil.

The relationships of inoculum density to infection incidence were

determined by experiments done in growth chambers. Two germinated

'Bonnie Best' tomato seeds were placed in a 100-ml polypropylene beaker

which contained 60 g of infested soil layered over 50 g of autoclaved

sand. A range of inoculum densities of the pathogen was used and a

composite of the antagonists was added at the constant concentration of

5000 conidia per isolate per gram of air-dried soil. Twenty-four

beakers of each inoculum combination then were placed in growth

chambers. After 2 wk the soil was washed from the roots; the plants

were soaked in 0.6% sodium hypochlorite for 1 min and rinsed in auto-

claved deionized water. The roots and lower stem were plated on

Komada's (16) selective medium for F. oxysporum and observed after 10

days for colonies of the fungus. Populations of the pathogen in the

soil from the beakers also were determined after 2 wk of incubation in

the growth chamber. The populations of the pathogen were quantified by





6
dilution plating of Komada's (16) medium. The pathogenic isolates were

identified by the technique of Sanchez et al. (29), in which the type

of lesion on tomato seedlings grown in pathogen-infested water agar is

used to differentiate the isolates of the pathogen from nonpathogenic

and wilt inducing F. oxysporum isolates.

In the greenhouse experiments, 5-wk-old tomato ('Walter') trans-

plants were placed individually in plastic pots (15-cm diameter) con-

taining fumigated soil. A 4-mil-thick plastic film was placed over the

soil to simulate the plastic mulch used in production fields. The

transplant was planted through a 3-cm hole in the center of the plastic

film. Fifty milliliters of a suspension containing 5 X 105 conidia of

each antagonist were poured into the planting hole and over the trans-

plant's crown and roots. Ten milliliters of a chlamydospore suspension

of the pathogen were injected into the soil at each of two points

approximately 7 cm from the plant. The plants were fertilized with

half-strength Hoagland's (12) solution every 2 wk and watered when

necessary. After 12 wk the root weight, infection incidence, disease

incidence, and lesion length were recorded.

The data presented in this section are means of experiments

repeated two times. Each replicate consisted of 48 plants per treat-

ment in the growth-chamber studies and 15 plants per treatment in the

greenhouse studies.


Results

Percentages of infection, percentages of diseased tomato plants,

and mean lesion lengths increased with increasing inoculum levels of the

pathogen (Fig. 1, Table 1). In the growth-chamber experiments, the





7
ratio of inoculum density to infection incidence was lowest in fumigated

soils and highest in fumigated soils with the antagonists added (Fig.

1-A).

Slopes of regression lines plotted with data from the growth-

chamber experiments of Log10 (Loge 1/1-X), where X is the proportion of

infected plants, on Log10 inoculum density were 0.82 (r = 0.98), 0.98

(r = 0.97), and 0.99 (r = 0.99) with fumigated soil, nonfumigated soil,

and fumigated soil plus antagonists, respectively (Fig. 1-B). The

inoculum densities required for 50% infection of plants (ID50) in each

soil were interpolated to be approximately 300, 900, and 6500 chlamydo-

spores per gram of air-dried soil in fumigated soil, nonfumigated soil,

and fumigated soil plus antagonists, respectively.

When the initial inoculum density of the pathogen was 500 chlamydo-

spores per gram of air-dried soil, populations increased after 2 wk to

4000 propagules per gram of soil in fumigated soil, remained constant

in nonfumigated soil, and decreased to 50 propagules per gram of soil

that had been fumigated and amended with antagonists.

In the greenhouse experiments, the antagonist amendment reduced

significantly (P = 0.05) the mean lesion length and the incidence of

disease (Table 1). The analysis of varience also showed that the inoc-

ulum density of the pathogen and the pathogen inoculum density-antagonist

interaction significantly affect disease incidence and mean lesion

length (P = 0.01). There were no significant correlations between

treatments and root weight or percent infection. All of the treatments,

including the controls, had infection incidences of over 90%.







































5 10 15 20 40 60 80
CHLAMYDOSPORES X 102/SOIL
Figure 1 A. The relationship of percentages of infection of tomato
('Bonnie Best') under growth-chamber conditions to densities of
chlamydospores of Fusarium oxysporum f. sp. radicis-lycopersici in
fumigated soil (0---0), nonfumigated soil ((- -K), and fumigated soil
amended with Trichoderma harzianum, Aspergillus ochraceus, and
Penicillium restrictum ---9 .. ).






























0 zz

R .

0.05


0.01"



0 .0 I i i I i i i i i i ,i i i i
20 100 1000 700C
CHLAMYDOSPORES /g SOIL

Figure 1 B. The relationship of percentages of infection adjusted
for multiple infections (logarithmic) of tomato ('Bonnie Best')
under growth-chamber conditions to densities of chlamydospores
(logarithmic) of Fusarium oxysporum f. sp. radicis-lycopersici in
fumigated soil (0---0), nonfumigated soil (- -K), and fumigated
soil amended with Trichoderma harzianum, Aspergillus ochraceus, and
Penicillium restrictum (- ...).












Table 1. Effect of initial inoculum density of Fusarium oxysporum f. sp.
radicis-lycopersici and a composite of five antagonists on mean lesion
length and percentage of plants with lesions under greenhouse conditions.

Percentage
Inoculum density Mean lesion of plants
(chlamydospores per pot)x Antagonists length (cm) with lesionsy

0 0.00 0

500 0.98 50

5000 1.73 69

50000 2.22 70

mean 1.23a 47a

0 +z 0.14 4

500 + 0.73 33

5000 + 0.68 33

50000 + 0.96 44

mean 0.63 b 28 b

20 ml of a chlamydospore suspension were injected into the soil 7 cm
from the plant.
YMeans in same column with different letters differ significantly
(P = 0.05) as determined by t test; percentage data analyzed after
transformation to arcsine4T
z+ = conidia of each of five isolates (three isolates of Trichoderma
harzianum, one isolate of Penicillium restrictum, and one isolate of
Aspergillus ochraceus) were added to the crown area of the transplant
at 5 X 105 conidia of each isolate per pot.









Discussion

The relationships of inoculum density to disease incidence have

been applied in the quantification of several soilborne diseases caused

by Fusarium spp. (1,7,11). In this study the ID50 in nonfumigated soil

was 900 chlamydospores per gram of soil, which is similar to the ID50s

found in other diseases caused by Fusarium spp. in nontreated soils

(7,11). The ID50 in the fumigated soil was 300 chlamydospores per gram

of soil; however the pathogen population increased from 500 chlamydo-

spores to 4000 propagules per gram of soil during the experiment. Guy

and Baker (11) reported a similar ID50 when the pathogen population

increased due to a chitin soil amendment. High ID50s (2000 or more

conidia per gram of soil) were reported by Abawi and Lorbeer (1) in

steam-treated and nontreated soils. However, the method for determining

disease severity was based on percent emergence rather than on the

percentage of infected or diseased tissue used in other investigations (1).

Also, conidia were used as the inoculum source rather than chlamydo-

spores, as in other studies. Their data, however, still indicate that

the lower ID50 occurred in treated soil. The position and slope of the

curve derived by the log-log transformation of inoculum density to

disease severity affect the ID50 of a particular disease system. Guy

and Baker (11) found that the addition of organic materials to the soil

altered the ID50 by shifting the position of the curve rather than

changing its slope, which they attributed to the relative changes in

infection rates being directly correlated with inoculum density. A

similar shift was noted in this study in the soils that were fumigated,






12
nonfumigated, or fumigated and amended with antagonists. In all soils

the slope was approximately 1.0.

The attenuation of isolates of Fusarium spp. grown on artificial

media must be considered in quantifying the relationship of inoculum

density to disease incidence and severity. As an isolate becomes less

virulent, more inoculum will be required to cause disease regardless of

the particular treatment. The axenic production of chlamydospores of a

pathogen reduces the chances of attenuation because the isolate does not

reproduce vegetatively between experiments; thus the possibility of

genetic variation is minimized. Changes in virulence of F. oxysporum f.

sp. radicis-lycopersici were not observed in any of the experiments.

The germination rate of chlamydospores of the fungus on potato dextrose

agar was not significantly different from 100% (P = 0.05) for up to one

year after their formation. French (8) reported that chlamydospores

formed from macroconidia of F. oxysporum f. sp. batatas remained viru-

lent for 7 yr when stored in water.

The application of a broad spectrum biocide to soil creates a

biological vacuum which disrupts the stability of the soil community.

The early recolonization pattern of treated soil involves a shift to an

early successional pioneer stage that consists of a few species which

occur in large numbers (42). Thus the community is of low diversity and

may be readily invaded by new species if the environmental conditions

are not severe. In this study the pathogen population remained stable,

as determined by dilution plating, in the more advanced seral stages of

nonfumigated soils. In the pioneer successional stage of fumigated soils,

however, the pathogen was able to compete and increase in population






13
density. The limited ability to compete as a saprophyte is charac-

teristic of other pathogenic Fusarium spp. (21,24,35).

The ability of other plant pathogens to compete as saprophytes in

treated soil also may be reduced with the reestablishment of the micro-

bial community (4,23,30,39). A decrease in the percentage of isolations

of Verticillium albo-atrum from seedlings of Senecio vulgaris was attrib-

uted to the recolonization of autoclaved soil by airborne propagules of

other microorganisms (30). This decrease is similar to that observed by

Rowe and Farley (27) with Fusarium crown rot of tomato.

The observed decrease in the population of the pathogen from an

initial inoculum density of 500 chlamydospores per gram of fumigated

soil amended with antagonists to 50 propagules per gram of soil may have

been caused by the production of toxins or by parasitism of the pathogen

by the antagonists. The increase in the ratio of inoculum density to

infection in the same soil may have been due to the adverse effects of

the antagonists on the growth of the pathogen or the successful competi-

tion of the antagonists at potential infection sites. The reduction in

the ability of the pathogen to infect roots is evident by the compar-

ison of the ID50s of 300 and 6500 chlamydospores per gram of soil in

fumigated soil and fumigated soil with antagonists added, respectively.

The reduced number of infected seedlings was correlated with the

decreased mean lesion lengths when antagonists were added as compared to

when the antagonists were not added in the greenhouse experiments. The

correlation of increased numbers of infections and the increase in

disease severity has been reported in other diseases caused by Fusarium

spp. (20,31,32).





14
The reduced infection in the growth-chamber experiments and the

reduced lesion length in the greenhouse experiments were due to the

partial restabilization of the treated soil and root environment by the

addition of the antagonists. The observation that nearly all of the

plants were infected, including the controls, in the greenhouse studies

was attributed to the long duration (12 wk) of the experiment and the

ability of the pathogen to spread as airborne inoculum. Rowe et al.

(28) found that under greenhouse conditions treated soil was rapidly

reinfested by the pathogen as airborne microconidia.

The success of reducing the severity of Fusarium crown rot of

tomato with biological control is dependent upon the reestablishment in

freshly treated soil of a microbial community that impairs the rein-

vasion by F. oxysporum f. sp. radicis-lycopersici. The host-pathogen

model employed in this study allowed the development of a system for the

selection of antagonists and the quantification of a biological control

procedure under growth-chamber and greenhouse conditions. This infor-

mation provides qualitative and quantitative bases for the application

of antagonists in the field in south Florida. In addition to the field

experiments, information is needed on the effects of the addition of

antagonists on recolonization by artificially and naturally introduced

microorganisms. The interactions of several microbial populations can

best be understood at the community level of the ecosystem and therefore

the concepts of community stability and succession will be applicable to

future studies on the quantification of biological control.














SECTION II
EFFECT OF FUNGAL COMMUNITIES ON THE PATHOGENIC AND SAPROPHYTIC
ACTIVITIES OF FUSARIUM OXYSPORUM F. SP. RADICIS-LYCOPERSICI


Introduction

The casual agent of Fusarium crown rot of tomato (Lycopersicon

esculentum Mill.), Fusarium oxysporum Schlecht f. sp. radicis-lycopersici

Jarvis and Shoemaker, is able to compete as a saprophyte in freshly

treated soils. Fusarium crown rot is associated with the rapid sapro-

phytic proliferation of the pathogen. Naturally and artificially

introduced soil recolonizers may reduce the severity of the epidemic by

impeding saprophytic proliferation (13,27).

The competitive saprophytic ability of a pathogen is affected by

the community of microorganisms which surrounds it. A soilborne plant

pathogen that grows poorly or not at all in nontreated soil may grow

readily as a saprophyte in soils in which the microbial community has

been disturbed recently, as by the application of a broad spectrum

biocide. The successional theory of community ecology states that a

series of communities, or sere, develop after such a perturbation (22).

Specific seral communities have certain characteristics in common,

regardless of whether they are plant, animal, or fungal communities.

The early successional communities are of low diversity and are domi-

nated by a few species, r-selected species, which grow rapidly and

quickly deplete the available resources. The success of a particular r-

selected species is dependent upon the absence of competitors (22).
15






16
In more diverse communities which develop later, the few r-selected

species are replaced by a number of k-selected species, which grow

slower and utilize the available resources over a longer period of time.

The later successional stages have communities of increased diversity

and stability (22). Although k-selected species coexist with other k-

selected species, the r-selected species are usually unsuccessful in the

presence of the more diverse k-selected communities. Control of an r-

selected pathogen may be possible by establishing other r-selected

species before the pathogen is introduced or by establishing k-selected

species before or after the introduction of the pathogen.

Since the early studies of biological control, biological agents

have been selected for their antagonistic properties toward the patho-

gen in various laboratory tests outside of the soil environment (2).

The results of these tests usually have not been repeatable under field

conditions. In the present series of experiments, antagonists were

selected for their abilities to proliferate in freshly treated soil, to

occupy the root environment of the host, and to increase the ratio of

inoculum density to infection incidence, as discussed in Section I. All

of these factors were evaluated using field soil, rather than agar, as

the growth medium. The selection for antagonists that were successful

competitors, rather than for those that produce toxins or show hyper-

parasitism on agar plates, may result in the successful control of other

plant diseases in which the epidemic is dependent upon the saprophytic

growth of an r-selected pathogen.

The objectives of this study were: 1) to determine the effect of

fungal communities on the saprophytic development of F. oxysporum f. sp.






17
radicis-lycopersici in soil, 2) to monitor the rate of fungal species

immigration into soils amended or not amended with the antagonists, and

3) to quantify the effects of fumigation and antagonists on the activity

of the pathogen in soil and on the host-pathogen interaction.


Materials and Methods

The ability of F. oxysporum f. sp. radicis-lycopersici to compete

as a saprophyte was quantified by monitoring the pathogen populations in

soils in the absence of the host. The pathogenic ability of the pathogen

in different soil communities was quantified as the percentage of host

plants infected after exposure to pathogen infested soil for 2 wk.

An isolate of F. oxysporum f. sp. radicis-lycopersici was obtained

from a diseased tomato plant collected in a south Florida field. Cul-

tures were stored in soil tubes according to the method of Toussoun and

Nelson (37).

Pompano fine sand was treated with methyl bromide-chloropicrin

(67/33% v/v) at the rate of 1 kg of fumigant to 50 kg of soil for 2 days

in a sealed container. The soil was aired in the greenhouse for 4 days

before further use.

Chlamydospores of the pathogen were formed from macroconidia under

axenic conditions and quantified by direct count with a standard hemo-

cytometer. The establishment of defined initial inoculum densities of

the pathogen in freshly fumigated soil was accomplished by methods

reported in Section I.

The antagonists used in the artificial infestation and recoloniza-

tion experiments included three isolates of Trichoderma harzianum Rafai,

one isolate of Penicillium restrictum Gilman and Abbott, and one isolate






18
of Aspergillus ochraceus Wilhelm. The antagonists were selected as

described in Section I.

Freshly fumigated soil was divided into five allotments of 20 kg

each (Table 2). The pathogen was added to three of the allotments at

1000 chlamydospores per gram of air-dried soil. Of the three allotments

infested with the pathogen, one was placed in plastic containers to

inhibit recolonization by airborne microorganisms, another was left

uncovered in the greenhouse to allow natural recolonization to occur,

and the third was left uncovered in the greenhouse and infested with the

antagonists at 1000 conidia of each of the five fungi per gram of air-

dried soil. The conidia were obtained as described in Section I. The

two allotments without the pathogen were left uncovered in the green-

house and one was infested with the antagonists as above. The pathogen

was also added at the above concentration to a sixth allotment which

consisted of nonfumigated soil. The water content of the soils was

maintained at approximately 10% by weight during the experiments.

The competitive saprophytic and pathogenic abilities of the patho-

gen in the different soil communities were quantified by monitoring its

population density in the soils and by determining its ability to infect

susceptible tomato seedlings, respectively. Every 7 days 1500 g of soil

from each allotment were removed. The population of F. oxysporum f. sp.

radicis-lycopersici then was determined in those soils previously

infested with the pathogen. The pathogen was added to those soil

samples which were not previously infested with the pathogen to determine

the saprophytic and pathogenic activities of chlamydospores of the

pathogen when introduced to previously recolonized soil. The population

of the pathogen in these soils was determined after 2 wk incubation in















Table 2. Treatments applied to 20-kg allotments of soil.

Soil Pathogen Natural
Treatment fumigated added AntagonistsY recolonizationz

1 + weekly +

2 + weekly + +

3 + initially

4 + initially +

5 + initially + +

6 initially N/A

+ = soil fumigated with methyl bromide-chloropicrin (67/33% v/v) at
1 kg of fumigant to 50 kg of soil for 2 days and then allowed to air
in the greenhouse for 4 days.
= soil not fumigated
Xweekly = Fusarium oxysporum f. sp. radicis-lycopersici was added at
1000 chlamydospores per gram of soil to a 1500 g soil sample obtained
every 7 days for the duration of the experiment.
initially = Fusarium oxysporum f. sp. radicis-lycopersici was added
to the entire soil allotment 4 days after fumigation at 1000 chlamydo-
spores per gram of soil.
Y+ = conidia of each of five antagonists (three isolates of Trichoderma
harzianum, one isolate of Penicillium restrictum, and one isolate of
Aspergillus ochraceus) added to the entire soil allotment at 1000 conidia
of each isolate per gram of soil 4 days after fumigation.
-= antagonists not added.
z+ = soils left uncovered to allow recolonization from naturally occur-
ring airborne inocula.
= soil placed in plastic containers to inhibit naturally occurring
recolonization.





20
growth chambers. The populations of the pathogen in the soils were

quantified by dilution plating on Komada's (16) medium, which is select-

ive for F. oxysporum. Pathogenic isolates were identified by the tech-

nique of Sanchez et al. (29), as described in Section I. The ability

of the pathogen to infect host plants grown in soil from the different

treatments was determined by placing two germinated 'Bonnie Best' tomato

seeds in a 100-ml polypropylene beaker which contained 60 g of soil

layered over 50 g of autoclaved sand. The beakers then were placed in

growth chambers at 20 C and watered every 48 hrs. After 2 wk, the soil

was washed from the roots; the roots and lower stem were soaked in 0.6%

sodium hypochlorite for 1 min, rinsed in autoclaved deionized water, and

plated on Komada's (16) medium. The plates were examined after 10 days

at 25 C. The plants were considered to be infected if F. oxysporum f.

sp. radicis-lycopersici grew from the crown area of the seedling.

The soil fungal communities were monitored by dilution plating of

soil samples on potato dextrose agar which contained 1 ml of Tergitol

NPX (Sigma Chemical Co., St. Louis, MO 63178) and 50 mg of chlortetracy-

cline hydrochloride (Sigma Chemical Co., St. Louis, MO 63178) per liter

of medium. The plates were incubated for 7 days at 25 C and 2000 lux of

fluorescent light. Benomyl (Benlate 50% WP, E. I. du Pont de Nemours

and Co., Wilmington DE 19898) was added to a replicate set of plates at

2.5 ppm when soil dilutions in water of 1:25 or lower were used to

inhibit the fast growing colonies of Trichoderma spp. Dilution series

ranged from 1:10 to 1:106 (wt:vol). The particular series of dilutions

used was dependent upon the expected populations of fungi.

The experiments were repeated at least twice; 10 petri plates were

used for each soil dilution and 48 tomato plants were used to determine

the incidence of infection for each experiment.









Results

The infection incidence of tomatoes by F. oxysporum f. sp. radicis-

lycopersici was correlated with the inoculum density of the pathogen in

nonamended (r = 0.99) and amended (r = 0.86) soils when the pathogen

was added to soil samples taken every 7 days during recolonization of

fumigated soils (Table 2, Treatment 1,2). Populations of the pathogen

and infection incidence were correlated (r = 0.93 and 0.90, respectively)

with the natural logarithm of the total number of fungal propagules

detected in the nonamended soils. The inoculum density of the pathogen

and infection incidence also were correlated (r = 0.89 and 0.94, respect-

ively) with the natural logarithm of the total number of fungal propa-

gules detected in the amended soils. No population of a single genus of

antagonists had a correlation greater than r = 0.85 with either the

infection incidence or inoculum density of the pathogen.

The proportion of infected tomato plants was directly related to

the ability of the pathogen to proliferate in soils which contained

different fungal communities (Fig. 2,3). In the recolonization experi-

ments, the highest infection incidence and the highest pathogen popula-

tions occurred 4 days after fumigation (Fig. 2,3). The populations of

naturally occurring recolonizers were low or not detected in the soil

at that time (Table 3,4). In both amended and nonamended soils,

saprophytic growth of the pathogen and infection incidence decreased

with time after fumigation. The incidence of infection eventually

stabilized at approximately 1% in amended and nonamended soils. The

rapid decrease in both the population of the pathogen and in the

incidence of infection in nonamended soils by 18 days after fumigation





22
occurred as natural populations of Trichoderma spp. increased 400-fold

between 11 and 18 days after fumigation (Table 4).

The rate of immigration of naturally occurring fungal species was

slower in soil amended with antagonists than in nonamended soil (Table

3,4). Thirty-nine days after fumigation, Cladosporium spp. and a wet

spored Mucor sp. were the only naturally occurring species isolated

from amended soils, whereas ten different naturally occurring species,

including Trichoderma sp. and Penicillium sp., were isolated from non-

amended soils. It was not possible to determine if the dominating popu-

lations in amended soils were from the original introduced antagonists,

but the species were the same as those introduced.

The frequency of isolation of any particular species varied with

time and treatment (Table 3,4). Aspergillus ochraceus dominated amended

soils during the early stages of recolonization, and it accounted for

46% of the total number of propagules isolated 4 days after fumigation.

The majority of the isolates from amended soils 11 and 18 days after

fumigation were T. harzianum. Beginning 18 days after fumigation, 85%

of the total number of colonies were either P. restrictum or T.

harzianum. In amended soils, the introduced species always accounted

for at least 98% of the total number of fungi isolated.

Some species of fungi were isolated frequently in freshly fumigated

soil but occurred less frequently with time. In nonamended soils,

Geotrichum sp., Cephalosporium sp., and Cylindrocarpon sp. were not

isolated past 18 days after fumigation. Pythium sp., Syncephalastrum

sp., Cunninghamella sp., and Rhizopus sp. were not isolated from non-

amended soils until 39 days after fumigation (Table 4).





23
The maximum number of fungal propagules (approximately 2 X 105

propagules per gram of soil) in amended and nonamended soils was

similar when naturally occurring recolonization was allowed. The

population of F. oxysporum f. sp. radicis-lycopersici in fumigated soils

which had been infested with the pathogen and placed in plastic contain-

ers to inhibit naturally occurring recolonizers was higher than the

total fungal populations in any of the other treatments (Fig. 4). Under

these conditions the pathogen population increased to 10 propagules per

gram of soil 25 days after fumigation. By direct observation it was

determined that the pathogen population consisted predominantly of

microconidia.

When the pathogen was added 4 days after fumigation (Table 2,

Treatment 3,4), its population density increased and then decreased with

time in nonamended soils (Fig. 4,5). The proportion of infected plants

was not related to the population density of the pathogen in nonamended

soils. When natural recolonization was inhibited, the pathogen popula-

tion increased and then decreased as the incidence of infection remained

relatively constant at 100o (Fig. 4). When natural recolonization of

fumigated soil was not inhibited, infection decreased with time and the

population of the pathogen increased and then decreased (Fig. 6).

When the pathogen was introduced into soils that were either non-

fumigated or fumigated and amended with the antagonists (Table 2, Treat-

ment 5,6), the pathogen populations remained relatively stable (Fig. 6,

7). In fumigated, amended soils the infection incidence increased to

44% 11 days after fumigation and then decreased to approximately 23% by

39 days after fumigation. In nonfumigated soils, the infection incidence

remained relatively stable, and varied from 40 to 50% (Fig. 7).
















Table 3. Populations of fungi that recolonized fumigated soils amended
with one isolate of Aspergillus ochraceus, one isolate of Penicillium
restrictum, and three isolates of Trichoderma harzianum at 1000 conidia
of each isolate per gram of soil.

Propagules X 102/g of soil at days after fumigationx
Fungi 4 11 18 25 32 39 46

Penicillium spp. 0.0 0.1 0.0 0.2 0.2 0.1 0.2

P. restrictum 12.9 200.0 130.0 1500.0 950.0 400.0 350.0

Trichoderma spp. 0.1 0.2 0.3 0.2 0.1 1.0 0.4

T. harzianum 7.3 333.0 400.0 1350.0 110.0 723.0 430.0

A. ochraceus 17.4 250.0 27.0 19.0 23.0 213.0 62.0

Cladosporium spp. 0.2 1.2 8.0 14.6 10.0 6.6 9.5

Mucor spp. 0.0 0.0 0.0 0.4 0.5 0.4 0.3

Total 37.9 784.5 565.3 2884.4 1093.8 1344.1 852.4


Xpropagules X 102 per gram of air-dried soil detected in potato dextrose
agar which contained 1 ml of Tergitol NPX and 50 mg of chlortetracycline
hydrochloride per liter of medium.














Table 4. Populations of fungi that recolonized fumigated, nonamended
soils.


Fungi

Penicillium spp.

Trichoderma spp.

Aspergillus niger

Cladosporium spp.

Mucor spp.

Fusarium roseum

Pythium sp.

Rhizopus sp.

Cunninghamella sp.

Geotrichum sp.

Cephalosporium sp.

Cylindrocarpon sp.

Fusarium solani

Syncephalastrum sp.

Total


Propagules X
4 11

1.0 45.0

5.0

0.1

5.0 12.5


102/g of
18

40.0

2000.0

0.5

6.5

0.2

0.1


soil at
25

46.6

1720.0

0.8

8.5

0.6

0.1


days after fumigationx
32 39 46

170.0 170.0 380.0

330.0 1010.0 1380.0

0.3 0.3 4.3

9.0 10.0 28.2

0.8 0.9 0.3

0.1 0.2 0.7

0.1 0.3

0.1 0.1

0.1 0.1


2.5


0.2


0.1

6.4 67.6 2050.0 1776.6 510.2 1191.8


1.0

1795.0


XPropagules X 102 per gram of air-dried soil detected in potato dextrose
agar which contained 1 ml of Tergitol NPX and 50 mg of chlortetracycline
hydrochloride per liter of medium.








100 71 10
oo-oo-o






80 8
90- -9




0 0
C.,


30 3x


S 18 2 3
-0 1,

20 s a d at 1
0




10





4 II 18 25 32 39 46

DAYS AFTER FUMIGATION

Figure 2. The relationship of percentage of infection of tomato ('Bonnie
Best') (0-4) and inoculum density of Fusarium oxysporum f. sp. radicis-
lycopersici (0111110) to time after fumigation of soils which were allowed
to recolonize naturally; the pathogen was added at 1000 chlamydospores
per gram to 1500 g of soil every 7 days and tomato plants were maintained
in the infested soil for 14 days under growth-chamber conditions before
infection incidence and the inoculum density of the pathogen were
determined.







30


20- -2

o 5 r






10 \ 0 -I0
0

00












0 I I I ,0
0

D



z Cn











DAYS AFTER FUMIGATION

Figure 3. The relationship of percentage of infection of tomato ('Bonnie
Best') (0-0*) and inoculum density of Fusarium oxysporum f. sp. radicis-
lycopersici (0111110) to time after fumigation of soils which were amended
with three isolates of Trichoderma harzianum, one isolate of Aspergillus
ochraceus, and one isolate of Penicillium restrictum at 1000 conidia of
each isolate per gram of soil; the pathogen subsequently was added at
1000 chlamydospores per gram to 1500 g of soil every 7 days and tomato
plants were maintained in the infested soil for 14 days under growth-
chamber conditions before infection incidence and the inoculum density of
the pathogen were determined.









100 i06

......0



80-





60-



40 a
-o a









20 -


0- 03



4 11 18 25 32 39 46 53 60 67 74
DAYS AFTER FUMIGATION

Figure 4. The relationship of percentage of infection of tomato ('Bonnie
Best') (0---) and inoculum density of Fusarium oxysporum f. sp. radicis-
lycopersici (0111110) to time after fumigation of soils in which recoloniza-
tion by other microorganisms was inhibited; the pathogen was added 4 days
after fumigation at 1000 chlamydospores per gram of soil.
--_____ ay 0
4 aI1 53 94 36 77
DAY AFEaMGTO
Fiue .Te eaiosi of pecnag ofifcio ftmto(B
Best) (-^)an inclmdniyoauaim xsou .s.rdcs
-yoesc aOIH)t ieatrfmgto fsisi hc eooia
tion ~~~ byohrmcoognsswsinbtd h ahgnwsadd4dy
afe uiato at10 C)yopre e rmo ol



















40 \


U-U
z 2



-00
o 0 3 o






20 -
0 o










S
00 (1 )


















lycopersici (0111110) to time after fumigation of soils in which recoloniza-
tion by other microorganisms was not inhibited; the pathogen was added 4
days after fumigation at 1000 chlamydospores per gram of soil.
days after fumigation at 1000 chlamydospores per gram of soil.







30
7 3


50


2 2
3o-U

-30-
w I I I C
U-4 25 32
z M -














DAYS AFTER FUMIGATION
20





10- 0








Figure 6. The relationship of percentage of infection of tomato ('Bonnie
Best') (---*) and inoculum density of Fusarium oxysporum f. sp. radicis-
lycopersici (011110) to time after fumigation of soils which were amended
with three isolates of Trichoderma harzianum, one isolate of Aspergillus
ochraceus, and one isolate of Penicillium restrictum at 1000 conidia of
each isolate per gram of soil; the pathogen was added 4 days after
fumigation at 1000 chlamydospores per gram of soil.









50 6





40

-Q
z 2"
0 0
I30
w C





2 00
I04r






0- r
0 I I I 0







4 II 18 25 32 39
DAYS AFTER INFESTATION
Figure 7. The relationship of percentage of infection of tomato ('Bonnie
Best') (0-4) and inoculum density of Fusarium oxysporum f. sp. radicis-
lycopersici (0111110) to time in nonfumigated soils; the pathogen was added
on day 4 at 1000 chlamydospores per gram of soil.








Discussion

Decreases in the saprophytic activity of F. oxysporum f. sp.

radicis-lycopersici due to competition from naturally recolonized and

artificially amended soils similar to those observed in this study have

been reported by other investigators (13,27); however, the compositions

of the communities have not been examined. In this study, the fungal

recolonization of soils was monitored by soil dilution plating, and the

theories of successional mechanisms were invoked to explain why the

pathogen population decreased as recolonization continued.

For comparison purposes, the pathogenic and saprophytic activities

of the pathogen also were monitored in nontreated soils. Saprophytic

proliferation of the pathogen did not occur in nontreated soils. Nash

et al. (19) reported similar results when they added chlamydospores of

F. solani f. phaseoli to nontreated soils. The lack of chlamydospore

germination in natural soils has been attributed to insufficient nutri-

ents and to the presence of inhibitory substances in the soil (41).

The ability of the pathogen to infect the host, as determined by

the ratio of inoculum density to infection incidence, was higher in

nonfumigated soils than in fumigated soils that had been allowed to

undergo recolonization for 25 days. This phenomenon was termed induced

antagonism by Welvaert (42). The practical aspect of induced antagonism

is that it prolongs the effect of a soil treatment after the treatment

is no longer active. The decreases in infection incidence of tomato and

saprophytic proliferation of the pathogen in fumigated soils were corre-

lated with the microbial population that formed during recolonization.

The relationship of the inoculum density of F. oxysporum f. sp.

radicis-lycopersici to the infection incidence of tomato was influenced






33
critically by the different soil treatments. In fumigated soils in

which the pathogen was added every 7 days and in nonfumigated soils,

pathogen population and infection incidence were related. The inoculum

density of the pathogen was not related to infection incidence when the

pathogen was maintained in fumigated, nonamended soils; in fumigated,

amended soils; or in fumigated soils in which recolonization by other

organisms was inhibited. The ability of the pathogen to infect the

host decreased with time in fumigated, nonamended soils. Although the

pathogen population increased, the incidence of infection did not in-

crease. In fumigated, nonrecolonized soils the incidence of infection

was nearly 100%o during the duration of the experiments. The high infec-

tion incidence was attributed to the high efficiency of the pathogen for

host infection in the absence of competing organisms. The decrease of

the pathogen population in the nonrecolonized soil probably was due to

the exhaustion of nutrient sources or to the accumulation of toxic metab-

olites. At present, it is not possible to explain the results obtained

when the pathogen was maintained in fumigated, amended soils. The path-

ogen population was relatively stable, but the infection incidence in-

creased and then decreased with time. The low incidence of infection 4

days after fumigation, when the pathogen and antagonists were first

added, may have been due to antagonisms by A. ochraceus, which was the

predominant antagonist at that time. Perhaps the population of T.

harzianum, which was dominant when the incidence of infection was high,

was not as capable of restricticting the pathogenic activities of the

pathogen. The decrease in infection incidence with time may have been

due to increased antagonism as the combination of populations of P.

restrictum and T. harzianum increased during the later stages of succes-

sion.






34
The total number of fungal populations detected increased rapidly

after fumigation and then decreased with time. Kreutzer (17) attributed

the rapid increase in populations of recolonizing fungi to the avail-

ability of nutrients. Wilson (43) hypothesized that the rapid increase

in organisms following fumigation or other severe perturbation was the

result of the growth of noninteractive populations, and that the

eventual decrease in populations was the result of interactive popula-

tion growth. In the noninteractive stages of recolonization, competi-

tion is low because of the large niche space available. In the inter-

active stages, however, the populations interact with each other and the

effect is an overall reduction in the total number of individuals.

Further evidence for the application of the noninteractive model to soil

recolonization was obtained when the pathogen was allowed to grow as a

saprophyte in the absence of other soil recolonizers. The highest num-

ber of total fungal propagules in any of the experiments (106 propagules

of the pathogen itself per gram of soil) was obtained when the pathogen

was maintained in a noninteractive environment by the exclusion of other

competing species. The noninteractive model may explain why the highest

inoculum density of total fungal propagules was not associated with the

lowest inoculum density of the pathogen or with the lowest infection

incidence of the host. Conversely, inoculum density of the pathogen and

the infection incidence of the host were closely related to the total

populations in amended soils; this was expected because the artificially

introduced isolates were selected specifically for their antagonistic

actions toward the pathogen, as described in Section I.

In fumigated soils to which the pathogen was added every 7 days,

there were higher correlations of infection or inoculum density with






35
the total number of fungi (r = 0.89 to 0.94) than with any single species

(r S 0.85). The presence of a specific organism was not as influential

on the activity of the pathogen as was the total, composite fungal

community. The higher correlations obtained when the activity of the

pathogen was compared to total number of detected fungi agrees with

Park's (25) conclusion that the probability of an organism coming in

contact with a single species is much less likely than the organism

interacting with the entire community. The lack of specific antagonism

of any one species towards the pathogen was further supported when

several isolates of the major recolonizing species did not show antago-

nism towards the pathogen on nutrient agar (unpublished results). The

changes in species composition and dominance in nonamended soils were

attributed to different successional stages of recolonization. Fungal

succession in treated soil has been the subject of several reviews (4,17,

42). The mechanism of succession usually is similar to that proposed by

Garrett (9) for microbial succession on plant debris, in which the avail-

able carbon source determines which organisms are dominant. In opposi-

tion to reports of fungal succession are instances in which single spe-

cies remain dominant during the entire exploitation of the substrate

(5,10,18,40). This occurs when a species is introduced artificially at

high inoculum densities to a substrate prior to colonization by other

organisms. Bruehl and Lai (5) reported that the advantage of prior colo-

nization of wheat straw increased the competitive saprophytic ability of

several fungi. The importance of prior colonization in soil systems is

evident in these studies by the decreased immigration rates by naturally

occurring fungal species in amended soils. In addition, when the patho-

gen was added to freshly fumigated soil, it was able to proliferate






36
for up to 25 days after fumigation; conversely, when added to fumigated

soils which had undergone recolonization for 25 days, it was unable to

compete as a saprophyte.

The apparent contradiction in the literature over the presence of

succession may be explained by examining different models of the mecha-

nisms of succession. Connell and Slatyer (6) have proposed three differ-

ent mechanisms of succession following a perturbation. In all three

models the earlier species cannot invade or grow after the site is fully

occupied by the same or later occurring species. In the facilitation

model, later species can become established only after earlier inhabi-

tants have suitably modified the environment. This model explains why

secondary invaders are detected only after a plant pathogen has invaded

the healthy tissues of the host. According to the tolerance model,

which is similar to the model proposed by Garrett (9), the later species

can establish themselves because they can utilize nutrients at lower

levels than earlier recolonizers. According to the inhibition model,

later species cannot grow in the presence of earlier species and their

establishment is dependent upon their ability to survive longer and

gradually replace the earlier species. The results of this study and

the literature indicate that succession in artificially infested sub-

strates follows the inhibition model, but that in nonamended substrates

succession follows the tolerance model.

A basic principle of community ecology is that the success of a

species, such as a plant pathogen, is dependent upon its ability to

interact successfully with the abiotic and biotic factors of its sur-

rounding environment. Because fumigation practices create an environ-

ment conducive to the proliferation of the pathogen, a possible means of






37
control is to establish a community which is inhibitory to the pathogen

in the treated soil. Fusarium oxysporum f. sp. radicis-lycopersici is

an r-selected species, as indicated by the rapid increase and then

decrease of its population density in the absence of other organisms;

therefore, its ability to compete as a saprophyte is dependent upon the

total propagule density of all interacting microorganisms in the soil

(22). The saprophytic proliferation of the pathogen was controlled by

adding r-selected antagonists to freshly fumigated soil before the re-

invasion of the pathogen could occur. By the application of the basic

concepts of community ecology to the control of plant disease and the

utilization of composites of antagonists, the success of biological

control investigations may increase, and disease control in the field

may be realized more frequently.













SECTION III
BIOLOGICAL CONTROL OF FUSARIUM CROWN
ROT OF TOMATO UNDER FIELD CONDITIONS


Introduction

Fusarium crown rot of tomato was first reported in south Florida

during the 1974-1975 growing season (33). Attempts to control the

disease with chemicals and host resistance have been unsuccessful (27).

At present, the only effective control measure is the application of a

captafol drench to greenhouse beds immediately after steaming (27). The

captafol drench selectively inhibits recolonization of the soil by the

pathogen, Fusarium oxysporum Schlecht f. sp. radicis-lycopersici Jarvis

and Shoemaker (15). When captafol was applied as a preplant or post-

plant drench to the transplant hole under south Florida field conditions,

the estimated yield was slightly higher, but there was a 1 wk delay in

plant maturation (34). Furthermore, a complete soil drench of the entire

bed is not practical to tomato production in south Florida because a

plastic mulch is maintained during the entire growing season.

The possibility of obtaining disease control with biological agents

was investigated. It was hypothesized that if selected soil antagonists

could decrease the rapid saprophytic development of the pathogen during

the early stages of soil recolonization, less infection would occur and

the severity of the epidemic would be reduced. In growth-chamber and

greenhouse experiments, infection incidence and mean lesion length on

tomato plants were reduced when antagonists were added to fumigated soil

38





39

(Section I). The purpose of this study was to test the potential of

antagonists to control Fusarium crown rot of tomato under field

conditions.


Materials and Methods

The field used for the experiments during the 1979-1980 season was

located near Delray, Florida, and contained Pompano fine sand with a pH

of 4.5 (measurement obtained from a 1:2 suspension of soil in 0.01 M

CaC12). The soil was fumigated with methyl bromide-chloropicrin

(67/33 v/v) at 1 kg of fumigant to 20 m2 of soil injected approximately

20 cm below the soil surface via three chisels. Plastic mulch (0.25 mm

thick) was placed over the bed immediately after injection of the fumi-

gant. Each bed was 1 m wide and the beds were separated by 1-m access

rows that were not fumigated. Two weeks after fumigation, 5-wk-old

tomato ('Walter') transplants were planted 30 cm apart in two rows which

were 50 cm apart on each bed. Subsurface irrigation was maintained at

approximately 40 cm below the bed surface. Cultural practices were

similar to those employed in the area.

Tomatoes had not been grown previously in the field, and the patho-

gen was not detected by soil dilution plating on a selective medium (16)

before planting. This situation allowed the establishment of field plots

with defined initial inoculum densities of both the pathogen and the

antagonists. The pathogen was added to the soil by injecting 10 ml of a

suspension of chlamydospores under the plastic mulch at opposite sides

of the transplant, 10 cm from the transplant hole. Macroconidia of the

pathogen were incubated at 28 C for 4 wk at 106 macroconidia per ml in

autoclaved deionized water to induce chlamydospore formation (Section I).






40
The population of antagonists consisted of three isolates of

Trichoderma harzianum Rafai, one isolate of Penicillium restrictum

Gilmanand Abbott, and one isolate of Aspergillus ochraceus Wilhelm. The

antagonists were selected for their abilities to increase rapidly in

freshly fumigated soil, to occupy the root environment of the host, and

to increase the ratio of inoculum density to infection incidence under

growth-chamber conditions (Section I). The antagonists were applied at

a concentration of 5 X 105 conidia per isolate per plant. One day

before use, conidia of each antagonist were harvested from petri plates

containing 14-day-old cultures grown on potato dextrose agar at 25 C

under 2000 lux of fluorescent light. The antagonists were added to the

soil by pouring 25 ml of a conidial suspension over the roots of each

transplant after it was positioned in the transplant hole and then

adding another 25 ml over the crown of the transplant immediately after

the roots were covered with soil. The treatments included infestation

of soil with pathogen populations of 0, 50, 500, or 5000 chlamydospores

per plant with or without the addition of antagonists.

Soil-dilution plating techniques were used to monitor populations

of the pathogen and antagonists during the growing season. Soil samples

were obtained by preparing composite samples of 5-g subsamples from the

crown areas of five plants from each plot. Komada's (16) medium, which

is selective for F. oxysporum, was used to isolate the pathogen from

soil dilutions of 1:25, 1:100, or 1:1000 (wt:vol). The pathogen was

identified further by a technique developed by Sanchez et al. (29) and

discussed in Section I. Potato dextrose agar, which contained 1 ml of

Tergitol NPX (Sigma Chemical Co., St. Louis, MO 63178) and 50 mg of






41
chlortetracycline hydrochloride (Sigma Chemical Co., St. Louis, MO

63178) per liter of medium was used to monitor the antagonist popula-

tions. Soil dilutions in water of 1:25, 1:100, and 1:1000 (wt:vol)

were employed and plates were stored at 25 C and 2000 lux of 12 hr of

fluorescent light per day for 7 days before examination for fungal

colonies.

A standard hygrothermograph was used to monitor the high and low

temperatures during the growing season.

The effect of the treatments on yield and disease incidence were

determined. Yield data were obtained by harvesting all of the fruit

that were past the mature green stage of ripeness each week for 4 con-

secutive weeks. Fruit weight and number were recorded for each plot.

Disease incidence, as determined by the presence of lesions on the crown

and lower stems of the plant, was determined at the last harvest date.

Sections of the stems at the edges of the lesions were plated on

Komada's (16) medium to confirm the presence of the pathogen.

A randomized, complete-block design with eight treatments repli-

cated five times was used. There were 20 plants in each plot. The

entire experiment was repeated in the field two times at 2 wk intervals.


Results

The incidence of Fusarium crown rot of tomato was affected signifi-

cantly (P = 0.05) by the initial inoculum density of F. oxysporum f. sp.

radicis-lycopersici, the antagonist amendment, and the time of planting

(Table 5). The increase in disease observed during the later planting

time as compared to earlier planting times was associated with cooler

prevailing temperatures. The mean low temperature during the first and






42
last planting times were 12 and 10 C, respectively. The mean high tem-

perature during the first month after planting was 34 C in the first

planting time, 32 C in the second planting time, and 30 C in the third

planting time.

Amendment with the antagonists reduced significantly (P 0.05) the

mean incidence of disease (Table 1). The analysis of varience also

showed that the inoculum density of the pathogen and the pathogen

inoculum density-antagonist interaction significantly affected disease

incidence. The mean disease incidence at the highest inoculum density

of the pathogen when the antagonists were not added was five times

greater than the mean incidence of disease at that inoculum density

when antagonists were added.

The population density of the pathogen decreased with time in soils

amended with antagonists and increased with time in nonamended soils

(Fig. 8). In soils amended with antagonists in the first planting, the

pathogen decreased from 600 propagules per gram of soil 3 wk after

planting to 200 propagules per gram of soil 19 wk after planting. In

nonamended soils the pathogen population increased from 1000 propagules

per gram of soil 3 wk after planting to 53000 propagules per gram of

soil 19 wk after planting. Similar results occurred in each of the

plantings.

In both amended and nonamended soils, the populations of antagonists

decreased until 11 wk after planting, and began to increase 15 wk after

planting (Fig. 9). The increase, however, in amended soils was approxi-

mately four times as great as in nonamended soils. The population of

Trichoderma spp. usually was much higher in amended than in nonamended

soils. Aspergillus ochraceus was not isolated from nonamended soils,

but was present during the entire season in amended soils.












Table 5. Effect of initial inoculum density of Fusarium oxysporum f.
sp. radicis-lycopersici and a composite of five antagonists on the
incidence of disease under field conditions.

Inoculum density Percentage of plants with lesionsY
(chlamydospores Planting date
per plant)x Antagonists 1 2 3 Mean

0 0.0 0.0 0.0 0.0

50 5.7 8.1 14.7 9.5

500 0.0 17.5 24.3 13.9

5000 15.0 42.8 53.9 37.2

mean 5.2a 17.1a 23.2a 15.1a

0 +z 0.0 1.0 1.2 0.7

50 + 5.1 5.2 6.7 5.7

500 + 3.8 1.3 8.6 4.6

5000 + 1.7 5.4 14.3 7.1

mean 2.6a 3.2 b 7.7 b 4.5 b


x20 ml of a chlamydospore suspension were injected into the soil 10
cm from the plant.
YMeans in same column with different letters differ significantly
(P = 0.05) as determined by t test; data analyzed after transformation
to arcsine I4.
ZConidia of each of five antagonists (three isolates of Trichoderma
harzianum, one isolate of Penicillium restrictum, and one isolate of
Aspergillus ochraceus) were added to the crown area of the transplant
at 5 X 10- conidia of each isolate per plant.















-.J
Oi
o \
L4






3


0
0 \ "0








0 11 1
I I I I---I

3 5 10 15 20
WEEKS AFTER PLANTING
Figure 8 A. Relationship of population density of Fusarium oxysporum
f. sp. radicis-lycopersici to time after planting in soils amended with
three isolates of Trichoderma harzianum, one isolate of Penicillium
restrictum, and one isolate of Aspergillus ochraceus at 5 X 105 conidia
per isolate per plant under field conditions at planting date one
(0....0), planting date two (X--(), and planting date three (.- --).
The pathogen was added initially at 5000 chlamydospores per plant.














-J
O




/


0_ .0


a4-
O



2-

0 .....

0 1 I I I I
3 5 10 15 20
WEEKS AFTER PLANTING
Figure 8 B. Relationship of population density of Fusarium oxysporum
f. sp. radicis-lycopersici to time after planting in nonamended soils
under field conditions at planting date one (0-....0), planting date two
(X--X), and planting date three (0--*). The pathogen was added
initially at 5000 chlamydospores per plant.









A
29 X




15 -


0 -
l5-
01

X
10 1. : -
C,,















WEEKS AFTER PLANTING
Figure 9 A. Relationship of population density of Trichoderma spp.
(X*****X), Aspergillus spp. (- ), and Penicillium spp. ( ) to
time after planting in soils amended with three isolates of T.
0


10- 5













harzianum, one isolate of A. ochraceus, and one isolate of P. restrictum
at 5 X 105 conidia per isolate per plant under field conditions.

















O

0
,4-



X

x w
U3
_J


0

a.

X. x-..





5 10 15 20

WEEKS AFTER PLANTING
Figure 9 B. Relationship of population density of Trichoderma spp.
(X.---X), and Penicillium spp. (@--4) to time in nonamended soils
under field conditions.










Discussion

The application of selected antagonists to soil reduced the inci-

dence of Fusarium crown rot of tomato under field conditions. Similar

results were obtained in experiments done under growth-chamber and

greenhouse conditions (Section I).

In the past, antagonists usually have been selected for their

ability to inhibit a pathogen under pure culture conditions (2). For

several reasons these antagonists failed to reduce disease when applied

under field conditions. Of the eight reasons that Baker and Cook (2)

presented for such failures, the most important is probably the fact

that the environmental conditions in agar are unrelated to those in the

soil. The success of reducing Fusarium crown rot of tomato under growth-

chamber and greenhouse conditions was attributed to the formation of a

microbial community which inhibited the saprophytic proliferation of the

pathogen, rather than to the detrimental interaction of the pathogen

with any one species of antagonist (Section I, II).

Yield was not affected by the different treatments because of the

atypically warm growing season. Fusarium crown rot of tomato is a cool

weather disease (14). Production operations in the area reported little

problem with the disease during the 1979-1980 growing season, when temp-

eratures rarely dropped below 10 C.

The lower inoculum densities of the pathogen in amended soils early

in the season probably were responsible for the reduction of disease

severity. Rowe and Farley (27) reported that the severity of Fusarium

crown rot of tomato is dependent upon the early infection of the tomato

plant. The absence of an increase in the population of the pathogen in






49
amended soils late in the season may have been due to increases in

populations of antagonists, which were greater in amended than in non-

amended soils. The increase in the pathogen population in nonamended

soils late in the season may have been due to sporulation on the above-

ground lesions or to saprophytic proliferation of the pathogen on fresh

plant debris. The lack of an increase in pathogen populations in

amended soils also may be important in reducing the amount of inoculum

available for infection in succeeding years.

The high populations of T. harzianum in amended soils and the

failure to detect A. ochraceus in nonamended soils indicate that the

addition of antagonists led to the establishment of populations of the

organisms in amended soils; however it was not possible to determine if

the populations of the specific isolates of the antagonists actually

originated from the added antagonists.

The applicability of the antagonist amendments to production

systems is realized when one considers that three 15-cm petri plate

cultures of each antagonist grown on potato dextrose agar produced

sufficient inoculum to infest approximately 10 tomato plants at the

prescribed rate of 5 X 105 conidia of each isolate per plant. The

application of the antagonists by a drench either before or after

planting could be effective in controlling the disease. It is impor-

tant, however, that antagonists recolonize the soil before reinfestation

by the pathogen occurs.

The increase in disease severity that follows the application of a

broad spectrum biocide to soils has been reported (4,17,26,38). The

usual explanation for this phenomenon is that the disturbance of the

microbial community increases the ability of a pathogen to proliferate






50
as a saprophyte during the early stages of recolonization. The applica-

tion of antagonists in these types of disease situations should be

successful in controlling the diseases, if the antagonists are properly

selected and administered.

In this study, the success of the biological control agents was

dependent upon several factors. The severity of an epidemic of Fusarium

crown rot of tomato is dependent upon the rapid proliferation of the

pathogen in treated soils (28). When the antagonists were applied to

soils before recolonization by the pathogen could occur, they were able

to effectively occupy the niche space created by the fumigation proce-

dures (Section II). The preoccupied niche space then was rendered

unavailable to the pathogen. The decrease in the saprophytic develop-

ment of the pathogen was due to its inability to compete in soils recol-

onized by antagonists. Severe disease expression requires infection of

the host early in the season (15); therefore, antagonists need to be

established mainly around the crown and roots of the transplant.

Successful control of Fusarium crown rot of tomato with biological

agents was dependent upon production practices, the biology of the path-

ogen, and the methods used for selection and application of the antago-

nists.












LITERATURE CITED


1. ABAWI, G. S., and J. W. LORBEER. 1972. Several aspects of the
ecology and pathology of Fusarium oxysporum f. sp. cepae.
Phytopathology 62:870-876.

2. BAKER, K. F., and R. J. COOK. 1974. Biological control of plant
pathogens. W. H. Freeman and Co., San Francisco. 433p.

3. BAKER, R. 1971. Analyses involving inoculum density of soil-borne
plant pathogens in epidemiology. Phytopathology 61:280-1292.

4. BOLLEN, G. J. 1974. Fungal recolonization of heat treated glass-
house soils. Agro-Ecosyst. 1:139-155.

5. B.IJEHL, G. W., and P. LAI. 1966. Prior-colonization as a factor in
the saprophytic survival of several fungi on wheat straw. Phyto-
pathology 56:766-768.

6. CONNELL, J. H., and R. 0. SLATYER. 1977. Mechanisms of succession
in natural communities and their role in community stability and
organization. Amer. Natur. 111:1119-1144.

7. COOK, R. J. 1968. Fusarium root and foot rot of cereals in the
Pacific Northwest. Phytopathology 58:127-131.

8. FRENCH, E. R. 1972. Supervivencia de Fusarium oxysporum f. batatas
en agua durante siete anos. Fitopatologia 7:30-31.

9. GARRETT, S. D. 1963. Soil fungi and soil fertility. Pergamon
Press, Oxford. 165p.

10. GARRETT, S. D. 1965. Towards biological control of soil-borne
plant pathogens. Pages 4-17 in: K. F. Baker and W. C. Snyder, eds.
Ecology of soil-borne plant pathogens. Univ. California Press,
Berkeley, Los Angeles. 571p.

11. GUY, S. 0., and R. BAKER. 1977. Inoculum potential in relation to
biological control of Fusarium wilt of peas. Phytopathology 67:
72-78.

12. HOAGLAND, D. R., and D. I. ARNON. 1950. The water-culture method
for growing plants without soil. Calif. Agric. Exp. Stn. Circ.
347. 32p.






52
13. JARVIS, W. R. 1977. Biological control of Fusarium. Canada
Agric. 22:28-30.

14. JARVIS, W. R., V. A. DIRKS, P. W. JOHNSON, and H. J. THORPE. 1977.
No interaction between root-knot nematode and Fusarium foot and
root rot of greenhouse tomato. Plant Dis. Rep. 61:251-254.

15. JARVIS, W. R., and R. A. SHOEMAKER. 1978. Taxonomic status of
Fusarium oxysporum causing foot and root rot of tomato. Phyto-
pathology 68:1679-1680.

16. KOMADA, H. 1975. Development of a selective medium for quantita-
tive isolation of Fusarium oxysporum from natural soil. Rev. P1.
Prot. Res. 8:114-125.

17. KREUTZER, W. A. 1960. Soil treatment. Pages 431-476 in: J. G.
Horsfall and A. E. Dimond, eds. Plant pathology, an advanced
treatise. Vol 3. Academic Press, New York. 675p.

18. MACER, R. C. F. 1961. The survival of Cercosporella herpotri-
choides Fron in wheat straw. Ann. Appl. Biol. 49:165-172.

19. NASH, S. N., T. CHRISTOW, and W. C. SNYDER. 1961. Existence of
Fusarium solani f. phaseoli as chlamydospores in soil. Phyto-
pathology 51:308-312.

20. NYVALL, R. F., and W. A. HAGLUND. 1972. Sites of infection of
Fusarium oxysporum f. pisi Race 5 on peas. Phytopathology 62:
1419-1424.

21. NYVALL, R. F., and T. KOMMEDAHL. 1973. Competitive saprophytic
ability of Fusarium roseum f. sp. cerealis 'Culmorum' in soil.
Phytopathology 63:590-597.

22. ODUM, E. P. 1971. Fundamentals of ecology. W. B. Saunders Co.,
Philadelphia. 574p.

23. OLSON, C. M., and K. F. BAKER. 1968. Selective heat treatment of
soil, and its effect on the inhibition of Rhizoctonia solani by
Bacillus subtilis. Phytopathology 58:79-87.

24. PARK, D. 1959. Some aspects of the biology of Fusarium oxysporum
Schl. in soil. Ann. Botany 23:35-49.

25. PARK, D. 1963. The ecology of soil-borne fungal diseases. Annu.
Rev. Phytopathol. 1:241-258.

26. RODRIGUEZ-KABANA, R., M. K. BEUTE, and P. A. BACKMAN. 1979. Effect
of dibromochloropropane fumigation on the growth of Sclerotium
rolfsii and on the incidence of southern blight in field-grown
peanuts. Phytopathology 69:1219-1222.






53
27. ROWE, R. C., and J. D. FARLEY. 1978. Control of Fusarium crown
and root rot of greenhouse tomatoes by inhibiting recolonization
of steam-disinfested soil with a captafol drench. Phytopathology
68:1221-1224.

28. ROWE, R. C., J. D. FARLEY, and D. L. COPLIN. 1977. Airborne
spore dispersal and recolonization of steamed soil by Fusarium
oxysporum in tomato greenhouses. Phytopathology 67:1513-1517.

29. SANCHEZ, L. E., R. M. ENDO, and J. V. LEARY. 1975. A rapid
technique for identifying the clones of Fusarium oxysporum f. sp.
lycopersici causing crown-and root-rot of tomato. Phytopathology
65:726-727.

30. SCHIPPERS, B. and A. K. F. SCHERMER. 1966. Effect of antifungal
properties of soil on dissemination of the pathogen and seedling
infection originating from Verticillium-infected achenes of
Senecio. Phytopathology 56:549-552.

31. SMITH, S. N., and W. C. S31riER. 1971. Relationship of inoculum
density and soil types to severity of Fusarium wilt of sweet
potato. Phytopathology 61:1049-1051.

32. SNYDER, W. C., S. M. NASH, and E. E. TRUJILLO. 1959. Multiple
clonal types of Fusarium solani phaseoli in field soil.
Phytopathology 49:310-312.

33. SONODA, R. M. 1976. The occurrence of a Fusarium root rot of
tomatoes in south Florida. Plant Dis. Rep. 60:271-274.

34. SONODA, R. M., J. MAROIS, and J. J. AUGUSTINE. 1978. Fusarium
crown rot of tomato in Florida. Proc. Fla. State Hort. Soc.
91:284-286.

35. STOVER, R. H., and B. H. WAITE. 1954. Colonization of banana
roots by Fusarium oxysporum f. cubense and other soil fungi.
Phytopathology 44:689-693.

36. THOMPSON, W. R. 1929. On natural control. Parasitology 21:269-
281.

37. TOUSSOUN, T. A., and P. E. NELSON. 1968. A pictorial guide to
the identification of Fusarium species. Pennsylvania State
Univ. Press. 51p.

38. VAARTAJA, O. 1964. Chemical treatment of seedbeds to control
nursery diseases. Botan. Rev. 30:1-91.

39. VAARTAJA, 0. 1967. Reinfestation of sterilized nursery seedbeds
by fungi. Can. J. Microbiol. 13:771-776.

40. WALKER, A. 1941. The colonization of buried wheat straw by soil
fungi, with special reference to Fusarium culmorum. Ann. Appl.
Biol. 28:333-350.






54
41. WATSON, A. G., and E. J. FORD. 1972. Fungistasis a reappraisal.
Annu. Rev. Phytopathol. 10:327-348.

42. WELVAERT. W. 1974. Evolution of the fungus flora following
different soil treatments. Agro. Ecosyst. 1:157-168.

43. WILSON., E. 0. 1969. The species equilibrium. Pages 38-47 in:
Diversity and stability in ecological systems. Brookhaven
Symposia in Biology No. 22. Brookhaven national laboratory,
Upton, New York. 264p.











BIOGRAPHICAL SKETCH


James J. Marois was born in Cincinnati, Ohio. He attended high

school and community college in Minnesota. He received the degree of

Bachelor of Arts in conservation biology in June, 1975, from Florida

Atlantic University. In November, 1975, Jim and Katherine L. Coleman

were married. After working for 2 years as a plant pathologist for

Yoder Brothers of Florida, Jim continued his studies in March, 1977,

towards the degree of Doctor of Philosophy in the field of plant

pathology. Presently he has no definite plans after graduation. Jim

is applying for research positions at state universities so that he

can continue his studies on the importance of soil microorganisms in

the host-pathogen interaction of soilborne plant diseases.













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 J. Mitchell
Chairman -
Associate 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.

Lee F. Jadkson
Assistant 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. .

Ronald M. Sonoda
Associate 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 tha-degee of
Doctor of Philosophy. / /

David H. Bubbll
Professor of Soil 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.

June, 1980 -

De College of Agriulture


Dean, Graduate School



















































UNIVERSITY OF FLORIDA

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