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This is to certify that the

thesis entitled

REDUCTION IN FUSARIUM POPULATIONS IN SOIL
BY OILSEED MEAL AMENDMENTS

presented by

MICHAEL ABDI ZAKARIA

has been accepted towards fulfillment
of the requirements for

Ph.D. degree inPlant Pathology

 

 

3‘” J ,
1&4! j/(fl’f‘ “/MM’HJ'

Major professor

Date March 23, 1978

 

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REDUCTION IN FUSARIUM POPULATIONS IN SOIL
BY_OlLSEED MEAL AMENDMENTS

By

Michael Abdi Zakaria

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology
1978

 

CC
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ABSTRACT

REDUCTION IN FUSARIUM POPULATIONS IN SOIL
BY OILSEED MEAL AMENDMENTS

By
'Michael Abdi Zakaria

A sandy loam soil was artificially infested with Fusarium oxy:

 

spgggm_and E, sglgni_and incubated fOr eight weeks to obtain a constant
chlamydospore concentration of about l05/g. The infested soil was
amended with 1% (w/w) of ten different plant and animal residues and
incubated in closed containers. Fusarium populations were estimated
at intervals using standard dilution plate techniques and a medium sel-
ective for the genus.

Linseed, cottonseed, and soybean meals were effective in reduc-
ing Fusarium populations to 0.1% or less of the original after 4-5
weeks of incubation. Such reduction was also obtained using rates as
low as 0.25% for soybean meal and 0.50% for linseed and cottonseed
meals after six weeks of incubation. The oilseed meal amendments were
also effective in reducing populations of five other Fusarium species
tested. Compared to Fusarium, however, the total fungal, bacterial,
and actinomycete populations were much less reduced.

Soil moisture levels influenced the effectiveness of linseed and
cottonseed meal amendments, but not of soybean meal. Using linseed
and cottonseed meals, Fusarium populations decreased most rapidly at
30-35% NHC; at 50% and 100% HHC, reduction was less rapid, and no re-
duction occurred at 15% HHC.

Fusarium populations decreased most rapidly and to a greater

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Michael Abdi Zakaria

degree in oilseed meal-amended soils incubated in closed containers
than in open containers. When infested soil samples were placed in
planchets and incubated on oilseed meal-amended soils in closed con-
tainers, Fusarium papulations in the soil samples were drastically re-
duced. These suggested that volatile substances were involved. The
volatile substances were trapped in H3803 solution, indicating that
ammonia and/or amines were possibly produced. Nith soybean meal amend-
ment, these substances appeared to be solely responsible for reducing
Fusarium populations, because the effectiveness of the amendment was
completely nullified in the presence of H3803 or H20. The trapping
solutions did not remove all of the toxic effect in the case of linseed
and cottonseed meals.

The oilseed meal amendments were not effective in uncovered plots
of Oshtemo-Boyer sandy loam or Conover loam soils in the field. In
the laboratory, the oilseed meal amendments showed less activity in
Conover loam than in the sandy loam soil, even when the soils were
covered; the amendments were without any effect in Brookston loam soil.
The pH of the sandy loam soil was increased following amendment with
oilseed meals, whereas the pH of the two loam soils was decreased.

Fusarium chlamydospores applied to Nuclepore membranes were
buried in oilseed meal-amended soils. At intervals, the membranes
were removed from the soils and the chlamydospores were transferred
onto agar discs fOllowed by immediate microscopic observation. During
the first three days of burial, most of the chlamydospores had germin-
ated, the germ tubes had lysed, and new chlamydospores had been formed.
However, prolonged exposure to the oilseed meal-amended soils resulted

in complete loss of chlamydospore viabliity, as verified by incubating

Michael Abdi Zakaria

them on discs of a selective agar medium for 24 hours.

Reduction in Fusarium populations as affected by oilseed meal
amendments was correlated with amount of Fusarium root rot of soybeans
and peas. Linseed and cottonseed meal amendments, however, showed
some phytotoxic residues even after the soils had been air-dried for
one week. Soybean meal amendment, beside being the most effective in
reducing Fusarium p0pulations and root rot, did not show any phytotoxic

residue after the period of air-drying.

To my wife and parents

~i'l

ACKNOWLEDGMENTS

I would like to express my deepest appreciation to my major
professor, Dr. J.L. Lockwood, for his guidance and constant encour-
agement, and for his patience and assistance in the preparation of
this manuscript.

To the members of my guidance committee: Dr. H.A. Imshaug,

Dr. A.N. Saettler, and Dr. A.R. Holcott, I convey my gratitude for
advice during the course of the research, and for critical evaluation
of the manuscript.

I would also thank Dr. H. Kamada for his useful suggestions and
advice, Dr. M.M. Mortland and Dr. C. Cress for technical assistance.

My sincere appreciation is also extended to the Indonesian
government for permission to study in the United States, and to the
U.S. Agency for International Development for financial support which

made this study possible.

iii

 

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TABLE OF CONTENTS

LIST or TABLES ’ .........................
LIST OF FIGURES ........................
INTRODUCTION ..........................
LITERATURE REVIEW .......................
The genus Fusarium ...................
Biological control using soil amendments ........
Influence of volatile substances on soil microorganisms.
Detrimental effect of decomposing plant residues on
plants .......................
MATERIALS AND METHODS .....................
Preparation of inocula .................
Soil preparation and infestation ............
Incorporation of organic residues into soil ......
Estimation of soil microbial populations ........
Field experiments ...................
Detection of volatile inhibitors ............
Identification of volatile inhibitory substances . . . .
Observation on the fate of Fusarium chlamydospores
in soil .......................
Bioassay using soybean (Gl cine max (L) )Merr. ) and
peas (Pisum sativum L. as host plants .......
Statistical analysis ..................
RESULTS ............................

Effect of different soil amendments on Fusarium
populations .....................
Effectiveness of different rates of oilseed meal
amendments in reducing soil Fusarium populations
Effect of soil amendments on soil microorganisms . . . .
Effect of increased soil pH on reduction in Fusarium
populations .....................
Effect of soil amendment with composted organic
materials on Fusarium populations ..........
Field experiments using uncovered plots ........
Effect of soil moisture content on reduction in
Fusarium populations ................

iv

Page

vi

3
8
16

34

36
38

4O

41
42

Page

Detection of volatile inhibitors ............ 47
Identification of volatile inhibitory substances . . . . 53
The fate of Fusarium chlamydospores in the amended ‘

soils ........................ 54
Effect of oilseed meal amendments on different .

species of Fusarium ................. 58
Relation of soil Fusarium populations to amount of

root rot ...................... 58
A field experiment using covered plots ......... 6l

Effect of different soils on effectiveness of oilseed
meal amendments in reducing Fusarium populations . . 62

DISCUSSION ................ ' ........... 65
LITERATURE CITED ........................ 72

Tab‘

 

 

-erE..

 

LIST OF TABLES
Page

Fusarium populations in soils four weeks after amend-
ment with 1% (w/w) ground plant and animal residues . . . 36

Soil microbial populations four to five weeks after
amendment with 1% (w/w) linseed and cottonseed meals,
as determined using selective media ........... 39.

Effect of increased soil pH on reduction in Fusarium
populations in soil six weeks after amendment with 1%
(w/w) alfalfa and oilseed meals ............. 41

Log Fusarium populations from soils after four weeks of
amendment using 1% (w/w) composted alfalfa and oilseed
heals ................ _ .......... 43

Fusarium populations in natural and artificially in-

fested field soils five weeks after amendment with al-

falfa and oilseed meals. The experiment was done near
Nottawa in St. Joseph county, Michigan, in the Spring,

1976 ........................... 45

Fusarium populations in naturally infested field soil

four weeks after amendment with 1% (w/w) alfalfa and

oilseed meals. The experiment was carried out near Burr

Oak, St. Joseph, Michigan, in early Summer, 1976 ..... 45

Effect of different soil moisture contents on reduction
of Fusarium populations four weeks after amendment with
1% (w/wl alfalfa and oilseed meals ........... 47

Fusarium populations from soil samples in metal plan-

chets placed on soil amended with 1% (w/w) alfalfa and
oilseed meals. The soil samples were incubated in the
presence of H20 and H3803 as trapping solutions in

closed containers for four weeks ............ 53

Populations of different Fusarium species in soil samples
placed in planchets after fOur week incubation on soils
either unamended or amended with 1% (w/w) alfalfa and

oilseed meals ...................... 59

vi

Table
10.

ll.

12.

 

Page
Severity of pea root rot caused by Fusarium solani f. sp.
pisi as affected by reduced populations of the patho-
genic propagules after fbur weeks of incubation in soil
amended with 1% (w/w) oilseed meals .......... ‘ . . 60

Soil pH and Fusarium populations in artificially infested
Conover loam soil eight weeks after amendment with 1% (w/w)
alfalfa and oilseed meals. The experiment was done at
Michigan State University farm in Spring of 1977 ..... 62

Soil pH and Fusarium populations in artificially Conover

loam and Brookston loam soils 10 weeks after amendment
with 1% (w/w) alfalfa and oilseed meals .......... 64

vii

Figure

1.

LIST OF FIGURES

Schematic diagram of the system used in the collection
of volatile substances evolved from treated soils for
identification using infra-red spectroscopy. The flask
containing water at the right end serves as a monitor
for flow rate ......................

Nuclepore membrane with Fusarium chlamydospores on the
upper surface. A nylon net was used to prevent direct
contact between the treated soil and the chlamydospores .

Fusarium populations in soils four weeks after amendment
with 1% (w/w) ground plant and animal residues .....

Fusarium populations in soils amended with different
concentrations (%, w/w) of alfalfa or oilseed meals
after two to six weeks of incubation. Least Significant
Range (L.S.R.) values were obtained using the Tukey's

'w' procedure ......................

Fusarium populations in soils fOur weeks after amendment
with 1% (w/w) linseed and cottonseed meals, as affected
by different soil moisture contents in closed containers.

Fusarium populations in soil placed in open and closed
containers six weeks after amendment with 1% (w/w) alfal-
fa or oilseed meals. Least Significant Range (L.S.R.)
values were obtained using the Tukey's 'wf procedure

Fusarium populations from soil samples in planchets

incubated on soil amended with 1% (w/w) alfalfa or oil-

seed meals in closed containers for four weeks. The
Least Significant Range (L.S.R.) was obtained using the
Tukey's {w} procedure ..................

Titration curves for the estimation of ammonia and/or
amines trapped by 2% H3803 solutions. The H3303 solu-
tions were placed in glass vials and incubated on soils
either unamended or amended with 1% (w/w) alfalfa and
oilseed meals in closed containers. An indicator was
added to the H3803 solutions before titrating with 0.01

N H2504 .........................

viii

Page

30

31

35

37

46

49

50

52

Figure

10.

Viability of Fusarium chlamydospores as determined by
the agar disc method. The chlamydospores prepared on
Nuclepore membranes were incubated in soil amended with
1% (w/w) alfalfa and oilseed meals for different lengths
of time. After each incubation period, membrane samples
were removed and the chlamydospores were transferred on-
to discs of a selective agar medium fOr microscopic ob-
servation of germination either immediately of following
a 24 hr incubation on the discs. Least Significant
Range (L.S.R.) values were obtained using the Tukey's

'w_' procedure ...................... I

Viability of Fusarium chlamydospores as determined by
the agar disc method. The chlamydospores were buried
seven days after soil treatment. Technical descriptions
fOr this experiment were as described in Figure 9.

Least Significant Range (L.S.R.) values for 7 and 10
days of burial were 13.5 and 42.3, respectively (Tukey's
'w} procedure, £.= 0.01) ................

ix

Page

56

 

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INTRODUCTION

Fusarium oxysporum Schlecht. and f, solani Mart. are often

 

found associated with an extensive seed and seedling rot, and later
a root rot on soybean grown in loamy sand soil in S.H. Michigan. The
first evidence of the disease is poor emergence over restricted or ex-
tensive (several acres) areas in the field. Symptoms, including seed
rot, curling and swelling of seedling hypocotyls, and necrosis are
typical of the disease.

Fusarium disease of soybeans was first reported by Cromwell (31)
in North Carolina in 1916. The disease, believed to be caused by f,

tracheiphilum Smith, occurred during the first eight weeks of planting;

 

symptoms included discoloration of root xylem tissue, followed by yel-
lowing and stunting of the plants. The disease was favored by high
temperature and sandy soils. Armstrong and Armstrong (6) found that
certain races of Fusarium isolated from wilted cowpeas and cotton

were pathogenic to soybeans and showed symptoms similar to those de-
scribed by Cromwell. Based on the Snyder and Hansen system of classi-
fication, they suggested that the specific name, f, oxysporum, be
used. They also fOund an isolate of E, oxysporum which caused a vas-
cular wilt, especially on soybean plants that had passed the succulent
stage.

In Iowa, a disease of soybean seedlings caused by f, orthoceras

 

Appel & Hr. has been observed since 1953 (36). The disease caused

w?

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2

poor germination and late emergence of seedlings in the field. Growth
of diseased seedlings were stunted and necrotic symptoms were found on
succulent root tissues of the seedlings. The root systems of severely
infected seedlings were sometimes completely destroyed. However, be-
yond the seedling stage, plants were rarely killed by the disease.

5, oxyspgrum was also found to be a potential threat to soybean
crops in Minnesota (42). Soybeans in approximately 90% of the fields
sampled in central and southern portions of the state were affected by
root rot. Poor stand, stunted growth, and necrotic root symptoms were
associated with the disease. 5, oxysporum was readily isolated from
diseased soybean roots and many of the isolates were highly pathogenic
when tested.

The purpose of this study was to investigate possible control
measures for Fusarium diseases in Michigan using organic amendments.
It was also of interest to investigate various parameters which might
influence the effectiveness of the amendments, and to elucidate the

mechanism of action of effective amendments.

LITERATURE REVIEW

The genus Fusarium

 

Biology in Soil. Species of the genus Fusarium are among the

 

most widely distributed in soil and on organic substrates (15) and
also among the most frequently isolated by plant pathologists, either
as pathogens or as saprophytes (154). As with many soil fungi members
of this genus are abundantly endowed with means of survival, one mechan-
ism of which is the capacity of rapid change morphologically, as well
as physiologically, to a new environment.

Some pathogenic Fusarium species are capable of colonizing root
systems of a wide range of non-host plants. For example, weeds belong-

ing to the genera Oryzopsis, Digitaria, Amaranthus, and Malva have

 

been known to harbor the tomato wilt pathogen, f, oxysporum f. sp.

1ycopersici without showing any symptoms (76). Crop plants such as

 

cotton, soybean, and cowpea were also found to carry the sweet potato
wilt Fusarium without showing any symptoms (7).

Dormant stages of Fusarium in soil may be encountered in the
form of mycelium in rotted tissues or humus particles and as chlamydo-
spores (167). The chlamydospores may be fbrmed by the direct conversion
of macroconidial cells, on the germ tubes of germinated macroconidia,
or from mycelia (40, 41, 43, 45, 68, 112).

Hhile Fusarium macroconidia are relatively insensitive to soil

fungistasis, the chlamydospores are more sensitive (4, 156).

4

Germination of the latter spore type occurs only if nutrients are
available (29, 138, 142, 163). Carbon and nitrogen sources are re-
quired for germination, although carbon exerts a greater limitation
than nitrogen. Forty-two percent chlamydospore germination was ob-
tained in the presence of glucose and (NH4)2504 while only 14-23% ger-
minated in the presence of glucose alone. In the absence of glucose,
(NH4)2504 did not stimulate any germination. Sterilized soil infested
with Fusarium also showed a much higher respiration rate when amended
with sucrose and NH4N03 than with sucrose alone (38). Respiration rate
in soil amended with NH4N03 alone was about the same as that in un-
amended soil.

During germination the chlamydospores produce germ tubes which
may grow extensively and form new chlamydospores, or may undergo ex-
tensive lysis before new chlamydospores were formed (30, 46, 159, 163).
Lysis of germ tubes occurs in the presence of C, if N is adequate.

Hhen N is low, lysis occurs very slowly, replacement chlamydospores
are formed, and the population will remain steady or slightly increase.

Taxonomy. The genus Fusarium was described by Link in 1809 for

species having fusiform non-septate spores borne on a stroma or sporo-

dochium, and was based on Fusarium roseum (15). It was validated by

 

Fries in 1821. According to the Sacardo system of classification, this
genus belongs to the family Tuberculariaceae of the order Moniliales
(13, 50). However, with the development of single-spore culture methods
for Fusarium identification, the presence of a stroma or sporodochium
is no longer accepted as an essential character of the genus.

All modern systems of Fusarium classification are based on the

work of Appel and Nollenweber as sunlnari zed by Hollenweber and

5

Reinking (172). In their system 143 species, varieties, and forms
were grouped in 16 sections mainly based on shape, septation, size,
color, and growth of macro- and microconidia; presence or absence of
sporodochia and pionnotes; production of chlamydospores, whether inter-
calary or terminal, singly, in clusters or in chains; and presence

or absence of sclerotia. The 16 sections were Arachnites, Arthro-
sporiella, Discolor, Elegans, Eupionnotes, Gibbosum, Lateritium,
Liseola, Macroconia, Martiella, Pseudomicrocera, Roseum, Spicari-
oides, Sporotrichiella, Submicrocera, and Ventricosum. Assignments

of species to sections were to some extent related to the discovery

of the perfect stages which consisted of the genera Calonectrja,

 

Gibberella, Hypomyces, Micronectriella, and Nectria. All of these

 

 

genera belong to a single order, Hypocreales.

Snyder and his co-workers (148, 149, 150, 151, 154) suggested
that only stable morphologic characters be used to delimit species
and all higher taxonomic categories. Shape of macroconidia, presence
and kind of microconidia and chlamydospores were suggested to be suit-
able characters for speciation. Size, septation, and pigmentation of
conidia have been found to vary with media and subculturing. Single-
spore subcultures originating from one perithecium may show natural
variability with respect to colony color, configuration, growth rate,
relative abundance of conidia, presence or absence of sporodochia and
of sclerotia, spore size, septation, and even their pathogenicity.
Even progenies of a single conidium have shown variations with respect
to presence or absence of sporodochia and size of macroconidia. They
also suggested that speciation should be based on similarities among

different individuals rather than on differences. Based on this

6

concept, all the 143 members of the genus Fusarium in the Hollenweber
and Reinking system were dramatically reduced to 9 species. For ex-
ample, all 40 members of the section Elegans which were separated on
the basis of the presence or absence of sporodochia and the width of

macroconidia were grouped into a single species, Fusarium oxysporum.

 

The other eight species comprising this nine-species system are: ‘5.

 

 

 

gpjsphaeria, f, lateritium, E, moniliforme, E, nivale, [. rigidius-

culum, F. roseum, F. solani, and f, tricinctum. This nine-species

_—w—

 

system has been widely accepted, especially by plant pathologists,
for over three decades. A pictorial guide to the identification of
Fusarium species, based on this nine-species system, was published
by Toussoun and Nelson (160).

To differentiate the pathogenic fonmsfrom the saprophytic forms

within a Fusarium Species appropriate designations (formae speciales)

 

were added to species names. For example, 5. oxyspgrum f. sp. lyco-
persici is actually a f, oxysporum which is pathogenic to tomatoes,
and f, oxyspgrum f. sp. pisi is pathogenic to peas. At first it was

believed that these physiological strains (formae speciales) were host

 

specific, but it has since been determined by cross-inoculation studies
that this is not necessarily the case (6, 7, 16, 114). For example,

5, oxysporum f. sp. 3911, a celery wilt pathogen, has been known to
attack sunflower and.f, oxysporum f. sp. batatas was also isolated
from naturally infected corn. More than one pathogenic race may also

exist within a forma specialis.

 

Forms with transitory or minor variations in conidial morphology
which were too unstable to find a place in botanical classification

were labelled as cultivars; e.g., f, roseum f. sp. cerealis cultivar

7

'Culmorum' if pathogenic, or merely E, rggggm_”Culmorum' if sapro-
phytic (152, 161).

Recent work in the taxonomy of the imperfect fungi is based in
large part on the studies of Mason as regards the importance of the con-
idiogenous cell and the ontegonyof the conidium. Production of phi-
alides with phialospores (micro-and macroconidia) and occasionally,
blastospores is characteristic of the genus Fusarium. This genus is
also characterized by the presence of a foot cell at the base of the
macroconidium. The recent taxonomic treatment oquusarium by Booth
(l5) recognizes 12 sections and 44 species and follows the concepts of
Mason. As a result, the sections Sporotrichiella, Arthrosporiella,
and Gibbosum of the Hollenweber and Reinking systemlwere modified and
section Roseum discarded. Two species with polyblastic conidiogenous
cells from section Sporotrichiella were transferred to section Arthro-
sporiella in which this spore formation type is the unifying character.
5, avenaceum from section Roseum was transferred to section Arthro-
sporiella for the same reason. The remaining species in section Roseum,
having simple phialidic conidiogenous cells, were transferred to sec-
tion Gibbosum. Species differentiation was based on 1) microconidia:
presence or absence, borne in chains or not, shape; 2) macroconidia:
shape, morphology of foot cells; siZe, septation; 3) conidiophore:
growth, whether polyblastic or simple- or poly-phialidic, borne in
sporodochia or not; 4) chlamydospores: presence or absence, borne ter-
. minal or intercalary or both; 5) cultures (based on potato-sucrose
agar): growth rate, color, presence or absence of aerial mycelium.
Keys to the sections and species, and a laboratory guide to the identi-

fication of the major species of Fusarium were published (15, 17).

8

Booth (15, 16), realizing the capacity of the genus Fusarium for
rapid change under different environmental conditions, proposed that
single-spore cultures and a standardized culture medium be used in
species identification. In his method, potato-sucrose agar with a pH
of 6.5-7.0 is used for growing the fungus. Growth is maintained at
22°-25°C, 10-14 inches below a daylight fluorescent tube with a photo-
period of 12 hours/day. For microscopic studies the spores_are mounted
in erythrosin disolved in 10% NH40H; the coverslip is sealed with two

layers of nail varnish.

Biological control usingflsoil amendments

 

Soil fungistasis,its importance fbr survival. Most fungi sur-

 

vive in soil in the fbrm of resting spores, hyphae, or sclerotia, which
are intimately associated with the phenomenon of soil fungistasis (54).
The importance of exogenous and endogenous nutrients on the germina-
tion of spores has been discussed in detail by Lockwood and his co-
workers (19, 67, 84, 99, 100, 101, 156, 157). The static state of
spores which is of a widespread occurrence in soil may be attributed
to lack of essential exogenous nutrients or to rapid loss of essential
endogenous nutrients by competing microorganisms. Although the pre-
sence of a low level of diffusible inhibitory substances has been
claimed to contribute to soil fungistasis, the involvement of those
substances occurred especially in alkaline(62, 63, 64, 81, 82, 83,
130) or limed soils (65, 66) and the fungistatic condition may or may
not be annulled by appropriate nutrients (11, 62, 130).

A pathogenic propagule is apt to survive longest in soil under

non-lethal conditions least favorable for germination (10, 35). Hhile

9

continued lack of activity of a population of resting stages may lead
to a gradual decline in numbers through senescence, there is a con-
verse situation that reversion to activity under the wrong sort of
conditions nay lead to an even more rapid disappearance (121). Stime
ulation of a pathogenic propagule into activity in the absence of its
host may waste its reserves and decrease its population (2, 58, 119,
145, 147).

Increased soil fungistatic level as a means of control. Control
of soil-borne plant pathogens can be achieved by modifying the bio-
logical equilibrium to the detriment of the pathogens or their activ-
ities (20, 70, 124, 168). This can often be attained by increasing the
activities of microflora through the addition of crop residues. Reduc-
tion in disease may or may not be associated with reduction in patho-
gen numbers. Reduction in root diseases due to increased fungistatic
level of amended soils have been reported (1, 2, 3, 91). Fusarium
root rot of beans in soil amended with 0.5-1.0% (w/w) spent coffee was
reduced to about 30% that of unamended control soil, although the Egg:
arium_population in the amended soil was increased by about 50%. In-
creased fungistasis was also obtained when cellulose was used to con-
trol the disease. The fungistatic level of both the amended and un-
amended soils was determined by adding certain amounts of glucose and
by observing their effect on chlamydospore germination or by measuring
the decomposition rate of the anthrone-positive substances. Gilpatrick

(52) found that production of sporangia by Phytophthora cinnamomfi_at

 

the surface of inoculated avocado roots was prevented in alfalfa-
amended soil, while similar roots showed abundant production of spor-

angia in unamended soil. Hhen inoculated roots from the alfalfa-amended

10

soil were used as inoculum in natural, uninfested-soil in which an
avocado seedling was growing, infection was delayed by 20 days. This
delay was overcome when the surface of the inoculated root was disin-
fected using HgClz. Microorganisms on the root surface were therefore
suggested as being responsible for the prevention of sporangial pro-
duction. Nest and Hildebrand (171) reported a shift in bacterial pop-
ulations in strawberry root rot soils caused by different amendments.
About 40 X more pathogenic isolates were obtained from unamended and
red clover-amended soils than from soybean-amended soil. They sug-
gested that incorporation of soybean plants into the soil resulted in
a pronounced selective stimulation of non-pathogenic bacteria and
inhibition of the pathogenic types. Ko (80) successfully untilized
the soil fungistatic phenomenon in the control of seedling root rot of

papaya caused by Pbytophthora palmivora. He used pathogen-free soil

 

to replace a core of pathogen-containing soil in the field as a growth
medium for the papaya during the susceptible stage. The pathogen was
excluded due to the fungistatic effect of the pathogen-free soil. .

Increased antagonism as a means of control. Reduction in disease

 

nay be associated with increased populations of antagonists to a par-
ticular pathogen as a result of soil amendment. Field soil amended
with 200-500 1b/acre of finely ground chitin showed 30-50% reduction

in bean root rot caused by Fusarium solani f. sp. phaseoli as compared

 

to unamended soil (108, 109). After two weeks of amendment, chitinase
producing microorganisms increased to five times that of the unamended
soil, 70% of which were actinomycetes and 30% bacteria. Complete
control of radish yellows caused by f, oxyspgrum f. sp. raphani was

also obtained in chitin-amended soil (73). Control of the disease was

11

associated with an increased population of antagonistic actinomycetes.
A similar relationship was shown in the reduction of pea wilt caused
by f} oxyspgrum f. sp. pisi (21, 77) and in reduced sclerotial germin-

ability of Sclerotium rolfsii in chitin-amended soils (60).

 

Beside chitin, plant materials have been used as sbil amendments

to control diseases. Reduction in Rhizoctonia disease of beans was

 

obtained in soil amended with 1% (w/w) immature buckwheat, corn, oat,
snapbean, and Sudan grass (118). Corn and oat amendments greatly stim-
ulated soil and rhizosphere streptomycetes antagonistic to R, sglggj,
By seven weeks after amendment the populations of the antagonists in
the amended soils were about six times that of the unamended soil.
Heinhold and Bowman (169) fbund that amendment of soil with green soy-
bean residues prevented build up of common scab of potatoes, whereas
incorporation of barley residues increased disease. Both amendments
resulted in three-fold increase in the number of antagonists, which

were predominantly Bacillus subtilis, as compared to the unamended

 

soil. However, when soybean and barley extracts were used as sources
of nutrients for antibiotic production, the former extract showed the
higher antagonism, indicating that it was a more suitable medium for
antibiotic production.

The effect of soil amendments or antagonists on the control of
soil-borne diseases could be augmented by application of the antagon-
ists together with the amendments. Menzies (106) was able to suppress
potato scab to about 25% of untreated soil by using a combined amend-
ments containing 10% suppressive soil and 1% alfalfa meal; neither of
the constituents was consistently effective alone. Soil amended with

1.5% (w/w) antagonist-inoculated chitin has also been shown to reduce

12

scab to less then 20% of that in unamended soil and in soil amended
with the antagonists alone (165). Suppression of Fusarium root rot
of beans was greater in soil amended with barley straw which had been

inoculated with a culture suspension of Chaetomium sp., than in soil

 

amended with barley straw alone (103). Fusarium wilt of cucumber was
also effectively controlled in soil amended with bark-sawdust compost
which had been fortified previously with antagonistic actinomycetes
(88). This method is now being commercially used in controlling Egg:
arjum_wilt of greenhouse tomatoes in Japan (H. Komada, personal commu-
nication). Backman and Rodriguez-Kabana (9) fOund that diatomaceous
earth granules impregnated with a 10% molasses solution were suitable

fer growth and delivery of Trichoderma harzianum to peanut fields.

 

Using this amendment, significant reductions in damage caused by

Sclerotium rolfsii and increases in yield were obtained over a three

 

year test period. An amendment rate of 140 kg/ha gave equivalent con-
trol to 10% PCNB treatment at a rate of 112 kg/ha.

Germinationelysis as a mechanism of control.' One mechanism which

 

may be involved in controlling soil-borne diseases using organic amend-
ments is propagule germination fbllowed by lysis of the germ tube and
death of the propagule (10). Organic residues supply nutrients that
stimulate germination of pathogenic propagules, but these nutrients are
also quickly utilized by other microorganisms, leading to starvation of
the pathogenic germlings, which in turn may cause (endolysis) autolysis
and death (85, 98). Soybean meal and ascorbic acid were found to

stimulate germination of Helminthosporium sativum conidia in soil dur-
ing the first 20 hours of amendment, but germination was soon fbllowed

by lysis of the germ tube, leading to the eradication of the conidia

13

(23, 24, 25). Alfalfa hay has been reported to control bean root rot
caused by Thielaviopsis basicola when incorporated into soil (117).
The alfalfa hay as well as its hexane- and water-soluble extracts were
stimulatory to germination of the fungus chlamydospores during the first
two days of amendment, but germination was f01lowed by lysis without
formation of secondary endoconidia or chlamydospores (2, 119, 145).

A similar mechanism has been shown to be responsible fer the re-
duction of Fusarium chlamydospore populations in amended soil. For
example, sugarcane amendment stimulated germination of f, oxyspgrum f.
sp. cubense chlamydospores which was soon fellowed by lysis of germ
tubes (136). Chitin amendment was stimulatory to germination of E.

solani f. sp. cucurbitae macroconidia but inhibitory to the fbrmation

 

of chlamydospores (131, 134). The general absence of Fusarium spp. in
conifer fOrest soils nay also be explained by the germination-lysis
mechanism (57, 159). Germination of Fusarium chlamydospores was stim-
ulated twice as much by a water extract of conifer litter as by 1%
glucose and 2.5% asparagine. Shikimic and quinic acids that occur in
pine needles were suggested to play a major role in stimulating the
germination of chlamydOSpores. In all cases, germination was followed
by lysis before new chlamydospores could be formed.

Effect of carbon to nitrggen ratio. Carbon to nitrogen ratio
associated with soil amendment has frequently been suggested to func-
tion in disease suppression. Control of root diseases using organic
materials of high C/N has been reported (92, 102, 120, 153). Organic
materials of high C/N, such as mature barley, wheat, corn, soybean,
and oat residues were effective in controlling Fusarium root rot of

beans, while green barley, soybean, and alfalfa residues that have low

14

C/N were not effective (92, 153). Suppression of disease was not
necessarily associated with reduction in inoculum density of the patho-
gen; fer example, oat straw, glucose, maltose, and dextran, although
effective in reducing disease, were fbund to increase inoculum density
(55, 116). It was suggested that control of root rot was due to in-
creased antagonism and to increased competition fer limiting N by soil
microorganisms. The effectiveness of high C/N amendments could be
negated by addition of supplemental inorganic N, especially ammonium-
N (104, 170).

High C/N by itself, however, could not be used to predict the
effectiveness of soil amendments in reducing diseases. Low C/N amend-
ments have also been found effective in controlling certain diseases.

For example, control of Phytophthora root rot of avocado was achieved

 

using alfalfa meal, the C/N of which was low (174). Alfalfa hay (C/N =
17) was also effective in suppressing take-all of wheat, and even
greater suppression could be obtained by addition of ammonium-N (139).
0at straw (C/N = 83) when incorporated into the soil at a rate of 1%
(w/w) reduced activity of Rhizoctonia solani by 50%, but so did soy-

 

bean hay (C/N = 29) (33). Suppression of Rhizoctonia activity was

 

also obtained using cellulose enriched with NH4N03 (C/N = 5); suppres-
sion was associated with a decrease in soil pH from 6.1 to 4.0.

The germination-lysis mechanism which was involved in the reduc-
tion of Fusarium chlamydospores in soil was enhanced by availability of

adequate N sources (30). Mixon and Curl (110), working with Sclerotium

 

rolfsii, reported that addition of nitrate-N to oat residue amendment
decreased germination of sclerotia in soil to half that caused by oat

residue alone.

15

Effect of inorganic N. Inorganic N sources like NH4N03, (NH4)2504,
and NaNO3 have been known to suppress saprophytic activity of sclerotia
in soil, and NH4N03 at a rate of 2000 ppm N was toxic to the sclerotia
(8). Huber and Watson (71) suggested that amendments modify disease
severity through their effect on nitrification, which in turn determines
the fbrm of N available. For example, accumulation of ammonium-N de-

creased root rot caused by Pythium, Phymatotrichum, and Qphiobolus,

 

while accumulation of nitrate-N decreased root rot caused by Rhizoc-

tonia, Fusarium, and Aphanomyces. Urea has also been known to suppress

 

 

Fusarium disease of banana through accumulation of nitrite (137). Tox-

icity of nitrite to Phytophthora cinnamomi was demonstrated in the form

 

of complete inhibition of zoospore germination (175).

Effect on plant parasitic nematodes. Incorporation of certain

 

plant residues has been found to reduce populations of plant parasitic
nematodes in soil. Chopped oat straw at a rate of 1% (w/w) resulted in

80% reduction of root knot of tomatoes caused by Meliodogyne incognita

 

when incorporated into soil (74). Stems and leaves of alfalfa and soy-
bean were fbund to inhibit hatching of M. incognita eggs in soil (75).
Linfbrd gt_al_(97) reported that incorporation of Panicum barbinode

 

and pineapple residues into Heterodera marioni-infested soils resulted

 

in more than 95% reduction of gall formation as compared to unamended
soil. Reduction in disease was associated with rapid increase in
saprophytic nematode populations.

Other beneficial effects. Beside stimulating microbial activi-

 

ties, soil amendment with organic materials improves soil structure
and physical condition, and may increase productivity of the soil (39,

45). More than 50% increase in yield of snap beans was obtained by

16

incorporation of 1-2% (w/w) mature or green buckwheat into soil (32).
Highly significant increases in potato yields and percent of No. l
tubers have resulted from incorporation of barley straw from the pre-
vious year into the soil, compared to baling off or removing the straw
from the soil (166). 'Hildebrand and west (61) obtained more than 50%
increase in aggregate weights of strawberry roots grown in soils amen-
ded with green soybean tissues, corn, or barnyard manure as compared
to those grown in unamended soil. Control of take-all of wheat has
been obtained in soil when the N and P levels were maintained high,
notwithstanding the presence of the pathogen. Improved nutrition of
the suscept was considered to augment its resistance to the pathogen

(26, 158).

Influence of volatile substances on soil microorganisms

Volatile substances in soil may be of abiotic or of biotic ori-
gin and their influence on soil microorganisms may be a stimulatory or
an inhibitory one. To microorganisms which have virtually every single
cell exposed to the surrounding medium and where consequently the sur-
face to volume ratiois very high, the influence of volatile substances
may be of a great significance (44).

As mentioned earlier, the presence of a low level of diffusible
inhibitory substances in soil has been claimed to be involved in soil
fungistasis, especially in alkaline and limed soil. Liming autoclaved
soil with Ca(0H)2 and CaCD3 at rates of 0.5% (w/w) suppressed germina-
tion of conidia of Trichoderma viride and sporangiospores of Zygorhyn-

 

chus vuilleminii conidia by more than 50% as compared with unlimed

sterilized soil, suggesting that volatile inhibitors could be produced

17

abiotically (65). K0 and his co-workers (83) found that ammonia was
released from limed soils known to produce volatile fungistatic fac-
tors.

All living things produce metabolites which are volatile at the
normal temperatures of their environments, and most produce at least
one that can have a significant effect on other members in the commu-
nity (72). Acetaldehyde has been suggested as being produced by dif-
ferent Trichoderma species grown on 2% malt extract agar. When incu-

bated together with the Trichoderma cultures, linear growth of Rhizoc-

 

tonia solani, Fomes annosus, and Fusarium oxysporum was inhibited by

 

 

49%, 75%, and 19%, respectively (34). Linear growth and sporulation
of different fungal species were also inhibited by volatiles produced
by different bacteria grown on nutrient agar (111). For example, when
the fungal cultures were incubated for six days on a bent glass rod
placed on the surface of nutrient agar inoculated with the bacteria,
6-85% inhibition in linear growth and 8-26% reduction in number of
spores/sample were obtained. Similar inhibition of linear growth of

GelasinOSpora cerealis was shown by the bacteria, and production of

 

perithecia was inhibited over 90%. Inhibition of linear growth and
sporulation was accompanied by an increase in pH of the fungal media
from 6.0 to 8.2-8.6, a1though pH 8.6 itself was not inhibitory.
Ethylene has been reported to be involved in soil fungistasis
(12.), especially in water-logged soil with high N and organic matter
content (141). More than 90% inhibition in germination of sclerotia

of Sclerotium rolfsii and conidia of Helminthosporium sativum was

 

 

shown on non-aerated soil known to produce ethylene as compared to

an aerated one. Bacteria isolated from water-logged soil were capable

18

of producing ethylene when grown on culture medium (129).

Involvement of actinomycetes in the productioniyfvolatile sub-
stances inhibitory to spore germination was demonstrated in sterilized
soils recolonized by mixed soil actinomycetes (63). Inhibition was
more prominent on soils with pH 7~8 than on those with pH 5.4-6.0.

Streptomyces spp. which produce earthy-smelling compounds have been

 

found to be abundant in soil between pH 6.5-8.0, and were favored by
liming (65).

Amendment of soils with certain organic materials may result in
production of volatile substances inhibitory to fungi. Cabbage and
other crucifers have been known to produce sulfur-containing volatiles
such as methanethiol, dimethyl sulfide, and dimethyl disulfide when
incorporated into soil (89). These compounds were inhibitory to hyphal
growth and to zoospore formation, motility, and germination in Aphagg:

myces euteiches (90). Production of volatile inhibitory compounds

 

in chitin-amended soil has also been reported. Reduction in sapro-

phytic growth and pathogenicity of Rhizoctonia solani was observed in

 

natural, chitin-amended soil within 42 days of incubation (144, 146).

In another case, germination of Aspergjllus flavus and Fusarium solani

 

 

f. sp. cucurbitae conidia was reduced by more than 40% in chitin-

 

amended soil as compared to germination in water (132). The volatile
inhibitory factor was completely absorbed by boric acid solution and
when the conidia were incubated on NH4C1 solution, similar inhibition
in germination was obtained, suggesting that ammonia might at least

be a component of the volatile inhibitory factor (133). A concentra-
tion of 60-197 ppm ammonia was also found in soil one week after amend-

ment with 5% (w/w) alfalfa meal (51). Complete elimination of

19

Phytophthora cinnomomi within avocado root tissues was obtained when
the infected roots were incubated for three to four days in a one-week-
old alfalfa-amended soil.’

Ammonia has been known to be effective in reducing plant path-
ogenic fungi and nematodes in soil (14, 37, 59. 60, 66, 113. 140).
For example, reduction in populations of Fusarium spp. to zero, or
nearly so, within the ammonia retention zone was obtained under field
conditions 14 days after injection with anhydrous ammonia at a rate of
730 mg ammonia-N (140). Inside the retention zone with a radius of
about 5 cm, Fusarium populations remained low or undetectable even
after 225 days of injection. Complete loss in germinability of S2135:

otium rolfsii sclerotia was also observed in soil amended with 0.2%

 

ammonia (60). Reduction in germinability was correlated with increased
numbers of antagonistic actinomycetes and with increase in soil pH.
Birchfield gt_al_(l4) reported that more than 80% reduction in popula-
tions of Rotylenchus reniformis was obtained in soil within the ammonia
retention zone following injection with about 20 ppm ammonia-N.

As mentioned earlier, volatile substances, especially at lower
concentrations, may have stimulatory effects on soil microorganisms.
Coley-Smith and his co-workers (27, 28, 78, 79) found that germination
of sclerotia of Sclerotium cepivorum was stimulated by volatile sub-
stances evolved from growing Alljum_species. Germination of sclerotia
in soil through which air was drawn after bubbling through Allium.ex-
tracts was five times that when air was bubbled through water. A
number of n-propyl and alkyl sulphides were identified from vapors of
chopped garlic and onion bulbs and their extracts. Volatile alkyl

sulphides, which were produced when alkyl cystein sulphoxides

20

originating from Alljum_roots were metabolized by soil bacteria, were
stimulatory to Sclerotium cepivorum.
Gilbert and his co-workers (47, 48, 49, 94, 95, 96, 107, 115) found

that volatiles from alfalfa hay and its distillate induced an immedi-

ate rise in respiration rate of soil microflora. The duration and
magnitude of respiration were dependent on the volume of the distill-
ate, one ml of which was yielded from 50 g alfalfa hay. The optimum
volume for stimulation was 4 ml; a distillate volume as high as 16 ml

was inhibitory. In addition to stimulating the respiration of soil
microflora in general, volatiles from the distillate were also stimu-

latory to germination and growth of Verticillium dahliae and Sclerotium

 

 

rolfsii sclerotia. At optimum concentration, two to five fold increase
in populations of V, dahliae were obtained, while at higher concentra-
tions decrease in populations occurred.

Detrimental effect of decomposing
plant residues on plants

 

Decomposing plant residues have many and diverse effects upon
soil, soil microflora, and living plants (45, 124). Although in gen-
eral the beneficial effects far outweigh the detrimental ones, the
latter may, under certain conditions, be of a considerable importance.

Compounds toxic to living plants may be produced in soil amended
with plant residues especially under heavy, poorly aerated, water-
logged soils and relatively low temperature (125). The compounds rep-
resent a great variety of chemical classes such as acids, bases, esters,
alcohols, aldehydes, nitrogenous compounds, and alkaloids (56, 105,
125). Soil amendment with plant residues, especially under the afore-

mentioned conditions, may result in depletion of 02 and N, and in

21

excessive production of C02, NH3, and other gases (124). Free ammonia
is known as a cell toxin; toxic effects on plants may be expected if
its concentration becomes sufficiently high (105). Ammonia was found
in soil at a concentration of 60-197 ppm after one week of amendment
with 5% (w/w) alfalfa meal (51). At a concentration of 10"3 M or more,
ammonia caused severe root and top stunting of avocado seedlings.
Extracts of soil amended with different plant residues have been
found toxic to seedlings. For example, extracts of soil amended with
timothy, rye, tobacco, and corn at a rate of 6% (w/w) for 15-20 days
were toxic to tobacco, timothy, and barley seedlings (122). Phyto-
toxicity was also exerted by barley-amended soil on lettuce and bean
seedlings during the first three weeks of amendment (126, 164). Phyto-
toxicity was associated with inhibition of seed germination and respir-
ation, root stunting, necrosis, and death of apical meristems.
Enhancement of soil-borne diseases as a result of phytotoxic
effects has also been reported (93, 123, 162). Exposure of tobacco
plants to phytotoxic extracts from decomposing rye and timothy resi-

dues for 10 minutes increased their susceptibility to Thielaviopsis

 

basicola root rot (123). Up to 50% of the root system of a wild Nico-
tiana sp., known to be highly resistant, became diseased when inocu-
lated with I, basicola spores after exposure to the toxic extracts.

Infection of bean stem segments by Fusarium solani f. sp. phaseoli was

 

also enhanced after exposure to water-soluble extracts from barley,
rye, wheat, timothy, broccoli, and broadbean residues (162). Chroma-
tographic analysis showed that exudation of ninhydrin-positive sub-
stances was greatly increased from areas of the stem in contact with

the various extracts and in the absence of the pathogen, suggesting

22

that the permeability of the exposed cells had been altered by the
extracts.

In burying fresh plant materials in soil there is always some
danger that such materials could be suitable substrates fOr non-obligate
pathogens (45). The semi-linging plant materials will selectively favor
colonization by these pathogens and retard colonization by obligate
saprophytes. Fresh papaya residues in soil were readily colonized by

Pythium aphanidermatum and Phytophthora parasitica, leading to a seed-

 

 

ling root-rot problem in papaya nurseries. Living weeds buried under

 

soil surface also served as food bases for Sclerotium rolfsii in pea-

nut fields.

MATERIALS AND METHODS

Preparation of inocula

 

The species of Fusarium used in the experiments were recognized
as in Snyder and Hansen system of classification (149, 150, 160).

They were E, lateritium Link, E, moniliforme Sheld., E, oxyspgrum

 

 

Schlecht., E, rigidiusculum (Brick) Snyd. 8 Hnas. [= F. decemcellulare

 

 

Brick], E, roseum Snyd. & Hans. [=F. concolor Reinking], E, solani
(Mart.) Sacc., E, solani f. sp. pisi (F.R. Jones) Snyd. & Hans., and

E, tricinctum (Corda) Sacc. E, oxysporum and E, solani were isolated

 

from diseased soybean seedlings, E, sglggj_f. sp. pi§i_was isolated
from pea, and the remaining five Fusarium species were obtained from
Dr. T. Kommedahl of the University of Minnesota. The fungi were main-
tained as stock cultures on potato-dextrose agar (PDA) slants at 4°C.
For production of inocula, the fungi were grown either on wheat bran—
sand medium or in potato-maltose broth.

The wheat bran-sand medium was prepared by mixing 200 9 ground .
wheat bran, 500 9, white silica sand, and 500 ml distilled water, fol-
lowed by 60 minutes of steaming: The wheat bran was ground in a Hiley
mill and passed through a 0.85 mm (20-mesh) sieve. The steamed mixture
was broken up with a fork before putting it into Erlenmeyer flasks for
steam sterilization. E, sglggj, E, sglggj_f. sp. Eiéi: and E, ogyspgr-
um_were individually grown in the medium for 4-6 weeks at 24°C before

use. The medium was inoculated using spore suspensions of individual

23

24

species at a rate of about 105 spores/g. The wheat bran-sand cultures
were vigroously shaken every other day during the first week of growth
to Obtain an even distribution of growth.

Potato-maltose broth was prepared by substituting maltose (20 g/
l) for dextrose in the potato-dextrose broth. One hundred ml portions
of the broth were poured into 250 ml Erlenmeyer flasks and autoclaved.

E, rigidiusculum, E, roseum, E, moniliforme, E, tricinctum, and E,

 

 

 

lateritium were individually grown in the medium for four weeks at

 

24°C with continuous shaking.

Soil preparation and infestation

 

Unless otherwise specified, Oshtemo-Boyer sandy loam soil (sand:
silezclay = 73:18:9, pH 5.5, 2.02% organic matter, water-holding cap-
acity (WHC) = 287 m1/kg), collected from St. Joseph county, Michigan,
was used throughout the experiments.

Soil was infested with E, sglggi, E, sglggi_f. sp. pjgj, and E,
oxyspgrum by mixing the wheat bran-sand cultures, previously passed
through a 1.7 mm (lo-mesh) sieve, with the soil at an approximate rate
of 10% (w/w). The soil was moistened to about 50% WHC and incubated
for eight weeks, during which time a constant chlamydospore concentra-
tion of 104-106/9 was achieved.

With the other Fusarium species, 100 ml portions of potato-
maltose broth cultures, previously homogenized in a Servall Dmni Mixer
(Ivan Sorvall, Inc.) at a rheostat setting of 50 for 5 minutes, were
mixed with 500 9 soil in plastic bags, followed by incubation at 24°C
for eight weeks. After the eight week incubation period, the infested

soils were air-dried and kept at 4°C until further use.

25

Individual WHC of soils was determined using a standard method
(128). A metal container with a porous bottom (diameter: 5cm, height:
3 cm) in which a moist filter paper was placed, was used. After placing
10 g of air-dried soil in the container, it was placed on a tray con-
taining water deep enough to touch the first 1 mm layer of the soil.
After two minutes on the tray the soil had absorbed an excessive amount
of water. The container was then transferred to a moist chamber, placed
on a piece of filter paper fbr about 10 minutes to drain the excessive
water. Care was taken so that the soil in the container did not touch
the filter paper sheet. The soil was then reweighed, the increase in '
weight from the initial indicating the WHC of the soil.

Soil pH, where necessary, was measured using a glass electrode
(127). TWenty g of soil was mixed with 20 ml distilled water in a 50
m1 beaker; the mixture was stirred at intervals for 30 minutes and let

stand for one hour before pH measurement.

Incorporation of organic residues into soil

Ten different kinds of plant and animal residues were tested:-
mature stems of barley, wheat, corn, and soybean collected from the
Michigan State University farm; commercial beet pulp, alfalfa meal,
linseed meal, cottonseed meal, and soybean meal; and ground crab
shells (supplied by H. Komada from Japan). The first five residues
were ground and passed through a 1.7 mm (lo-mesh) sieve before used.
Unless otherwise specified, these materials were mixed with air-dried
Fusarium-infested soil at a rate of 1% (w/w) by shaking in a plastic
bag for 30 seconds. The moisture content was then adjusted to 30-35%

WHC using tap water, and the moistened soil was further shaken for

26

another 30 seconds to obtain an even moisture distribution. The soil
was then transferred into a 550 m1 plastic container (200—300 g per
container), covered with a sheet of polyethylene and secured with a
rubber band. The treated soils were incubated at 24°C on a laboratory
bench.

In some experiments, the organic materials were composted for
different lengths of time before incorporation into soil. Composting
was done by mixing individual organic materials with 20% (w/w) soil
and 1% (w/w) urea. TWo hundred 9 portions of the mixtures were mois-
tened to about 30% WHC and incubated in sealed (Parafilm 'M', American
Can Co.) 500 ml Erlenmeyer flasks fbr 4, 6, and 10 weeks at 28°C. Dur-
ing incubation, the mixtures were stirred weekly using a spatula. The

composted materials were air-dried for 24 hrs before use.

Estimation of soil microbialpopulations

Soil microbial populations were estimated at intervals using
serial dilutions of soils from different treatments in 0.1% water agar,
and plating them on different selective media. '

For Fusarium, a modified selective medium developed by Komada
(86, 87) was used. The basal medium contained 1.0 g KZHPD4, 0.5 9 KCl,
0.5 g M9504.7H20, 10.0 mg Fe-EDTA, 2.0 g asparagine, 20.0 g galactose,
15.0 g Difco agar, and 1000 ml distilled water. The antimicrobial
supplement consisted of 1.0 g PCNB (75% wp), 0.5 g oxgall, 1.0 g
Na2B407. 10 H20, and 0.25 g chloramphenicol. This supplement was
added after steaming the basal medium for one hour. Further steaming
fbr another 30 minutes was required to completely dissolve the antimi-

crobial ingredients. The medium was then cooled to about 60°C in a

27

water bath befbre the pH was adjusted to 3.9 using 2 M H3P04. After
pouring onto sterile Petri plates (12 ml/plate) the medium was kept in
plastic bags at 4°C fur future use.

One-half ml of appropriate soil dilutions was pipetted into each
plate, spread over the surface of the agar and incubated for one week
at 24°C before Fusarium colonies were counted.

For fungi in general, PDA with 0.5 m1 TMN detergent (Union Car-
bide Co.) and 0.25 g chloramphenicol per liter was prepared, poured in
5-7 ml portions into test tubes. After autoclaving for 15 minutes the
agar medium was cooled to 45°C in a water bath and 0.5 m1 of soil sus-
pension was added, mixed fbr 5 seconds using a Vortex Jr. mixer, and
plated. For bacteria a similar technique was employed except that a
medium containing 100 ml soil extract, 1.0 9 glucose, 0.5 g KZHP04,
0.05 g PCNB (75% wp), 20 g DifCo agar, and 900 ml distilled water was
used. The soil extract was prepared by autoclaving 1 kg soil in 1
liter tap water, followed by repeated filtration until clear. For
actinomycetes the selective medium contained 4.0 g colloidal chitin,’
20 g Difco agar, and 1000 ml distilled water (69). Plates containing
the three different media were incubated for one week at 24°C before

colonies were c0unted.

Field experiments

 

Three separate experiments were conducted in the field using
alfalfa and oilseed meals as organic amendments.

In the first two experiments, soil was either without supplement-
al infestation or was artificially infested with wheat bran-sand cul-

tures of E, ogyspgrum and E, solani. In the third experiment, soil

28

was artificially infested with wheat bran-sand culture of E, §913p1_

f. sp. pjsj, Infestation was done by incorporating each culture at a
rate of ca. 1% (w/w) into the soil using a rototiller. Two weeks after
infestation, soil samples were collected for estimation of the initial
Fusarium populations.' At the same time, the organic amendments were
applied to the soil surface, and incorporated by rototilling. Changes
in Fusarium populations were followed by collecting soil samples every

two weeks.

Detection of volatile inhibitors

 

Several experiments were designed to determine if volatile in-
hibitors were involved in reducing Fusarium populations in the oilseed
meal-amended soils. In one experiment, containers with or without
polyethylene sheet covers were used for incubating treated soils. In
another experiment, stainless steel planchets containing Fusarium-
infested soil were incubated on the surfaces of treated soils in
closed containers.

Possible presence of ammonia and/or amines in the volatiles was
examined using 2% H3B03 as a trapping solution (22,133) placed in
glass vials and incubated on treated soils in closed containers. Per-
iodically, the solutions were removed from the vials and titrated
using 0.01llH2304 to estimate the ammonia and/or amine equivalents
(22). An indicator prepared by dissolving 0.33 g bromocresol green and
0.165 9 methyl red in 500 ml ethyl alcohol was used in the titration.

Possible involvement of ammonia and/or amines in reducing Fusarium
populations was tested by incubating Fusarium-infested soil in planchets

together with H3803 solutions on treated soils in closed containers.

29

Fusarium populations were estimated at the beginning of appropriate

experiments and bi-weekly thereafter.

Identification of volatile inhibitory substances

Four sets of 100 9 portions of amended and unamended soils were
prepared. The soils were maintained in plastic bags fbr fbur days,
mixed every other day, before transferring to closed containers.

One set of the treated soils was used to verify that the vola-
tile substances reduced Fusarium populations in soil. This was done
by incubating planchets containing Fusariumbinfested soil in the sur-
faces of amended and unamended soils in closed 500 ml containers.

The remaining three sets were utilized in the identification of
the volatile substances using infra-red spectroscopy. The treated
soils were incubated in 250 ml Erlenmeyer flasks sealed with rubber
stoppers. Each stopper was equipped with a connecting glass tubing,
0.5 cm in diameter, which in turn was sealed at its terminus using a
clamped tygon tubing (Figure 1). After three weeks of incubation, the
gases evolved in each set of treated soils were handled in three dif-
ferent ways. In one, the gases were transferred directly to an evac-
uated infra-red cell. In the second and the third, the gasses were
trapped by passing through a coiled glass tubing immersed in liquid
nitrogen for a period of five minutes, either directly or indirectly
via a column of anhydrous CaSO4 (10 cm long, 1.7 cm diameter) (Figure
l). The two ends of the coiled glass tubing were then sealed using
clamped tygon tubings, and the trapped gases were subsequently trans-

ferred to an evacuated infra-red cell fbr identification.

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31

Observation on the fate of Fusarium chlamydospores in soil
Chlamydospores of E, splgg1_were prepared according to the method
of Hsu and Lockwood (68). Conidia were germinated for 16 hours in a
shaking liquid medium containing the same ingredients as the basal
portion of the Fusarium selective medium described earlier. The germ-
lings were aseptically separated from the ungerminated conidia by file
tration through 0.15 mm (loo-mesh) sieve. The retained germlings
were aseptically washed several times, then suspended in sterile dis-

tilled water to obtain a density of approximately 103

germlings/ml.
Two ml portions of the suspension were applied onto Nuclepore membrane
squares (1 cm2, pore size: 0.4 um) by suction. The membranes were
then floated on 0.03 M Na2504 solution for 10 days, during which time
chlamydospores were formed. The membranes were buried in amended and
unamended soils with a nylon net separating the chlamydospores from

direct contact with the soils (Figure 2). Petri plates were used as

the containers, and these were enclosed in small plastic bags.

NYLON AMENDED
NET \ /\ SOIL

|
PETRI \ MEMBRANE
PLATE WITH SPORES

Figure 2. Nuclepore membrane with Fusarium chlamydospores on the
upper surface. A nylon net was used to prevent direct
contact between the treated soil and the chlamydospores.

 
 

32

The membranes were removed from the soils at intervals and the
chlamydospores were transferred onto discs of the selective agar me-
dium to observe germination (143).

Bioassay usingfisoybeans (Glycine max (L.) Herr.)
andlpeas_(Pisum sativum LT)_as host plants

 

To observe whether reduction in Fusarium populations would cor-
relate with reduction in root rot, soils infested with either a mix-
ture of E, 05ysp9rum and E, sglgpj_(isolated from diseased soybean
roots) or E, splgp1_f. sp. pi§i_were amended with 1% (w/w) alfalfa
and oilseed meals. Unamended but infested control soils were included
fbr comparison. After four weeks of incubation in closed containers,
all treated soils were spread on plastic trays to form a layer of '
about 1 cm thick and air-dried for one week at 24°C. After air-drying,
the soils were individually remoistened to about 30% WHC and four 200
g-portions of each soil were transferred into 200 ml styrofoam cups.
Soybeans (var. Hark) or peas (var. Miragreen) were planted, six in
each cup, and watered every day using 20 ml tap water per cup. The
cups were incubated in a growth chamber at 27°C, a photoperiod of 12
hrs/day, and a light intensity of about 20,000 lux.

Three weeks after planting, the plants were uprooted and the roots
were washed with running tap water. Root rot was then assessed on a
scale of 0-6, with 0 indicating a disease-free root system and 6, most

severely diseased (173).

Statistical analysis
In experiments using the dilution plate method, data were pre-

sented as averages of 2-3 replications, each consisting of 3-6 plates.

‘33

Analysis of variance was done using data transformed to log (x + l)
to maintain homogeneity of the variances of different treatments (155).
In the other experiments, 3 replications were used, and data were an-
alyzed without transformation. Most experiments, where necessary,
were repeated to verify the consistency in results. Significant dif-
ferences among treatments were estimated using least significant

ranges (L.S.R.) obtained from the Tukey's '3} procedure.

RESULTS

Effect of different soil amendments
on Fusarium pppfilitions

 

Three hundred 9 portions of soil infested with E, oxysporum and
E, gplggj_were individually amended with nine different kinds of ground
plant and animal residues, each at 1% (w/w) concentration. The soil
moisture level was adjusted to 30% WHC and the treated soils were in-
cubated in closed containers at 24°C. Estimation of Fusarium popula-
tions was done at the beginning of the experiment and at weekly inter-
vals.

Linseed and cottonseed meal amendments were the most effective in

1402/9

reducing the Fusarium population (Figure 3). Reduction to 10
was obtained using these amendments, and to 103-104/g with crabshell,
as compared with more than 105/g in the control and other treatments.
Fusarium population reductions by cottonseed meal, linseed meal, and
ground crabshell were significant at the 1% level (Table 1). However,
since the Fusarium population in soil amended with ground crabshell

was still high, this amendment was not used further in the work. Later

it was found that soybean meal was equally or more effective in reducing

Fusarium populations.

35

 

0

INHIAL POPULATION

/ a «at
a“ :5

nsnmum Kxutxnow
51., 5a

an

 

 

 

Figure 3. Fusarium populations in soils four weeks after
amendment with 1% (w/w) ground plant and aninal
residues.

36

Table l. Fusarium populations in soils four weeks after amendment
with 1% (w/w) ground plant and animal residues.

 

 

Log Fusarium Log Fusarium

Soil treatment population? Soil treatment populations/
9 soila g soila
Unamended control 5.74 Wheat straw 5.76
Soybean stalk 5.92 Corn stalk 5.64
Alfalfa meal 5.85 Crabshell 3.85
Barley straw 5.80 Linseed meal 2.14
Beet pulp 5.77 Cottonseed meal 1.57

 

aInitial log Fusarium population was 6.49/g soil. Least Signif-
icant Range (L.S.R.) for the final log Fusarium populations was 1.51
(Tukey's '3] procedure, E,= 0.01).

Effectiveness of different rates of oilseed meal
amendmentsTih reducing soil FGSariumppopuTations

Soils were amended with oilseed meals at rates of (w/w) 1%, 0.50%,
and 0.25%, and incubated in closed containers. Unamended control
soil and soil amended with 1% (w/w) alfalfa meal were included for
comparison.

Generally, soil Fusarium populations decreased with the increase
in amendment rates and with time over a six week period (Figure 4).
However, soybean meal applied at a rate as low as 0.25%(w/w) still re-
duced Fusarium populations to less than 0.1% of the unamended control
after six weeks of incubation. More than 1000 fold reduction was also
obtained in soils amended with linseed and cottonseed meals at a rate
as low as 0.50% (w/w).

When the experiment was completed, the soils containing 1% (w/w)

37

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imam ammo; .coFHmnsucw we mxmmz xwm 0» oz» Logan mpoms comm—yo so mm_mm_o mo
Azxx .wv mcovumsacmucou ucmsmmepu spa: caveman m—Pom cw mcovumpaaoa savanna;

 

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38

amendments were incubated for another four weeks without covers; mois-
ture contents were adjusted weekly by weighing. During this period,
Fusarium populations in the oilseed meal-amended soils remained in the

1 propagules/g. Fusarium populations in the alfalfa meal-

order of 10
amended and unamended'soils were still more than 104 propagules/g at
the end of the experiment. This result suggested that the oilseed
meal-amended soils maintained their fungistatic properties against

recolonization by the Fusarium.

Effect of soil amendments on soil microorgapjsns

 

Three hundred 9 portions of soil, either unamended or amended
with 1% (w/w) linseed and cottonseed meals were incubated in closed
containers after the moisture content had been adjusted to 30% WHC.
Estimation of soil microbial populations was done by employing fOur
different media selective to either Fusarium, fungi in general, bac-
teria, or actinomycetes. Soil microbial populations were estimated
at intervals using dilution plates containing the selective media.

After four weeks of incubation, Fusarium populations in oilseed
meal-amended soils were reduced to 0.1% or less of the unamended con-
trol (Table 2). After five weeks of incubation, the total fungal
populations in the amended soils were also highly significantly re-
duced, but less than that of Fusarium; the densities were still in the

order of about 104 propagules/g compared to about 106

propagules/g in
the initial population. The most frequently isolated fungi were from

the order Mucorales and from the genus Trichoderma. Similar reductions

 

were observed in soybean meal-amended soil. The numbers of actinomy-
cetes and bacteria were only slightly (but significantly) lower in

the amended soils than in unamended control.

39

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a

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em.. ee.~ .e.~ me.m mm.m a.eem e=.Laa=.

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no. .a.e.e.

 

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A3\zv RF saw: ucmsucmsm emumm mxmmz m>p$ on snow mcovpepaaoaipewnosume ppom .N ano»

4O

Effept of increased soilppH on reduction
in Fusarium pgpulations

Oilseed meal-amended soils consistently showed an increase in pH
up to 2.3 units as compared to alfalfa meal-amended and unamended
.soils. Therefore, an experiment was carried out to test whether arti—
ficially raising the soil pH would enhance the effectiveness of oilseed
meal amendments .

Artificially infested soil was treated with 0.17% (w/w) Ca(0H)2
to increase the natural pH (5.5) to 8.1. Three hundred 9 portions of
this soil were amended with 1% (w/w) alfalfa and oilseed meals. In-
fested soils with natural pH were similarly amended for comparison.
After adjusting the moisture content to 30% WHC, the treated soils
were incubated in closed containers.

Linseed and cottonseed meal-amended soils with increased pH
showed less reduction in Fusarium populations than those with natural
pH after four weeks of incubation. However, soybean meal-amended soil
with increased pH showed as much reduction as that with natural pH.
After six weeks of incubation, highly significant reduction in Eggegf
jug populations was obtained in oilseed meal-amended soils whether or
not treated with Ca(0H)2, as compared to the alfalfa meal-amended
soil (Table 3). Except fbr soybean meal, however, Fusarium popula-
tions were always higher in the Ca(0H)2-treated soils than in soils
with natural pH. The pH of the natural, but oilseed-meal amended
soils, was increased by 1.2-2.7 units as compared to that of unamended
soil; the pH of the alfalfa meal-amended soil remained nearly constant
(Table 3). In the Ca(OH)2-treated soil, the pH of unamended soil and

of soils amended with alfalfa, linseed, and cottonseed meals dropped

41

from 0.7-1.2 units (from pH 8.1 at the start of incubation). In con-
ltrast, soybean meal-amended soil showed an increase of 1.3 pH units.
Table 3. Effect of increased soil pH on reduction in Fusarium popu-

lations in soil six weeks after amendment with l% (w/w)
alfalfa and oilseed meals.

 

 

 

Log Fusarium pOpulationsa/g soil Final pr
501‘ treatment Natural Ca(OH)24weated Natural Ca(OH)
soil soil soil -treate
soil
Alfalfa meal 3.85 4.13 5.3 6.9
Linseed meal 1.04 2.38 6.5 7.4
Cottonseed meal 0.82 1.58 7.0 7.0
Soybean meal 0.48 0.48 8.1 9.4

 

 

 

aInitia1 log Fusarium population was 4.7/g soil. Least Signif-
icant Range for the final log Fusarium populations was 1.70 (Tukey's
'gf procedure, E_= 0.01).

bSoil was treated with 0.17% (w/w) Ca(OH)a to raise the pH from

5.5 to 8.1. The final pH of the natural and th Ca(OH)2—treated con-
trol soils were 5.4 and 7.4, respectively.

Effect of soil amendment with composted orgpnic
mateFials'on FusariUm populations

Three hundred 9 portions of Fusarium-infested soil were individ-
ually amended with 1% (w/w) composted oilseed meals to observe whether
reduction in the pooulations could be hastened. Soil amended with com-
posted alfalfa meal and unamended soil were included for comparison.
Incubation of the treated soils was done in closed containers.

Only slight reduction in Fusarium populations was obtained during

42

the first 10-14 days of amendment. Fusarium populations were drastic-
ally reduced by four weeks after amendment with oilseed meal composts
4, 6, and 10 weeks old (Table 4). However, significantly less reduc-
tion occurred in soils amended with 6 and 10 week old cottonseed com-
posts and 10 week old soybean compost. Soil amendement with 4 and

6 week old alfalfa composts exerted no effect on Fusarium populations,
but soil amended with 10 week old alfalfa compost had significant
higher Fusarium populations than the unamended control. These results
indicated that composting neither hastened nor enhanced the effective-

ness of the oilseed meal amendments in reducing Fusarium populations.

Field experiments usingEncovered plots

To test whether the oilseed meal amendments effective under lab-
oratory conditions would also be effective in the field, two experiments
were carried out in St. Joseph county, S.W. Michigan, in Spring and
early Summer of 1976.

One experiment was done near Nottawa at a site where Fusarium
seed and seedling disease of soybeans was fbund extensively during the
previous year. Natural soil and soil artificially infested with wheat
bran-sand cultures of E, 05yspgrum and E, gplepj_were amended with al-
falfa, linseed, and cottonseed meals at rates of 1%, 0.5%, and 0.25%
based<n1the weight of soil in rows, 3 meters long, 15 cm wide, and
15 cm deep. Unamended controls were included, and each treatment was
replicated three times.

The other experiment was done near Burr Oak where soybean seed-
lings failed to emerge after the Spring planting and Fusarium was

associated with the root rot symptoms. The treatments consisted of

43

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1. poepcou
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n.¢um.m mmmm umoasou
Fwom m\:owmmpzaoa aneomsm mom

 

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44

1% (w/w) amendments of alfalfa meal, linseed meal, cottonseed meal,
and unamended control; each was replicated three times. The plot size
for each treatment was the same as that in the earlier experiment.

Fusarium populations were estimated immediately befbre soil
amendment and bi-weekly thereafter. Most of the amended plots showed
higher Fusarium populations after 4-5 weeks as compared to the unamen-
ded controls (Tables 5 and 6). By 9-12 weeks after amendment, Eusegr
jum_populations in all amended soils were higher than those in the un-
amended control soils. Therefore, the results obtained from these
field experiments did not support those obtained under laboratory
conditions. Under the laboratory conditions the treated soils were
maintained at about 30% WHC and incubated in polyethylene sheet-covered
containers. In the field, the soil moisture may fluctuate and the
plots were not covered with polyethylene sheets.

Effect of soil moisture content on reduction
in Fusarium pppulations

 

Three hundred 9 portions of artificially infested soil were
either unamended or amended with 1% (w/w) alfalfa, linseed, and cotton-
seed meals. The moisture contents of the treated soils were adjusted
to 15%, 30%, 50%, and 100% WHC before incubation in closed containers
at 24°C. Fusarium populations were estimated at the beginning of the
experiment and bi-weekly thereafter.

Four weeks after amendment reduction in Fusarium populations was
greatest in linseed and cottonseed meal-amended soils with soil mois-
ture maintained at 30% and 50% WHC; reduction was not as great at 100%
WHC (Figure 5). Fusarium populations were increased slightly in those

soils at 15% WHC. In the unamended soils, Fusarium populations were

45

Table 5. Fusarium populations in natural and artificially infested
field soils five weeks after amendment with alfalfa and
oilseed meals. The experiment was done near Nottawa in
St. Joseph county, Michigan, in the Spring, 1976.

 

 

 

Ame Log Fusarium population/g soila
. ndment,
So11 treatment %(w/w)
Natural Artificially
soil infested
Unamended control 3.32 4.08
Alfalfa meal 1.00 4.28 4.53
0.50 4.28 4.48
0.25 3.97 4.20
Linseed meal 1.00 4.34 4.20
0.50 4.08 4.20
0.25 3.80 3.89
Cottonseed meal 1.00 4.25 4.40
0.50 4.34 4.30
0.25 3.96 4.30

 

 

 

aLog Fusarium populations at the time of soil amendment in
natural and artificially infested soils were 3.60 and 4.84, respec-
tively.

Table 6. Fusarium populations in naturally infested field soil four
weeEs after amendment with 1% (w/w) alfalfa and oilseed
meals. The experiment was carried out near Burr Oak, St.
Joseph county, Michigan, in early Summer, 1976.

 

 

Soil treatment Log Fusarium population per g soila
Unamended control 3.45
Alfalfa meal 5.18
Linseed meal 5.20
Cottonseed meal 5.04

 

aInitial log Fusarium population was 3.28.

46

affected only slightly by soil moisture.

 

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Figure 5. Fusarium populations in soils four weeks after amendment
with l% (w/w) linseed and cottonseed meals, as affected
by different soil moisture contents in closed containers.
In another experiment, soybean and alfalfa meals were included.
Similar trends in reduction of Fusarium populations were shown by
linseed and cottonseed meal amendments. However, soybean meal was
effective at all moisture levels tested, although reduction in Fusar-
ipm_populations at 15% WHC was significantly less than that at other

soil moisture levels (Table 7). Fusarium populations in the alfalfa

meal-amended soil, like those in the unamended control soil, were not

47

much affected by soil moisture.

Table 7. Effect of different soil moisture contents on reduction of
Fusarium populations four weeks after amendment with 1%
(w7wl alfalfa and oilseed meals.

 

 

 

Log Fusarium population/g soil at
indicatEH'EhTT—moisture content (% WHC)a

Soil treatment ___

15 30 50 100
Unamended control 4.86 4.78 5.08 5.00
Alfalfa meal 4.71 5.02 4.95 4.48
Linseed meal 5.08 2.15 2.23 3.76
Cottonseed meal 5.23 1.30 2.62 4.49
Soybean meal 1.21 0.52 0.52 ,0.52

 

 

aInitial log Fusarium population was 5.25. The Least Signifi-
cant Range (Tukey's '3] procedure, E.= 0.01) for the soil treatment
X soil moisture effect was 0.68.

Detection of volatile inhibitors
. An experiment was done to compare the effect of oilseed meal-
amended soils incubated in open and closed containers on Fusarium pop-
ulations. Soils were either unamended or amended with 1% (w/w) alfal-
‘fa, linseed, and soybean meals; the moisture content was adjusted to
:30% WHC. Two sets of such soil treatments were prepared; one was in-
cubated in polyethylene sheet-covered containers, the other in open
<:<>ntainers. The moisture contents of the soils in the open containers
Iveere maintained constant by addition of water every day. Fusarium

F><>l>ulations were estimated at the beginning of the experiment and

48

bi-weekly thereafter.

Oilseed meal-amended soil kept in open containers showed less
reduction in Fusarium populations than those kept in closed containers
(Figure 6). Six weeks after amendment, Fusarium populations in the
oilseed meal-amended soils in closed containers were reduced to about

2 4 propagules/g

10 propagules/g or less, as compared to more than 10
in open containers. Fusarium populations in alfalfa meal-amended and
in the unamended control soils showed little or no reduction in either
type of containers. The much greater reduction in closed containers
indicated that volatile substances might be involved.

To further test this possibility, 300 9 portions of soil either
unamended or amended with 1% (w/w) alfalfa and oilseed meals were pre-
pared. Planchets, each containing 2 g of Fusarium-infested soil, were
incubated on the surfaces of the treated soils in closed containers.i
Four planchets were incubated on each soil treatment. Fusarium pop-
ulations were estimated initially and bi-weekly thereafter.

After four weeks of incubation on oilseed meal-amended soils
Fusarium p0pulations in soils from the planchets were reduced to less
than 0.1% of those incubated on the unamended and alfalfa meal-amended
soils (Figure 7). Fusarium populations from planchets incubated on
“the unamended and alfalfa meal-amended soils were not reduced. This
l"&SUlt clearly showed that reduction in Fusarium populations was ob-

tained in soil samples separated from direct contact with the amended

ssciils, and strongly indicated the presence of volatile substances.

To test whether ammonia and/or amines were present in the vola-

ti les, 2% of H3803 solution was used as the trapping agent. The sol-

ut'ion was placed in glass vials and incubated on treated soils in

5

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49

 

 

 

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: —
Unamended Alfalfa Linseed Soybean
control meal meal meal

Fusarium papulations in soil placed in open and
closed containers 6 weeks after amendment with 1%
(w/w) alfalfa or oilseed meals. Least Significant
Range (L.S.R.) values were obtained using the
Tukey's 'gf procedure.

50

 

Initial Log Pbpulation

: Unamended control
: Alfalfa meal

: Linseed meal

: Cottonseed meal

: Soybean meal

(3:0.01)

UJOL">><

Log Fusarium pOpulation / g soil

 

 

   

Figure 7. Fusarium populations from soil samples in planchets
incuhated on soil amended with 1% (w/w) alfalfa or
oilseed meals in closed containers for four weeks.
The Least Significant Range (L. S. R. ) was obtained
using Tukey' s 'w' procedure.

51

closed containers. The soils were either unamended or amended with 1%
(w/w) alfalfa and oilseed meals, and the moisture contents were adjust-
ed to 30% WHC. At intervals the trapping solutions were removed from

the vials and three dr0ps of indicator were added. The solutions were

then titrated with 0.01 N H $04 to estimate the ammonia or amine equiv-

2
alents. The H3803 solutions were replenished immediately after each
removal.

The lartest amount of titratable materials was derived from soil
amended with soybean meal, followed by those amended with linseed and
cottonseed meals (Figure 8). The H3B03 solutions collected from the
alfalfa meal-amended and the unamended control soils did not change
the color of the added indicator.

An experiment was carried out to examine whether ammonia and/or
amines trapped by the H3803 solutions contributed to reduction in
Fusarium populations in soil. Four planchets, each containing 3 g
of Fusarium-infested soil and four small Petri plates (5.5 cm diame-
ter), each containing 10 ml of either 2% H3B03, H20, or nothing at
all, were incubated together on 500 9 treated soils in closed con-
tainers. Square plastic containers, (19 X 19 x 16) cm3, individually
enclosed in polyethylene bags were used as the incubation chambers.
The four planchets were placed in the center, and the Petri plates at
the four corners of each containers. Fusarium populations were esti-
mated at the beginning of the experiment and bi-weekly thereafter.

After four weeks of incubation, drastic reduction in Fusarium
populations occurred in soils in planchets incubated on linseed and
cottonseed meal-amended soils, regardless of the presence of the trap-

ping solutions (Table 8). However, no reduction was obtained during

Figure 8.

52

 

 

 

 

at! T 1 I I 1
.
./
i201 -
8v
3"
.
z
5. umuo
" 10" -
/A A
.oonowsuo
:;;/V’ —‘_-
o ........... 1-2?1'2." i133: t .1
‘9

Titration curves for the estimation of ammonia and/or amines
trapped by 2% H3803 solutions. The H3BO3 solutions were
placed in glass vials and incubated on soils either unam-
ended or amended with 1% (w/w) alfalfa and oilseed meals in
closed containers. An indicator was added to the H3803 so-
lutions before titrating with 0.01 N H2804.

53

incubation on soybean meal-amended soil in the presence of the trap-

ping solutions, although drastic reduction occurred in their absence.

Fusarium populations in soils incubated on the unamended control and

the alfalfa meal-amended soils were not affected by the presence of

the trapping solutions.

Table 8. Fusarium populations from soil samples in metal planchets
placed on soil amended with 1% (w/w) alfalfa and oilseed
meals. The soil samples were incubated in the presence of

H20 and H3803 as trapping solutions in closed containers
for four weeks.

 

 

 

Log Fusarium populationa/g soil sample

Trapping
solution

Unamended Alfalfa Linseed Cottonseed Soybean

control meal meal meal meal
None 4.90 4.43 2.24 2.02 1.48
H20 4.74 4.51 1.74 1.48 -5.00
H3803 4.87 4.87 2.66 1.48 5.00

 

 

aInitial log Fusarium population was 5.30. The Least Signifi-
cant Range (Tukey's 'w} procedure, P = 0.01) for the trapping solu-
tion X soil treatment effect was 1.38.

Identification of volatile inhibitory substances

 

Infra-red spectroscopy was used in attempting to identify the
volatile inhibitory substances. Four sets of amended and unamended
soils were prepared. In one set Fusarium-infested soil samples placed
in planchets were incubated on the surfaces of treated soils in closed
containers. After four weeks of incubation, Fusarium populations in

soil incubated on oilseed meal-amended soils were reduced to less than

54

0.1% of those incubated on alfalfa meal-amended and unamended control
soils, indicating that volatile substances inhibitory to the fungus
were produced.

The other three sets of treated soils were incubated in sealed
Erlenmeyer flasks. After three weeks of incubation the gases evolved
in each soil were transferred to an evacuated infra-red cell, either
directly or by first trapping them in liquid nitrogen. Gases origin-
ated from all amended soils showed formation of crystals upon passing
through coiled glass tubings immersed in the liquid nitrogen. However,
when the transferred gases were scanned with wavenumbers ranging from
850-2400 cm'], no indication of the presence of substances other than
CO2 (at 2360 cm']) was observed on the infra-red spectrograms. In
view of the fact that oilseed meal amendments showed substances which
were trapped in H3803 and titratable by 0.01 N H2504 (Figure 8), the
method of obtaining samples of the volatile substances in the present '
experiment may not be an appropriate one.

The fate of Fusarium chlamydospores in
the amended soils

 

To study the mode of reduction in Fusarium p0pulations in the
oilseed meal-amended soils, Fusarium chlamydospores were placed on
membranes and buried in amended soils for different lengths of time.
Ten membranes were buried in each 100 9 portion of treated soil in a
Petri plate. Two membranes were removed from individual soil treat-
ments after each incubation period and the chlamydospores were trans-
ferred onto discs of a selective agar medium fer microscopic observa-
tion on germination, either immediately or following a 24 hr incuba-

tion period on the agar discs. Cotton blue in lactophenol was used

55

to stain the chlamydospores and the germ tubes. Percent germination
was estimated based on 100 counts from four different microscope
fields on a single membrane.

In one experiment, the chlamydospores were buried at the same
time as the soils were amended. When the membranes were taken out 24
hours after burial and immediately observed under a microscope, 95%
or more of the chlamydospores from all amended soils had germinated,
while none had germinated in the unamended soil. Production of coni-
dia and some lysis of hyphae had occurred in all amended soils by 48
hours of burial. Lysis of hyphae was very extensive and production of
new chlamydospores was frequently seen after three days burial in the
amended soils. When membranes were removed from amended soils 7-21
days after burial, many new chlamydospores were present but none had
germ tubes. No hyphae were present.

After seven days of incubation in the oilseed meal-amended soils,
less than 30% of the chlamydospores germinated after 24 hour incubation
on the agar discs, whereas the germinability of the chlamydospores bur-
ied in the unamended and the alfalfa meal-amended soils was more than
80% (Figure 9). After 21 days of incubation in the oilseed meal-amended
soils, the germinability of the chlamydospores was completely lost,
while in the unamended and the alfalfa meal-amended soils 65% or more
of the chlamydospores were still viable.

In another experiment, the chlamydospores were buried seven days
after soil amendment. After seven more days of burial in the oilseed
meal-amended soils, the germinability of the chlamydospores was prac-
tically zero, whereas those buried in the unamended and the alfalfa

meal-amended soils still showed 80% or more germination (Figure 10).

F1 gure 9.

GERMINATION: 9‘

56

 

'o . "NAMEND.

Aalaiza _

 

 

13:34reoou

    

LINSEED

 

 

 

7 1.4 21
ms in son

Viability of Fusarium chlamydospores as determined by the
agar disc method. The chlamydospores prepared on Nuclepore
membranes were incubated in soil amended with 1% (w/w) a1-
falfa and oilseed meals fbr different lengths of time. Af-
ter each incubation period. membrane samples were removed
and the chlamydospores were transferred onto discs of a
selective agar medium fbr microscopic observation of germ-
ination either immediately or fbllowing a 24 hr incubation
on the discs. Least Significant Range (L. S. R. ) values were
obtained using Tukey' s 'w' procedure.

Figure 10.

57

 

KJ- \wmu‘
* .
~w _ Llflgubl -(
z o
2
<
3!
£40 . scrum» «man .1

o

Oil-sou. M
mm
J

 

 

" l ' 17
DAYS

Viability of Fusarium chlamydospores as determined by

the agar disc method. The chlamydospores were buried
seven days after soil treatment. Technical descriptions
for this experiment were as described in Figure 9. Least
Significant Range (L.S.R.) values for 7 and 10 days of
burial were 13.5 and 42.3, respectively (Tukey's '3} pro-
cedure, E_= 0.01).

58

Similar results were obtained when the membranes were removed from

the treated soils after 10 days of burial.

Effect of oilseed meal amendments on
different species of Fusarium

 

Two 9 portions of soil infested with either E, lateritium, E,

 

moniliforme, E, rigidiusculum, E, roseum, or E, tricinctum were incu-

 

 

 

bated on the surface of 500 9 soil either unamended or amended with
1% (w/w) alfalfa and oilseed meals in closed containers, (19 X 19 X
6) cm3.

After four weeks of incubation, all five Fusarium species tested
were drastically reduced on the oilseed meal-amended soils (Table 9).
None of the Fusarium species were reduced on the unamended and the
alfalfa meal-amended soils.

Relation of soil Fusariumppppulations
to amount of root rot

 

 

Oilseed meal-amended soils showed typically reduced Fusarium
populations after four weeks. Using soybeans as host plants, a positive
correlation between root rot and Fusarium populations/g soil was ob-
tained. Soybean meal amendment seemed to be the most effective, as
compared to linseed and cottonseed meal amendments, based on the fact
that disease was reduced and no phytotoxic symptoms were observed.
Phytotoxicity occurred in cottonseed meal- and linseed meal-amended
soils; especially in the latter, germination of soybean seeds was
completely inhibited. More root rot was associated with the unamended
and alfalfa meal-amended soils. Because of the difficulty in obtain-
ing root rot disease symptoms with soybean, due to apparently specific

environmental requirements which are not yet completely worked out,

59

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60

Fusarium root rot of peas was used in further work.

Reduction in root rot of peas was also associated with the de-
crease in Fusarium populations. Log E, splepj_f. sp. pj§j_populations/
g of unamended soil and soils amended with 1% (w/w) alfalfa, linseed,
cottonseed, and soybean meals were 3.90, 3.76, 2.00, 1.70, and 1.40,
respectively; and the disease indices of peas after a three week
growth were 3.0, 2.7, 2.1, 1.6, and 0.9, respectively (Table 10). How-
ever, significant reduction in root rot severity was only obtained in
soybean meal-amended soil as compared to the alfalfa meal-amended
(E_= 0.05) and the unamended control (E_= 0.01) soils.

Table 10. Severity of pea root rot caused by Fusarium solani f. sp.
pޤj_as affected by reduced populations of the pathogenic

propagules after four weeks of incubation in soil amended
with 1% (w/w) oilseed meals.

 

 

 

Soil treatment pophlgtddh7diddila Disease indexb
Unamended control 3.90 3.0
Alfalfa meal 3.76 2.7
Linseed meal 4 2.00 2.1
Cottonseed meal 1.70 1.6
Soybean meal 1.40 0.9

 

aInitial log Fusarium population/g soil was 4.35.

bRoot rot was rated on a scale of 0-6, with 0 indicating no
disease and 6, most severe disease. The Least Significant Range
(Tukey's 'wf procedure) values for the disease indices were 1.5
E.= 0.05) and 2.0 (E_= 0.01).

61

In this experiment, the least range required to show significant
differences among treatments was relatively wide, presumably due to
the great variations in root rot indices in the presence of linseed
and cottonseed meal amendments. Both treatments caused some phyto-
toxic symptoms on the root systems, like root stunting and necrosis
which, though not characteristic of the disease, complicated disease
evaluation. Moreover, the disease indices for the four replications
varied from 1.2-2.5 and 0.5-2.7 for cottonseed and linseed meal amend-
ments, respectively. No phytotoxicity was observed in plants grown in

soybean meal-amended soils.

A field experiment usipgppoveredplots

In Spring of 1977, an experiment was carried out at Michigan
State University farm to examine whether the volatile inhibitory .
effect of oilseed meals could be maintained under field condition
by covering the experimental plots with polyethylene sheets.

Field soil (Conover loam, pH 6.5, organic matter content: 31.5%,
sand:si1t:clay = 50:24:26, WHC: 390 ml/kg) artificially infested with

Fusarium solani f. sp. pisi was either unamended or amended with

 

about 1% (w/w) alfalfa and oilseed meals. Amendments were incorporated
in rows 1.5 m long, 15 cm wide, and 15 cm deep. The rows were then
covered individually with polyethylene sheets which extended 10 cm
beyond the edges of the rows, and were secured by applying soil over
them. Unoovered rows, amended with 1% (w/w) alfalfa and soybean meals
were included for comparison. Assignment of treatments to rows was
completely randomized, and each treatment was replicated three times.

No reduction in Fusarium populations was obtained in all amended

62

soils, whether or not they were covered with polyethylene sheets. By
eight weeks after amendment, soil samples collected from all amended
plots showed higher Fusarium populations as compared to those from
unamended control plots (Table 11). When the pH of the soils in co-
vered plots were measured, it was f0und that linseed, cottonseed, and
soybean meal amendments reduced the natural pH of 6.5 to 4.5, 4.5, and
5.2, respectively. The pH of the unamended soil was 6.3 and that of
soil amended with alfalfa meal was 5.8.
Table 11. Soil pH and Fusarium populations in artificially infested
Conover loamfsdTT-Eight weeks after amendment with 1%

(w/w) alfalfa and oilseed meals. The experiment was done
at Michigan State University farm in Spring of 1977.

 

 

 

 

50,] treatment Log Fusarium population/ Soil pr
g 5011

Covered plots

Unamendéd control 3.95 6.3

Alfalfa meal 4.73 5.8

Linseed meal 4.74 4.5

Cottonseed meal 4.62 4.5

Soybean meal 4.72 5.2

Non-covered plots

Alfalfa medT 4.47

Soybean meal 4.75

 

aLog Fusarium population at the time of soil amendment was 4.25.

bSoil pH before soil amendment was 6.5.

Effect of different soils on effectiveness of oilseed
meal amendments in reducipgTFusarium populations

The failure of oilseed meal amendments to reduce Fusarium popu-

lations under covered field conditions in Conover loam soil led to an

63

experiment to test whether the effectiveness of those amendments could
be affected by the types of soil used.

Conover loam and Brookston loam (pH 7.5, sand:si1t:clay = 48:
33:19, 6.46% organic matter, WHC: 535 ml/kg) soils were artificially
infested with wheat bran-sand cultures of E, gplegj_f. sp. pjgj,

Three hundred 9 portions of each soil were individually amended with
1% (w/w) alfalfa and oilseed meals. After adjusting the moisture
level to 30% WHC, the amended soils were incubated in closed contain-
ers.

No reduction in Fusarium populations was obtained in Brookston
loam soil. Even after 10 weeks of incubation, the Fusarium popula-
tions in the alfalfa and the oilseed meal-amended soils remained in
the order of about 105 propagules/g soil (Table 12). In Conover loam
soil, slight reduction in Fusarium populations were shown in the oil-
seed meal-amended soils by six weeks of incubation. However, by eight
weeks of incubation Fusarium populations in such soils were still in
the order of about 103 propagules/g soil. After 10 weeks of incuba-
tion the Fusarium populations in oilseed meal-amended soils were re-
duced to less than 102 propagules/g (Table 12). The Fusarium popula-
tions in the alfalfa meal-amended soil remained in the order of about
105 propagules/g throughout the experiment.

When the soil pH was measured after 10 weeks of incubation, all
amended soils showed a reduction of 1.3-2.0 pH units from the original
in Conover loam soil, and 1.4—1.8 pH units in Brookston loam soil. A
reduction of 1.9-2.1 pH units from the originals was also obtained
when unamended Conover loam and Brookston loam soils were incubated

for 10 weeks in closed containers.

64

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DISCUSSION

Estimation of Fusarium populations in soil was based mainly on
the use of a selective medium. The efficiency of the medium in sup-
porting germination and growth of Fusarium appears to be very high.
Direct microscopic observation of soils naturally infested with E,

oxyspgrum f. sp. lycppersici revealed that two or more chlamydospores

 

in soil were often seen aggregated and adhering to soil particles (87).
When each such aggregate was considered as a unit, this medium showed
a plating efficiency of more than 80%. Regardless of the plating ef-
ficiency of the medium, appropriate controls were always included in
the experiments, and plate counts were correlated with spore viability
as determined microscopically and with a disease bioassay, as will be
discussed in the following.

Linseed, cottonseed, and soybean meals incorporated into a
sandy loam soil at a rate of 1% (w/w) were effective in drastically
reducing Fusarium populations in closed containers in the laboratory.
These amendments, however, did not reduce Fusarium populations under
natural field conditions (Tables 5 and 6). Further work in the labor-
atory indicated that a much greater reduction in Fusarium populations
was obtained in oilseed meal-amended soils incubated in closed con-
tainers than in those incubated in open containers (Figure 6). Re-
duction was also obtained in soil placed in planchets and incubated

on the surfaces of oilseed meal-amended soils in closed containers

65

66

(Figure 7). These results provide strong evidence that volatile in-
hibitory substances were involved.

Volatile amines and/or ammonia were apparently produced by the
oilseed meal-amended soils, as indicated by the titration curves of
H3803 solutions using 0.01 N H2504 (22, 133, Figure 8). The fact
that oilseed meal-amended soils showed phytotoxicity and an increase
in pH up to 2.6 units also is consistent with the possible production
of ammonia (5, 105). In soybean meal-amended soil, the volatile sub-
stances trapped in the H3803 solutions and water were apparently
solely responsible for the reduction in Fusarium populations, at least
insofar as volatile activity is concerned, since their effects were
nullified by the presence of those solutions (Table 8). The fungitox-
ic effect of linseed and cottonseed meal amendments, however, were
not nullified when similarly treated, suggesting that other volatile
toxic substances nay also have evolved from the linseed and cotton-
seed meal amendments.

Attempts to identify the volatile inhibitory substances using
infra-red spectroscopy have not been successful. Except for C02, the
presence of other volatile substances was not detected. This was pre-
sumably due to very low equilibrium concentrations present in the gas
phase at any given time over the soils. CO2 could not be responsible
(for the reduction in Fusarium populations, because even in alfalfa
meal-amended soils high concentrations of CO2 were produced.

A The fact that reduction in Fusarium populations in oilseed meal-
amended soils was governed by production of volatile inhibitory sub-
stances may explain why those amendments were not effective in the

field experiments. Moreover, the effectiveness of the amendments,

67

especially linseed and cottonseed meals, was affected by soil moisture
levels (Figure 5, Table 7). Laboratory experiments in closed containers
showed that soil types also influenced the effectiveness of the oilseed
meal amendments. Reduction in Fusarium populations occurred at a much
slower rate in the Conover loam soil than in the Oshtemo-Boyer sandy.
loam soil; no reduction was obtained in the Brookston loam soil (Table
12). The Brookston loam soil contained twice as much organic matter as
the Conover 10am, and three times as much as the sandy loam soils.

The reduced or nullified effectiveness of the oilseed meal amendments

in the two loam soils nay be due to immobilization of the inhibitory
substances by the soil organic matter and clay minerals, the concentra-
tions of which were high in these soils, and also to microbial degra-
dation (18). These considerations also may explain why no reduction

in Fusarium populations was detected within eight weeks in the field
experiment using Conover loam soil, even though the plots were covered
(Table 11).

Activities and growth of nitrifying bacteria are known to be fa-
vored by neutral to alkaline soil environments (5). In acid soils nit-
rification proceeds very slowly even in the presence of adequate nutri-
ents and the responsible species are rare or may be totally absent.
Oilseed meal amendments in the sandy loam soil (pH 5.5) resulted in an
increase in pH (to pH 8.1 in case of soybean meal); in the two loam
soils (pH 6.5-7.5), however, these amendments decreased the soil pH
by 1.3 units or more after 10 weeks of incubation. This decrease in pH
is probably due to nitrification of nitrogenous compounds originated
from the organic amendments, and from the indigenous organic matter in

the case of unamended loam soils. It would be of interest to

68

investigate how the soil pH would change and how Fusarium populations
would be affected if 2-ch1oro-6-(trichloro-methyl)pyridine (N-serve)
is applied together with the amendments in these soils. This compound
is an inhibitor of nitrification of ammonium-N (53).

The volatility of the toxic substances may limit the practica-
bility of the oilseed meal amendments. However, this may be compensa-
ted in part by their broad range of effectiveness against different
species of Fusarium and the magnitude of the reduction achieved (Table
9). Moreover, drastic reduction in Fusarium populations was obtained
at amendment rates as low as 0.25% (w/w) fer soybean meal and 0.50%
(w/w) fer linseed and cottonseed meals after six weeks of incubation
(Figure 4). These results are important not only from the economic
standpoint, but also in minimizing the hazard of phytotoxicity. Whe-
ther or not the oilseed meal amendments would be effective in reduc-
ing populations of other pathogenic fungi is still to be looked into.

Another positive feature of the oilseed meal amendments was
their selective action against different groups of soil microorganisms.
In spite of their strong inhibitory effects against Fusarium spp., they
affected the actinomycete and bacterial populations relatively slight-
ly (Table 2). Fungi from the order Mucorales and Trichoderma spp.

 

were also feund plentiful in the oilseed meal-amended soils. The pre—
sence of these microorganisms will maintain the fungistatic levels of
the soils (99, 100, 101) which will in turn minimize recolonization of
the soil by Fusarium.

Raising the pH of Oshtemo-Boyer sandy loam soil prior to amend-
ment with oilseed meals did not enhance reduction in Fusarium popula-

tions (Table 3). In fact, linseed and cottonseed meal-amended soils

69

with increased pH showed less reduction in Fusarium populations than
those with natural pH; effectiveness of soybean meal was not affected
by the soil pH. This approach, therefore, would not appear to be a
useful means of enhancing the effectiveness of the amendments. The
identities of the volatile substances evolved from the first two oil-
seed meal amendments need to be known before the effect of pH can be
explained.

Composting the oilseed meals prior to incorporation into the
soil offered no advantages over non-composted materials in reducing
Fusarium. Decreased reduction in Fusarium populations occurred with
the increase in composting periods (Table 4).

Amendment of soil with either alfalfa or oilseed meals tempor-
arily nullified the fungistatic properties of the soil.' In freshly
amended soils 95% or more of Fusarium chlamydospores on buried meme
branes germinated after one day, while no germination occurred in
unamended soils. Germination was followed by production of conidia,
lysis of hyphae, and production of new chlamydospores during the first
three days of burial. The fungistatic properties of the soils, how-
ever, were restored by seven days after amendments; no germination of
chlamydospores was observed in soil after this period. Germination
occurred only after incubating the chlamydospores on discs of the sel-
ective agar medium for 24 hours. Prolonged incubation in oilseed
meal-amended soils led to a reduced viability of the chlamydospores
(Figures 9 and 10). The viability of the chlamydospores in alfalfa
meal-amended and unamended control soils remained above 60% even after
21 days of burial. These results indicated that the mechanism of pop-

ulation reduction was not associated with germination-lysis as has

70

been reported in several cases (3, 23, 24, 25, 119, 145), but rather
a direct killing of the newly f0rmed, but ungerminated chlamydospores
by toxic substances produced during incubation in the oilseed meal-
amended soils.

Reduction in viable populations of pathogenic Fusarium in oil-
seed meal-amended soils was correlated with reduction in root rot of
_ soybeans and peas (Table 10). However, of the three oilseed meals
used, soybean meal was the most effective in reducing root rot sever-
ity. It also showed no phytotoxicity following one week of air-drying,
whereas root stunting and necrosis were associated with linseed and
cottonseed meal amendments, even after this period. Phytotoxicity
exerted by soil organic amendments has been known to enhance several
soil-borne diseases (93, 123, 162).

In conclusion, non-specific and drastic reduction in Fusarium
populations could be obtained in certain soils amended with linseed,
cottonseed, and soybean meals, through production of volatile sub-
stances lethal to the chlamydospores of the fungus. This mode of re-
duction in Fusarium populations as a result of soil amendments using
plant residues apparently has never been reported befbre. Reduction
in pathogenic Fusarium populations was correlated with reduction in
I root rot, although with linseed and cottonseed meal amendments the
severity of root rot was obscured by their residual phytotoxicity.
Soybean meal seems to be the most promising among the three oilseed
meals tested; it showed the greatest reduction in Fusarium populations,
its phytotoxic effect in soil was completely removed within one week
of air-drying, and its effectiveness could be maintained even at a

rate as low as 0.25% (w/w). Reduction in Fusarium populations also

71

correlated with the results obtained from direct microscopic observa-
tions on chlamydospore viability. Therefore, the fact that oilseed
meal amendments are effective in reducing Fusarium populations in soil
is beyond any doubt. Because volatile substances are involved, if
these amendments were to be used effectively in the field, the soil
should be sealed and kept moist f0r a period of time, depending on

the type of soil involved, enough time should be allowed for disappear-
ance of phytotoxic residues of the amendments after the Fusarium popu-

lations have been reduced, befbre crops are planted.

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