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THE DISEASE-SUPPORTING CAPABILITY OF
SOILS OF DIFFERENT FUNGISTATIC CAPACITY

By

Thomas Siegfried Isakeit

A THESIS

Submitted to

Michigan State University
in partial fulfillment of the requirements‘
for the degree of

MASTER OF SCIENCE

Department of Botany and Plant Pathology

1984

ABSTRACT

THE DISEASE-SUPPORTING CAPABILITY 0F SOILS
OF DIFFERENT FUNGISTATIC CAPACITY

By

Thomas Siegfried Isakeit

Disease incidence in three soils artificially infested
with root-infecting fungi was compared with the degree of
fungistasis in the soils. Incidence of tomato wilt, caused

by Fusarium oxysporum f. sp. lycoPersici, was in the order

 

Capac loam >Boyer sandy loam >Colwood loam. This ranking
was inversely correlated with that of fungistasis to the
pathogen, as determined by nutrient titration. By contrast,

incidence of radish wilt, caused by E. oxysporum f. sp.

 

conglutinans was in the order Boyer sandy loam >Colwood

 

loam > Capac loam. However, fungistasis was in the order
Colwood loam > Boyer sandy loam > Capac loam. Total
microbial biomass was twice as high in Colwood loam as in
the other soils. The nutrient sinks of the soils, as
measured by the rate of respiration of added glucose, was in
the order Colwood loam > Capac loam > Boyer sandy loam.
These two soil characteristics were partially correlated
with fungistasis in the soils, and were inversely correlated
with tomato wilt but not radish wilt development in the
soils. These results suggest that factors in addition to

fungistasis are involved in disease expression.

To my parents.

ii

ACKNOWLEDGEMENTS

I sincerely appreciate the guidance, patience,
understanding, and especially the encouragement of my major
professor, Dr. J.L. Lockwood. In the tumult that marked my
three years in Michigan, his laboratory was truly my "home".

I also thank the other members of my committee, Dr.
M.L. Lacy and Dr. G. Safir for their suggestions and
especially their criticisms.

I thank the participants of "The Alex Filonow Memorial
Seminar Series", especially the founder, Dr. Alex B.
Filonow, for their contributions to my education.

I am extremely grateful to my typist par excellence,
Ms. Janet Besaw, for her devotion and diligence. Thanks to
you, Janet, I made the deadline.

Finally, I am grateful to the Michigan Agricultural
Experiment Station for the funds that made my studies at MSU

possible.

iii

TABLE OF CONTENTS

LISL Of Tables 0 o o o o o o o o o o o o o o o o o o 0 Vi

LiSt Of Figures 0 o o o o o o o o o o o o o o o o o o Viii

Literature Review . . . . . . . . . . . . . . . . . . 1
I Introduction . . . . . . . . . . . . . . . . 1
II Disease-suppressiveness in soil . . . . . . . 3
III Fungistasis in disease-suppressive soils . . 14

IV Role of the microflora in suppression . . . . 23
V Competition for nutrients in suppressive soils 29

List of References . . . . . . . . . . . . . . . . . . 32
Introduction . . . . . . . . . . . . . . . . . . . . . 40

Materials and Methods . . . . . . . . . . . . . . . . 42

 

 

 

 

 

 

 

 

 

 

 

 

 

Characterization of soils . . . . . . . . . . . 42
Microbial characteristics of soils . . . . . . . 42
Maintenance of fungi . . . . . . . . . . 45
Production of inoculum . . . . . . . . . . 46
Cochliobolus sativus . . . . . . . . . 46
Fusarium app. . . . . . . . . . . . . . . . 46
Assay of disease . . . . . . . . . . . . . . . . 47
Fusarium oxysporum f. sp. lycopersici . . . 47
Fusarium solani f. sp. pisi . . . . . . . . 48
Fusarium oxysporum f. sp. conglutinans . . . 49
Cochliodeus sativus . . . . . . . . . . . . 50
Quantitative determination of fungistasis . . . . 52
Soil solution assay . . . . . . . . . . . . . . . 53
Bioassay for volatile inhibitors . . . . . . . . 53
Soil respiration of glucose . . . . . . . . . . . 54
Results . . . . . . . . . . . . . . . . 55
Disease incidence in three soils in relation to
their level of fungistasis . . . . . . . . . . . 55
Fusarium oxysporm . sp. conglutinans . . . 55
Fusarium oxysporum f. sp. _ycopersici . . . 61
COchliobOIus sativusp . . . . . . . . . . . . 67
Fusarium solani‘f. .pisi . . . . . . . . 70

 

iv

Page

Germination of propagules on non-sterilized

and sterilized soil . . . . . . . . . . . . . . . 74
Assay of soil solutions for germination

inhibitors . . . . . . . . . . . . . . . . . . . 74
Assay of soils for volatile inhibitors . . . . . 77
Microbial biomass and microbial composition

of the soils . . . . . . . . . . . . . . . . . . 77
Nutrient sinks of three soils . . . . . . . . . . 79

DiscuSSion . O O O O O O O O O O O O O O O O O O O O O 84

List of References . . . . . . . . . . . . . . . . . . 95

Table

Table

Table

Table

Table

Table

Table

Table

LIST OF TABLES

Characteristics of soils

Incidence of Fusarium wilt of radish in three
non—sterilized and sterilized soils, after 25
days.

Recovered populations of Fusarium oxysporum
f. sp. con lutinans from three soils infested
with 20,050 and 100,000 propagules per gram
(ppg), at the time of seeding and 24 days
after seeding to radish.

 

 

Incidence of radish wilt in two soils
infested with two concentrations of Fusarium
ox s orum f. sp. conglutinans and maintained
at JO EPa (—0.1 bars) matric potential, and
recovered populations of the pathogen, both
three weeks after planting.

Effect of Fusarium Bxyspogum f. sp.
lycopersici at three-inoculum dbnsities (ppg)
on tbmato shoot length, fresh shoot weight,
and vascular necrosis in three soils, 6 weeks
after transplanting.

 

Effect of Cochliobolus sativus at three
inoculum densities on severity of sub-crown
internode disease and numbers of leaf lesions
on wheat in three soils, 4 weeks after
seeding.

 

Effect of Cochliobolus sativus at three
inoculum densities on severity of sub-crown
internode disease on wheat in three soils,
four weeks after seeding.

 

Effect of Fusarium solani f. sp. isi at two
inoculum densities on root, shoot, an lesion
lengths, fresh shoot weights and disease
severity on pea seedlings in three soils,
four weeks after transplanting.

 

vi

PAGE

43

56

60

62

66

69

71

73

r1
P. 1.

 

Table 9.

Table 10.

Table 11.

Effect of Fusarium solani f. sp. isi at two
inoculum densities on shoot and les on length
and fresh shoot weights on pea seedlings in
three soils, four weeks after transplanting.

Biomass and microbial populations in three
soils.

Ranking of soils with respect to their
microbial biomasses, nutrient sinks, levels
of‘fungistasis, and relative incidences of
radish and tomato wilt.

vii

PAGE

75

78

85

 

Figur

\JJ

Figure:

LIST OF FIGURES

Effect of chlamydospores (2 X 105 per
gram of soil) of Fusarium oxysporum f.
sp. conglutinans on wilt incidence of
radish in three soils. Data represent
mean values, based on 4 replicates of 12
plants each per treatment. Wilt
incidence after 13 days was significantly
greater (P=0.05) in Boyer sandy loam than
in the other soils.

 

 

 

Effect of five inoculum densities of
Fusarium oxysporum f. sp. conglutinans on
wilt incidence 6f'radish in three soils,
24 days after planting. Data represent
mean values, based on 4 replicates of 12
plants each per treatment. Wilt
incidence at 100,000 propagules per gram
of soil was significantly greater
(P=0&XM) in Boyer sandy loam than in the
other soils.

 

Germination of chlamydospores of Fusarium
oxysporum f. sp. conglutinans on three

 

 

soils amended with different
concentrations of 5:1 (w/w) glucose:
peptone, after 12 hours. Data represent
mean values, based on 4 replicates per
treatment; 100 spores were counted per
replicate. Duncan's multiple range test
showed the following differences amogg
means: 150 ug glucose/soil, Boyer ,
Capacc, and Colwooda; 300 ug
glucose/soil, Boyerb, Capacc, and
Colwooda. Mean values of soils followed
by the same letter did not differ
significantly (3:0.05).

viii

PAGE

57

59

63

Effect of three inoculum densities of
Fusarium oxysporum f. sp. lycopersici on
wilt incidence of tomato in three soils,
6 weeks after transplanting. Data
represent total incidence in 20 plants
per treatment. Wilt incidence was
significantly lower (3:0.05) in Colwood
loam than in the other soils using chi-
square analysis.

 

 

Germination of chlamydospores of Fusarium
oxysporum f. sp. lycopersici on three
soils amended with different
concentrations of 5:1 (w/w)
glucose:peptone, after 12 hours. Data
represent mean values, based on 4
replicates per treatment; 100 spores were
counted per replicate. Duncan's multiple
range test showed the following
differences among eans: 150 ug
glucose/soil, Boyer , Capacc, and
Colwooda; 300 pg glucose/soil, Boyera,
Capacb, Colwood . Mean values of soils
followed by the same letter did not
differ significantly (2:0.05).

 

 

Germination of conidia of Cochliobolus
sativus on three soils amended with
diff§?3ht concentrations of 5:1 (w/w)
glucose:peptone, after 6 hours. Data
represent mean values, based on 4
replicates per treatment; 100 spores were
counted per replicate. Duncan's multiple
range test showed the following
differences among means: 600, 120 , and
2400 ug glucose/soil, Boyera, Capac , and
Colwooda. Mean values of soils followed
by the same letter did not differ
significantly (2:0.05).

 

ix

PAGE

65

68

72

1O

Germination of chlamydOSpores of Fusarium
solani f. sp. isi on three soils amended
with differen concentrations of 5:1
(w/w) glucose:peptone, after 12 hours.
Data represent mean values, based on 4
replicates per treatment; 100 spores were
counted per replicate. Duncan's multiple
range test showed the following
differences among means: 150 an%)300 ug
glucose soil, Boyera, Capac , agd
Colwood ; 600 ug glucose/soil, Boyera ,
Capac , and Colwooda. Mean values of
soils followed by the same letter did not
differ significantly (P=0.05).

Cumulative 14002 respiration by three
soils at -1 kPa (-0.01 bars) matric
potential“: pulsed with 0.05 1101 (1.85
MBq) of -glucose. Data represent mean
values, based on 3 replicates per soil.
No significant differences (P=0.05) were
observed among the soils‘during 2-8
hours.

C0 respiration by three soils at —1 kPa
(- .01 bars) matric potential measured
12 and 24 hours after the addition of 1%
(w/w) glucose. Data represent mean
values, based on 4 replicates per soil.
Duncan's multiple range test showed the
following differeqpes amon means after
24 hours: Boyer , Capac , Colwooda.
Mean values of soils followed by the same
letter did not differ significantly
(3:0.05).

C0 respiration by three soils at -30 kPa
{-GJ bars) matric potential measured 6,
12, and 24 hours after the addition of 1%
(w/w) glucose. Data represent mean
values, based<n14-replicates per soil.
Duncan's multiple range test showed the
following difgerence among means: 12
hours, Boyera , Capacb, Colwooda; 24
hours, Boyer a, Capac , and Colwoodc.
Means values of soils followed by the
same letter did not differ significantly.

PAGE

76

80

81

83

FUNGISTASIS IN RELATION TO DISEASE-SUPPRESSIVENESS OF SOILS:
A LITERATURE REVIEW

I. INTRODUCTION

The phenomenon of fungistasis in soils has been known
for over three decades as a mechanism by which microbial
propagules do not germinate and grow at an inopportune
locations or times. Recent work, however, suggests that
soil fungistasis imposed over a long term can also affect
pathogen survival and virulence (Bristow and Lockwood,
1975; Filonow et al., 1983). Thus, soil fungistasis may
play a greater role in the ecology of soil-borne plant
pathogens than merely germination inhibition. The
relationship between fungistasis and disease development
in soils is the subject of this review.

As a prerequisite for examining the relationship of
soil fungistasis and disease development, two concepts
must be considered. First, although most soils are
fungistatic, they differ in their ability to impose
fungistasis, as shown by different germination rates when
identical concentrations of nutrients are added to soils
to annul fungistasis (Filonow and Lockwood, 1979).
Second, soils differ in their ability to support disease
development, as shown by differences in disease incidence

or severity when identical quantities of inoculum of plant
1

pathogens are used to infest soils (Alabouvette et al.,
1982). .A narrow perspective is applied to soils differing
in their ability to support disease when they are labelled
either "disease-suppressive" or "disease-conducive". Much
of the current research on "suppressive soils" implies
that disease expression in soil is an "all or nothing"
phenomenon and fails to acknowledge that disease
differences are only relative. If all soils are
considered, however, it should be seen that there is a
continuum of response. In this review, fungistasis and
disease development in soils will often be discussed using
"disease-suppressive" and "disease-conducive" as convenient,
comparative labels for soils which are far apart on the
continuum of disease response.

The approach of this review will be to examine
fungistasis as a component of disease-suppressiveness of
soils. First, the occurrence and characteristics of
disease-suppressive soils will be briefly described. This
discussion will illustrate the diversity of the soils
involved and serve as a basis for presenting
generalizations. More inclusive reviews have been
published (Cook and Baker, 1983; Hornby, 1983; Rovira and
Wildermuth, 1981; Schneider, 1982; Schroth and Hancock,
1982). Next, fungistasis will be compared among soils
with different degrees of disease suppression. Since

fungistasis and disease-suppressiveness are primarily

induced by biotic factors the relation of the microflora
involved in disease-suppression to the imposition of soil
fungistasis will be discussed. The mechanisms by which
the microflora exert their disease-suppressive effect will
be compared to the mechanisms by which microflora impose
fungistasis. Although it is recognized that fungistasis
and disease-suppression are occasionally induced by
abiotic factors, these factors will be discussed only when
they influence the biotic factors in soil. llzis hoped
that this review will reveal generalizations about the
level of fungistasis in soils relatively suppressive to
disease'and the deficiencies in knowledge that preclude

generalizations.

II. DISEASE-SUPPRESSIVENESS IN SOIL:

Recognition that soil-borne diseases do not occur
with the same incidence or severity in different soils
arose originally from field observations made early in the
history of plant pathology, as early investigators
examined thewmany factors that affect disease. Atkinson
(1892) is often credited with first recognizing "disease-
suppressive" and "disease—conducive" soils. He noticed

that cotton wilt, caused by Fusarium oxysporum f. ap.

 

vasinfectum, was absent in certain soil types, but

 

research on the nature of the differences was not done.

Later workers can be credited with systematic examination

of conditions in soils leading to differences in disease.

These workers performed crop monoculture experiments
in pathogen—infested soils to monitor disease development,
rather than rely solely on field observations, as Atkinson
did. Normally, monoculture should exacerbate disease
because it provides conditions that favor inoculum
increase. However, these early workers found that this
did not occur in certain soils. After three seasons of
monoculture, Jones (1923) observed that root rot of peas,

caused by Fusarium solani f. sp. pisi, disappeared in a

 

well-drained gravelly loam, while it remained severe in a
nearby muck soil. Walker and Snyder (1934) also performed
pea monoculture experiments with certain soils where pea
wilt, caused by F; oxysporum f. sp. plea}, was absent, in
spite of continuous pea cropping. In their greenhouse
experiments, after the third monoculture, pea wilt
incidence increased to 61 % in a sandy loam, but only to 8%
in a red clay. Thus, these workers established that
certain soils suppress disease development, in spite of
conditions such as monoculture that favor pathogen
increase.

One consequence of suppressive conditions in soil is
a less effective inoculum. The suppressive soils required
a higher inoculum density to obtain a level of disease
that would be present with lower inoculum densities in

relatively conducive soils. In the case of muskmelon

wilt, caused by F; oxysporum f. sp. melonis, Wensley and

 

McKeen (1963) found a direct relationship between
population of the pathogen in soil and wilt incidence.
However, they also observed more wilt in a Fox sandy loam
than in a Colwood loam, even though the population of the
pathogen was lower in the sandy loam.

Enumeration of pathogen populations in soils, in
conjunction.with disease evaluation.from field surveys,
can identify suppressive soils. However, such
identification should be considered only tentative, since
apparent population differences among soils, as revealed
by differences in colony counts on agar media, could
reflect differences in recovery of inoculum. Therefore,
evaluation of disease after addition of a known quantity
of inoculum to soils and monitoring survival of this
inoculum is a better method for identifying suppressive
soils. Differences in the amounts of inoculum required to
cause disease in two different soils can be dramatic, if
the soils are widely separated from each other on the
continuum of response to plant pathogens. For example,
chlamydospores of F; oxysporum f. sp. batatas gave 96%
disease incidence when 5,000 prOpagules per gram (ppg)
were incorporated into conducive Elkhorn sand, while 5,000
ppg gave~a 67% disease incidence in suppressive Chualar
sandy loam (Smith and Snyder, 1971).

However, there usually is a threshold at which

disease suppression in soil can be overcome by very high
inoculum densities (Burke, 1965; Alabouvette et al.,
1982). To prevent suppressiveness from being masked by
excessive inoculum, soils should be compared using several
Ilevels of inoculimL With this approach, Alabouvette et

al. (1982) found that curves of L oxysporum f. spp. wilt

 

incidence plotted against inoculum density were different
for different soils. Disease incidence differed among
soils over all inoculum densities, although differences
were smaller at higher inoculum densities. Different
concentrations of inoculum were also used to determine

suppressiveness of soils to Rhizoctonia solani (Kinsbursky

 

and Weinhold, 1983). This approach could be applied to
evaluate suppressiveness of soils to other soil-borne
plant pathogens. In most cases, plants growing in
relatively suppressive soils can tolerate higher inoculum
densities than plants in relatively conducive soils.

The decline, low incidence, or absence of disease in
suppressive soils can often be attributed to a decline in
inoculum. After 12 weeks, 58% of added chlamydospores of

Phytophthora cinnamomi were recovered from Kioloa soil

 

conducive to & cinnamomi on many hosts, but only 14% of

 

added chlamydospores were recovered from suppressive
Tallaganda soil (Halsall, 1982a). Inoculum decline also

occurs in some soils suppressive to Fusarium. As an

 

example, two months after the addition of 3,000 ppg of E:

oxysporum f: Sp. lini to two soils planted to flax, the

 

population increased to 4,100 ppg in a soil with 97% wilt
incidence, but declined to 300 ppg in a soil with 3% wilt
incidence (Tu et al., 1978). This trend was Observed in

another suppressive and conducive "pair" of soils with

 

three other form species of F; oxysporum (Komada, 1976).
Some types of propagules die more rapidly than other
types in suppressive soils. After two months, numbers of

viable macroconidia of Lsglani f. sp phaseoli were 25%

 

of that added to a soil conducive to bean root rot,
compared to 0.1% in a soil suppressive to bean root rot.
When chlamydospores were tested instead of macroconidia,
80% survived in the conducive soil, while 62% survived in
the suppressive soil (Furuya, 1982). This great
difference 111 survival. between macroconidia and
chlamydospores illustrates the importance of using
survival propagules such as chlamydospores to compare
population dynamics between soils that differ in disease
suppressiveness, as they would better reflect the survival
capability of the pathogen as it would occur in the field.

There are also many soils in which suppression occurs
without pathogen decline; disease is absent in spite of
the presence of a sufficient number of propagules to cause
symptoms in other soils. Eh) illustrate this point,
populations of F; oxysporum f. sp. 23223 in certain

organic soils in which basal rot of onion did not occur,

were often the same as those in similar soils where basal
rot was present (Abawi and Lorbeer, 1971). Additionally,
a survey of 24 pea fields with a population range of 275-
6,175 ppg of F; £21333 f. sp. pig; revealed no
relationship between population and root rot severity in
previous pea crops (Burke et al., 1970). These studies
may not be sufficient to demonstrate suppression in spite
of overwhelming inoculum, because differences among soils
in propagule recovery for enumeration may distort the true
population ranking of the soils. Addition of a known
quantity of propagules and subsequent enumeration could be
used to determine persistence of inoculum in suppressive
soil, rather than relying solely on enumeration of

indigenous pathogens. When this was done by adding 300

ppg of I; oxysporum f. ap. melonis to Chateaurenard

 

suppressive soil planted to muskmelon, the population of
the pathogen remained at about the same level at the end
of the experiment without causing the level of disease
that would occur in a conducive soil with the same amount
of inoculum (Louvet et al., 1976).

On the other hand, pathogen populations generally
increase in conducive soils, as in the case of F;

oxysporum f. sp. batatas, which increased in the presence

 

of sweet potato to twice the added 500 ppg (Smith and
Snyder, 1971). The persistence of pathogens in some

suppressive soils and their decline in other suppressive

soils suggests either different modes of suppression or
different degrees of suppression.

In the majority of suppressive soils, the disease-
suppressive factor is biological. Evidence for this is
given by the elimination of suppression after biocidal
treatments of soil (Burke, 1954; Menzies, 1959; Furuya,
1982). Sterilization, however, does not eliminate
suppressiveness induced by abiotic factors, such as
montmorillonite clays (Stotzky, 1972). It is important to
use the least disruptive sterilization procedure to
demonstrate that loss of suppression is associated with
loss of the soil microflora and not with a change in the
abiotic components of soil. Gamma irradiation is often
thought to be the least disruptive, yet the changes in
levels of extractable nutrients were greater in gamma-
irradiated soils than in soils heated to 60°C for two
hours (Mantylahti and Ylaranta, 1981). Thus, with
sterilization techniques that alter soil characteristics,
it becomes difficult to rule out abiotic contributions to
suppressiveness.

Suppressiveness can be re-established in sterilized
soils by introducing into them a small quantity of
unsterilized suppressive soils (Menzies, 1959; Louvet et
al., 1976; McCain et al., 1980), or only the microflora
isolated from suppressive soil (Lin and Baker, 1980).

waever, a non-transferrable biologically-mediated‘

10

suppres—sion also has been described (Burke, 1954),
suggesting that in some cases, suppression occurs through
an interaction of the microflora.and abiotic components
unique to the soil.

In fact, certain abiotic soil components are closely
associated with biotic components in suppressive soil.
Montmorillonite clays, for example, are associated with
soils suppressive to Panama wilt of banana, caused by F;

oxysporum f; Sp. cubense (Stover, 1962). Fine—textured

 

soils are more suppressive to Pythium ultimum than coarse-

 

textured soils CHancock,‘1979). In addition to texture,
soil reaction can contribute to suppression. An acidic

soil, which favored the growth of Trichoderma spp. was

 

more suppressive to IL solani than an alkaline soil (Liu
and Baker, 1980). Since abiotic soil components can
affect pathogen behavior directly, these factors, as well
as effects of the soil microflora, should be comparatively
evaluated.

An under-appreciated aspect of suppressiveness is
that it can be manipulated. A well-known example of this
is "take-all decline," where soils with initially severe

take-all disease become suppressive to Gaeummanomyces

 

graminis var. tritici with continuous cereal monoculture.
Take-all-suppressive soils arise from a change in soil
microflora induced by cereal monoculture (Hornby, 1979).

but unlike other cases of pathogen suppression which

 

 

 

11

appear to result from constitutive soil properties, take-
all suppression can only arise in the presence of the
pathogen.(Wildermuth, 1980). Thus, take-all decline is
probably a special case of an inducible suppression which
involves a three-way interaction of host, pathogen, and
microflora.

Suppressiveness to R; solani was induced in soils by
weekly radish monoculture (Chet and Baker, 1980), but not
by monoculture with other crops (Liu and Baker, 1980).
The suppression was correlated with an enhanced population

of Trichoderma harzianum. But while it is significant

 

that a particular crop can influence the development of a
suppressive microflora in, perhaps, any soil, the
longevity of the suppressiveness in not known. Does the
suppressiveness, once established, persist with crop
rotation, or is it lost, like take-all suppressiveness is,
once a non-cereal is planted?

There is also evidence that the microflora of soils
can be manipulated to make soils suppressive to Fusarium
wilt diseases by such procedures as cropping. Repeated
planting in a conducive soil amended with 10% suppressive
soil resulted in a decline of muskmelon wilt, caused by E;

oxysporum f. sp. melonis (Alabouvette et al., 19800).

 

However, the presence of "suppressive" soil is not
necessarily a prerequisite for inducing suppression, as

suggested by a decline of carnation wilt, caused by F;

12

oxysporum f. Sp. dianthi, in unamended conducive soil by

 

repeated planting of carnation (Tramier et al., 1979).

Nor does the induction of suppressiveness to a Fusarium

 

wilt pathogen necessarily require previous cropping with
its host. For example, a continuously-cropped Chualar
sandy loam, which had never been planted to sweet

potatoes, was more suppressive to wilt of sweet potatoes,

 

caused by F: oxysporum f. sp. batatas, than the same soil
that had never been cropped at all (Smith and Snyder,
1971). In addition to the plant pathogens discussed, the
induction of disease-suppression by cropping or other
agricultural practices which alter the soil microflora may
also be applicable to other soil-borne plant pathogens.
While treatments that stimulate suppressiveness
should be sought and incorporated into a cropping program,
other treatments that decrease the suppressiveness of soil
should be identified and avoided in a cropping program.
For example, soil amendments of peat and manure increased
incidence of carnation wilt (Tramier et al., 1979).
Alternatively, treatments may affect the pathogen and the
soil microflora, but have no effect on disease incidence.
Monthly cropping of barley, followed by turning the crop
under, increased populations of“;; ultimum in suppressive.
soil, but the degree of suppressiveness did not change
(Hancock, 1979). It can be concluded, based on the

complexity of the plant host-pathogenic fungus-soil

13

system, that any attempts to manipulate suppressiveness of
soil for biological control must be made on a somewhat
individualized basis.

While knowledge of conditions that enhance
suppressiveness is scant at best, the question of the
range of species a given soil is suppressive to has, for
the most part, remained unasked. Soils from five
continents have been described as suppressivteto a wide
variety of plant pathogens, but few workers have evaluated
suppressiveness of a particular soil to more than one
plant pathogen. This tendency of specialization could
lead to the incorrect assumption that a soil suppressive
to one pathogen also is suppressive to others. This is
known not to be true with the Chateaurenard, melon—wilt
suppressive soil, which apparently suppresses only form

species of'fi oxysporum, but not the other Fusarium app.

 

 

or other genera of plant pathogens (Alabouvette et al.,

1980a). Are other soils suppressive to F; oxysporum

 

specifically suppressive to that fungus, or is there a
wider range of pathogens inhibited? The identification of
the range of pathogens affected by suppressive soils is an
important step ikn' developing approaches for
advantageously manipulating suppressiveness.

From the above discussion, disease-suppressiveness of
soils can be defined as a prOperty of soil whereby

inoculum effectiveness is reduced; this is reflected in

14

less disease relative to other soils. This property is
best identified by adding a known quantity of propagules
of a pathogen to soils and comparing among them disease
incidence or severity of a susceptible host under
environmental conditions favorable to disease development.
Since the primary determinant of suppressiveness is the
soil microflora, it follows that all natural soils are
more or less suppressive. Suppressiveness is eliminated
by destroying the microflora and is restored by re—
establishing the microflora. The range of suppressiveness
of soils is related not only to the microfloral
composition of the soil, but also to the physical and
chemical components of soil and their interaction with the
soil microflora. Evidence of different microflora among
soils comes from the enhancement of suppressiveness by the
transfer of microflora from a relatively suppressive soil
to a relatively conducive soil (Alabouvette, et al.,
1979). The suppressive microflora is dynamic and soil
treatments such as cropping can alter it and lead to
changes in disease expression. The requirement for
successful manipulation of this microflora for biological
control is an understanding of its active components and

how these operate.

III. FUNGISTASIS IN DISEASE—SUPPRESSIVE SOILS:
The consequence of fungistasis in soils is dormancy

of fungal propagules. Additionally, macroconidia of

15

Fusarium, which are not survival propagules, convert to

 

chlamydospores under fungistatic conditions (Nash et al.,
1961). Fungistasis occurs primarily because of a
nutrient-deficient state of soil which is imposed by the
soil microflora (Lockwood, 1977). The amounts of carbon
and nitrogen required for germination of chlamydospores of

Fusarium solani f. ap. phaseoli were reduced by the

 

 

simultaneous addition of antibiotics to inhibit the
microflora (Cook and Schroth, 1965). Although this
decrease in nutrients required for germination suggests
that the inhibition of the microflora reduced competition
for nutrients, there is also the less likely possibility
that microflora producing antifungal substances were
inhibited.

Fungistasis is not imposed to the same degree in all
parts of a soil. In the rhizosphere, it is reduced
because of a greater supply of nutrients, as shown by

germination of Fusarium macroconidia to produce tropic

 

hyphal growth to the root, instead of forming
chlamydospores (Jackson, 1957).

While aflLL soils exhibit fungistasis, differences in
fungistasis among soils can be observed by the addition of
nutrients in concentrations that partially annul the
effect (Filonow and Lockwood, 1979). Quantitative
determination of nutrient concentrations that annul

fungistasis is termed "nutrient titration". In practical

16

terms, propagules of plant pathogens in a highly
fungistatic soil need to be placed closer to the root in
order to germinate and grow to it; in effect, the inoculum
potential is lowered. Quantitative differences in
nutrient annulment of fungistasis are reflected not only
by decreased germination of propagules, but also, by
inhibition of hyphal growth, including germ tubes produced
by propagules (Hsu and Lockwood, 1971).

Soils could be suppressive either because of an
elevated level of fungistasis that prevents germination
or, paradoxically, germination is promoted at a time or
location that is inopportune for the pathogen.
Germination promotion as a.mechanism of suppression has
not been widely reported. Burke (1965) described a
Sagemoor sandy loam suppressive to L _s_c_1_l__a_r_1_i f. sp.

phaseoli which apparently was less fungistatic to

 

macroconidia than a conducive Ritzville sandy loam.
Macroconidia are less sensitive to fugnistasis than
chlamydOSpores and will germinate more readily on soils in
the absence of nutrients. growth from macroconidia was
greater in the suppressive soil than in the conducive
soil. However, this increased growth in the suppressive
soil was ephemeral and was associated with relatively late
formation of small chlamydospores. In contrast, germ tube
growth was suppressed in the conducive soil. While there

was no difference in the rapidity of lysis of mycelia and

17

germ tubes between the two soils, chlamydospores were
larger and more numerous in the conducive soil. Thus, in
the disease-suppressive soil, ‘the pathogen germinated in
the absence of hosts or nutrients that would sustain it.
The end result was a depletion of nutrient reserves,
resulting in a failure to form survival structures,
leading to decreased survival. The contribution of the
microflora, either as stimulants of germination or
aggressive antagonists of germinated propagules, has not
been determined.

A stimulation of germination was associated with the

suppression of Fusarium in a coniferous forest soil.

 

Biocidal treatments led to a decrease in macroconidial

germination of Fusarium in the soil, suggesting the

 

contribution of the microflora to this "stimulatory"
suppression (Schisler and Linderman, 1984). However,
abiotic soil components may play an important role in the

stimulation of germination of Fusarium in coniferous

 

forest soils. Evidence for this was stimulation of

chlamydospore germination of F; oxysporum f. sp. lilii in

 

nonsterile soil by the addition of five organic acids that
were leachable from pine needles. Germination was
followed by germ tube lysis with no new chlamydospore
formation (Hammerschlag and Linderman, 1975). The role of
soil microflora therefore is unclear. If they are not

involved in germination stimulation, they could be

18

involved in preventing the reformation of chlamydospores.
More research is needed to understand this mode of
suppression in coniferous forest soils. The prevalence of
germination stimulation as a mode of pathogen suppression
in agricultural soils is not known, but it appears to be
uncommon.

In contrast to germination stimulation, enhanced
fungistasis apparently occurs in some disease-suppressive
soils. This is shown by 42% germination of conidia of £1

oxysporum f. Sp. vasinfectum on water agar discs on "wilt-

 

free" Kovilpatti soil» .as compared with 84% and 87%
germination on "wilt-sick" Palladam and Udamalpet soils,
respectively (Arjunarao, 1971). Germ tubes produced in
the suppressive soil were short and produced terminal
chlamydospores, while germ tubes in the conducive soils
were longer and produced intercalary chlamydospores.
Ungerminated chlamydospores on the suppressive soil were
not killed, as shown by their subsequent germination when
agar discs bearing chlamydospores were transferred from
soil to a glucose solution and root extract.
Sterilization <xf the suppressive soil. nullified
germination inhibition, suggesting that the soil
microflora are involved.

Enhanced fungistasis also was observed in a soil
suppressive to bean root rot (Furuya, 1982). There was

less germination of F; solani f. Sp. phaseoli macroconidia

19

in suppressive Kitami soil, as compared with conducive
Tokachi soil. Germ tubes in the suppressive soil were
abnormal and lysed more quickly, and hyphal growth and
formation of chlamydospores was less than in the conducive
soil. Germination of chlamydospores on the laimosphere of
detached bean hypocotyls was inhibited in the Kitami soil,
suggesting that plant exudates could not overcome the
inhibition. Suppression was biological in origin, as
shown by nullification of inhibition by biocidal
treatments, although a residual abiotic inhibition was
suggested by a slower germination rate and abnormal germ
tube morphology in these treated soils.

Fungistasis also appeared to be elevated in the
Chateaurenard wilt-suppressive soil, as evidenced by
greater inhibition of germination of chlamydospores of

several form species of F; oxysporum and F; solani in this

 

suppressive soil as compared with wilt—conducive soil
(Alabouvette et al., 1979; 1980b) . The fungistasis could
be annulled with biocidal treatments and nutrient
amendments, suggesting involvement of the microflora. The
requirement of'more glucose to promote germination of};1

oxysporum in wilt-suppressive than in conducive soils has

 

been shown by other workers (Hwang et al., 1982; 1983).
This differential germination also applies to the
rhizosphere of the soils. Germination of the

chlamydospores of F; oxysporum f. sp. tracheiphilum by

 

20

cotton root tips was 18% in conducive soil, but only 8% in
suppressive soil (Smith, 1977). In suppressive soils,
elevated fungistasis results in more intense competition
for nutrients, which may restrict development of the
pathogen.

There have been fewer studies on the relationship of
fungistasis levels and disease suppression to pathogens

other than Fusarium. In a suppressive soil, Pythium

 

splendens sporangia coated with cucumber root extract

 

germinated 15% compared with 90% germination in a soil

 

conducive to P; splendens damping-off (Kao and Ko, 1982;
1983). However, this differential effect did not apply to
R; solani, whose growth and the amount of mustard cabbage

damping-off it caused, were identical in the P; splendens

 

suppressive and conducive soils (Kao, 1982). Five out of
30 samples of soil were found to inhibit hyphal growth to
less than 50% of that occurring in a conducive control
soil, but disease suppressiveness of these soils to R;
gglgni disease was not assessed (Ko and Ho, 1983).

Chlamydospore germination of’P; cinnamomi was 56% in an

 

aqueous extract of P; cinnamomi—conducive Kioloa soil, but

 

only 17% in an extract of suppressive Tallaganda soil
(Halsall, 1982a). It appears, then, that soils
suppressive to pathogens other than Fusarium are also more
fungistatic to these pathogens than conducive soils,

although the fungistatic effects of soils suppressive to

21

other pathogens are not yet known.

The long-term effects of elevated fungistasis in
suppressive soils are not known. Recent work suggests
that a greater loss of endogenous nutrient reserves in
propagules maintained under fungistatic conditions was
associated with a decrease in propagule germinability and
virulence (Bristow and Lockwood, 1975; Filonow et al.,
1983). This phenomenon could partially account for
reduced disease in suppressive soils with a persistent,
high population of pathogens. It could also account for
disease Suppression in soils in which the inoculum density
declined, although this may reflect the operation of other
types of antagonism, such as predation or hyperparasitism,
in addition to competition.

Fungistasis elevation may occur with soil amendments
used to induce suppression. One example is the reduction

of tomato wilt, caused by'fh oxysporum f. Sp. lycopersici,

 

with chicken manure. The pathogen population was not
reduced, but its germination and growth were inhibited in
the rhizosphere and the rhizoplane (Homma et al., 1979).
The above examples suggest that fungistasis in
disease-supressive soils is either very strong or very
weak. In soils with high fungistasis, the response of
pathogen spores to nutrients is reduced. If these
nutrients originate from a root, the consequence is a less

effective inoculum which results in reduced disease. 0n

22

the other hand, a very low level of fungistasis allows
precocious germination, with a consequent lower inoculum
density because the pathogen becomes debilitated from
unsustainable growth. If germination stimulants are
involved, the identification of these chemicals and the
microflora that produces them, suggest the potential for
exploiting these for disease control. Soil fungistasis
and disease suppression are both dependent on the soil
microflora, yet the connection between disease suppression
and fungistasis has not been explicitly evaluated.

With all the attention given to soil fungistasis, it
must be emphasized that this phenomenon is only one aspect
of a general microbiostasis operating in soil. So, while
there is a relationship between the level of fungistasis
imposed by a soil and disease expression in it, an
intriguing question arises: can there not also be a
relationship of inhibition of germination and growth in
soil to suppression of diseases caused by prokaryotic as
well as fungal soil-borne pathogens? An answer to this
question is not possible at this time simply because there
has been scant research in this area. While there are
many reports of soils suppressive to fungal pathogens,
there is only one report of a soil suppressive to an

actinomycete, namely, Streptomyces scabies on potato

 

(Menzies, 1959). and no reports of soils suppressive to

soil-borne bacterial pathogens. As with fungi, inhibition

23

of germination and growth, primarily from a microbial-
induced scarcity of nutrients in soil, has been shown with
bacteria (Davis, 1976) and actinomycetes (Mayfield et al.,
1972). It is conceivable that an enhanced microbiostasis
in some soils may explain disease-suppression or inoculum
decline of prokaryotic plant pathogens, but since these
pathogens are of relatively minor importance as compared
to fungal pathogens, this area of research has not so far
garnered much attention. This is unfortunate. While such
research may be of minor importance to the problems of
plant pathology, it could make a significant contribution

to the understanding of microbial interactions in soil.

IV. ROLE OF THE MICROFLORA IN SUPPRESSION:

It is clear that in the majority of cases of
suppressiveness to a disease, as well as the elevated
fungistasis associated with it, the soil microflora plays
a primary role. Differences between suppressive and
conducive soils usually consist of identifiable
quantitative or qualitative soil microflora differences.
There are exceptions, however. Burke (1965) found no
specific microfloral differences between.bean root rot-
suppressive and -conducive soils. Rombouts (1953) found
no difference between antagonism of banana rhizosphere
microflora from conducive or suppressive soil. However,

these negative results do not carry much.weight because

24

the isolation techniques used by these workers may not
accurately reflect active portions of the microflora that
affect the pathogen.

Many workers found higher populations of a specific
portion of the microflora - that which is antagonistic to
the pathogen - in suppressive soils. Chet and Baker
(1981) found that a soil in Colombia, suppressive to R;

solani, contained 8 X 105 propagules (ppg) of Trichoderma

 

hamatum. The same concentration of T; hamatum, when added
to a conducive soil, rendered it suppressive. In cotton
wilt-suppressive Kovilpatti soil, there was a greater
number of antagonistic bacteria, fungi, and especially
actinomycetes than in conducive soils (Arjunarao, 1971).
Halsall (1982b) found more actinomycetes and antagonistic

Streptomyces in a soil suppressive to P; cinnamomi than in

 

 

a conducive soil. A soil suppressive to take—all disease
supported higher populations of myc0phagous amoebae in the
rhizospheres and rhizoplanes of wheat plants growing in it
than in conducive soil (Chakraborty, 1983). Indigenous

bacteria, especially a species of Pseudomonas, were

 

thought to be responsible for suppressiveness of a Metz

sandy loam to _F_‘_._ oxysporum (Scher and Baker, 1980). This

 

Species of Pseudomonas, when used to infest a conducive

 

Fort Collins clay loam, made it suppressive to flax wilt,
caused by F; oxysporum f. Sp. lini. ‘Yellows of celery,
caused by f; oxysporum f-Eflh apii, was inhibited in the

25

presence of Corynebacterium sp. in soils of pH greater

 

than 6.2 (Opgenorth and Endo, 1983). A sterilized soil,

made suppressive to GLgraminis by recontamination with

 

non-sterilized soil, contained greater numbers of

Trichoderma viride than its non-sterilized, take-all-

 

conducive counterpart (Ludwig and Henry, 1943). In soil

suppressive to P; cinnamomi, its host, Eucalyptus

 

 

marginata, had fewer mycorrhizae associated with it than

 

in a conducive soil (Malajczuk, 1979). Rbot microflora
differences between suppressive and conducive soils were
thought to be related to differences in the quantity of
mycorrhizal roots between the two soils (Malajczuk and
McComb, 1979).

In other cases, suppressiveness associated with
greater numbers of microorganisms - a quantitative
difference, rather than the presence of certain species, a
qualitative difference - suggests enhanced nutrient
competition from a larger biomass as the mode of action.
Higher populations of bacteria and actinomycetes were
found in Tamborine Mountain soil suppressive to P;

cinnamomi root rot of avocado than in a conducive soil

 

(Broadbent and Baker, 1974). Fungi, actinomycetes, and
bacteria, when added to a sterilized soil, were all

effective in reducing infection of wheat by Bipolaris

 

sorokiniana and F; graminearum (Henry, 1931 ). Increased

 

suppressiveness to Verticillium dahliae, induced by chitin

 

26

amendments, was associated with higher populations of
bacteria and actinomycetes in the rhizosphere of
strawberry (Jordan et al., 1972).

The opposite relationship, that disease suppression
decreases with a decline inlnicroflora, was shown with a
suppressive soil stored for six months. A decline of the
microbial population from 25 to 8 X 106 colony forming
units per gram of soil was correlated with a 14% to 61%

increase in germination rate of §;_§plendens sporangia

 

coated with cucumber root extract and £1 concomitant
decrease in disease suppression (Kao, 1982). A lower
level of germination inhibition in subsoil as compared
with topsoil was correlated with a lower total microbial

population, but size of the population alone did not

 

determine suppressiveness to P;_§plendens, since a re—
infested conducive soil withtan elevated population.was
still not rendered suppressive (Kao and Ko, 1983). Thus,
critical analysis of the role of biomass size in disease
suppression should be undertaken with only one soil, in
which only the biomass is changed, while other soil
characteristics are kept constant. However, this may be
an unachievable ideal and so, disease and biomass
comparisons between different soil types should not be
condemned, as they may still illustrate valid
relationships, especially with extreme differences in

soils.

27

Interactions between a pathogen and a specific
component of the microflora include not only
hyperparasitism, predation, and antibiosis, but also,
competition between closely-related species for the same
ecological niche. This has been observed among Fusarium
species. Fifty-four percent of isolations from roots of

celery in areas suppressive to Fusarium yellows were

 

infected with non-pathogenic strains of F; oxysporum while
roots from areas conducive to disease had 6% incidence of
saprophytes, suggesting exclusion of pathogenic Fusarium

from infection sites by saprophytic Fusarium in

 

suppressive soils (Schneider, 1983). Additional evidence
for this is given in pmeliminary results, where the
incorporation of some of these saprophytes into soil gave
almost complete control of'yellows of celery. Saprophytic
F. gxyspgrum were also considered important in

Chateaurenard soils suppressive to Fusarium wilts

 

(Alabouvette et al., 1979). Melon root microflora in
suppressive soil consisted primarily of Fusarium Species,

especially 2; oxysporum (Alabouvette, 1983). Additional

 

evidence for the role of non-pathogenic analogs of

pathogens was the re-establishment of Fusarium wilt

 

suppression in steamed soil with addition of saprophytic

F; oxysporum and F; splani, but not with other fungi

 

(Rouxel et al., 1979). One attribute of a successful

competitor is a superior survival rate. In soil

28

suppressive to g; solani root rot of bean, macroconidia of

F. solani f. Sp. phaseoli declined to CL1% of the original

 

concentration in suppressive Kitami soil, but macroconidia
of non-pathogenic:§; solani declined to only 25% of the
original in the same time period (Furuya, 1982). This
suggests that in a suppressive soil, there will possibly
be more saprophytic than pathogenic Fusaria to compete for
the same infection sites. Competition between L roseum
and other fungi for nutrients in a Specific niche was
thought to be the mechanism of lentil disease suppression,
as the introduction of actinomycetes and bacteria had no
effect on disease (Lin and Cock, 1979). Apparently,
saprophytic Fusarium spp. are better competitors for low
concentrations of nutrients than pathogenic Fusarium spp..
For example, germination of chlamydospores of saprophytic

isolates of E; oxysporum in unamended suppressive soil

 

occurred more readily than germination of pathogenic F;
oxysporum form species (Alabouvette et al., 1980b). In

another wilt—suppressive soil, Fusarium saprophytes

 

germinated better than pathogens in the presence of
glucose and asparagine amendments (Smith and Snyder,
1972)-

While there are many examples of a Specific portion
of the microflora, such as hyperparasites of the pathogen,
responsible for disease suppression, competition for

nutrients between the pathogen and either the general

29

microflora or a Specific portion of it is probably a
common mechanism of suppressiveness. Knowledge of the
specific or general microflora that imposes
suppressiveness, of the location of the pathogen-
microflora interaction and, in the case of competition,
the nature of substrates that generate the competition,

are prerequisites for manipulation of suppression.

V. COMPETITION FOR NUTRIENTS IN SUPPRESSIVE SOILS:
The importance of nutrient competition on disease
incidence was considered as long ago as 1931 by Henry who

ascribed reduced disease incidence of L sorokiniana on

 

wheat in recontaminated sterilized soil to "exhaustion of
food supplies". In natural soils, suppression of Fusarium
wilt diseases has been attributed to unsuccessful

competition of pathogenic E; oxySporum with soil

 

microflora for carbon sources (Hwang et al., 1982; Smith
and Snyder, 1972). Suppression 0f.E; roseum on lentils in
cellulose—amended soil is probably a result of competition
for limiting nitrogen (Lin and Cook, 1979). Suppression

of E; solani f. Sp. phaseoli root rot of bean in

 

cellulose-amended soil is also thought to occur through
competition for limiting nitrogen (Baker and Nash, 1965).,

Much attention has been given to iron as a limiting
substrate. Successful competition for iron is thought to
hinge on the ability to produce a siderophore»that has a

greater affinity for Fe3+ than the siderophore of

30

another competitor. In this respect, pathogenic Fusaria
are Ixmn' competitors, while certain. fluorescent
Pseudomonads are better competitors. Thus, with the

addition of a fluorescent Pseudomonas sp., or its

 

siderophore, pseudobactin, a soil was made suppressive to

L oxysporum f. Sp. lini and Lgraminis (Kloepper et al.,

 

 

1980). The pseudobactin bound available iron making it
unavailable for pathogen utilization, although the
pseudobactin-bound iron can be readily utilized by the

Pseudomonas Sp.

 

Metal chelators have been used instead of
siderophores to manipulate suppressiveness. For example,
the addition of Fe-EDDHA to soil conducive to radish

wilt, caused by F; oxysporum f. sp. conglutinans, rendered

 

 

it suppressive (Dupler et al., 1982). On the other hand,
EDTA made a flax wilt-suppressive soil more conducive,
presumably because it had less affinity for iron than the
siderophore of L oxysporum f. sp. 1.121 (Scher and Baker,

1983).

 

The relationship of disease suppression manifested by
iron competition and soil fungistasis is not clear.

Germination of microconidia of L oxysporum f. Sp. lini

 

was not inhibited by iron chelators, but germ tube
elongation was inhibited. This growth inhibition was
reversible with FeCl3 amendments (Scher and Baker, 1982).
NaFe-EDDHA had no effect on germination of Cochliobolus

 

31

victories and Botrytis cinerea in soil (Lockwood and

 

Schippers, 1984). These results suggest that iron
competition is not involved in propagule germination
inhibition, but the role of iron in other phases of
pathogen development should not be excluded. The
contribution of competition for other elements to disease
suppression has not been investigated.

The most important - and unanswered - question is
whether the same nutrients are involved in competition
leading to disease suppression as are involved in the
competition resulting in fungistasis. If the same
nutrients are involved, fungistasis assay, such as by
nutrient titration, could be a short-cut for testing soils
for relative disease suppressiveness (Ko and Ho, 1983).
If different nutrients are involved, this would provide an
explanation for a lack of correlation between amount of

disease and levels of fungistasis.

32

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Abawi, GuS., and J.W. Lorbeer. 1971. Populations of
Fusarium oxysporum f. Sp. ce as in organic soils in New
York. Phytopathology 61:1 — 048

 

Alabouvette, C. 1983. La réceptivité des sols aux
fusarioses vascularies. Role de la competition
nutritive entre microorganisms. PhJL dissertation,
Universite de Nancy. 168 pp.

Alabouvette, C., Y. Couteaudier, and J. Louvet. 1982.
Comparaison de la réceptivité de différents sols et
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Alabouvette, C., F. Rouxel, and J. Louvet. 1979.
Characteristics of Fusarium wilt-suppressive soils and
prospects for their utilization in biological control.
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Alabouvette, C., F. Rouxel, and J. Louvet. 1980a.
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Alabouvette, C., F. Rouxel, and J. Louvet. 1980b.
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vascularies. Ann. Phytopathol. 12:21-30.

 

Alabouvette, C., R. Tramier, and D. Grouet. 1980c.
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Arjunarao, V. 1971. Biological control of cotten wilt. I.
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Baker, R., and S. M. Nash. 1965. Ecology of plant
pathogens in soil. VI. Inoculum density of Fusarium
solani f. sp. phaseoli in bean rhizosphere as affected
5y cellulose and supplemental nitrogen. Phytopathology
55:1318—1382.

 

Bristow, P.R., and J.L. Lockwood. 1975. Soil fungistasis:
role of Spore exudates in the inhibition of nutrient-
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Broadbent, P., and K.F. Baker, 1974. Behavior of
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conducive to root rotl Aust. J. Agric. Res. 25:121-
137.

 

Burke, ILW. 1954. Pathogenicity of Fusarium solani f.
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hytopathology 44:483.

 

Burke, ILW. 1965. Fusarium root rot of beans and behaviour
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55:1122-1126.

Burke, D.W. D.J. Hagedorn, and J.E. Mitchell. 1970. Soil
conditions and distribution of pathogens in relation to
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60:403-406.

Chakraborty, S. 1983. Population dynamics of amoebae in
soils suppressive and non—suppressive to wheat take-
all. Soil Biol. Biochem. 15:661-664.

Chet, I., and R. Baker. 1980. Induction of suppressiveness
of Rhizoctonia solani in soil. PhytOpathology 70:994-
998-

Chet, I., and R. Baker. 1981. Isolation and biocontrol
potential of Trichoderma.hamatum from soil naturally'
suppressive to Rhizoctdnia Sdlani. Phytopathology
71:286-290.

Cook, R.J., and K.F. Baker. 1983. The nature and practice
of biological control of plant pathogens. American
Phytopathological Society, St. Paul, MN. 539 PP.

Cook, RJL, and M.N. Schroth. 1965. Carbon and nitrogen
compounds and germination of chlamydospores of Fusarium
solani f. phaseoli. Phytopathology 55:254—256.

34

Davis, IRJL 1976. Soil.'bacteriostasis: relation to
bacterial nutrition and active soil inhibition. Soil
Biol. Biochem. 8:429-433.

Dupler, M., FKM. Scher, and R. Baker. 1982. Effect of
synthetic iron chelates on population densities of
Fusarium ox s orum and Pseudomonas putida. (Abstr.)
Phytopathology 72:1011.

Filonow, AHB., 0.0. Akueshi, and J.L. Lockwood. 1983.
Decreased virulence of Cochliobolus victoriae conidia

after incubation (n1 soils or (”1 leached sand.
Phytopathology 73:1632-1636.

 

 

Filonow, A.B., and J.L. Lockwood. 1979. Conidial exudation
by Cochliobolus victoriae on soils in relation to soil
mycostasis. ‘In Soil-borne plant pathogens. Edited Ly
B. Schippers and.W. Gams. Academic Press, Londons pp.
107-119.

Furuya, H. 1982. Studies on Fusarium root rot of bean.
(In Japanese.) Bull. Akita Pref} Coll. Agr. 8:1—49.

 

Halsall, D.M. 1982a. A forest soil suppressive to
Phytophthora cinnamomi and conducive to Phytophthora
cryptogea. IL Survival, germination, an nfectivity
0g mycelium and chlamydospores. Aust. J. Bot. 30:11-
2 .

Halsall, D.M. 1982b. A forest soil suppressive to
Phytophthora cinnamomi and conducive to Phytophthora

crytogea. II. Suppression of sporulation. Aust. J.

 

Hammerschlag, F., and R.G. Linderman. 1975. Effects of
five acids that occur in pine needles on Fusarium
chlamydospore germination in non-sterile soil.
Phytopathology 65:1120-1124.

 

Hancock, J.G. 1979. Occurrence of soils suppressive to
Pgthium ultimum. in Soil—borne plant pathogens.
1 e By B. Schippers and W. Gams. Academic Press,
London. pp. 183-189.

Henry, AJL 1931. The natural microflora of the soil in
relation to the foot-rot problem of wheat. Can. J.
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Homma, Y., C. Kubo, M. Ishii, and K.I. Ohata. 1979.
Mechanism of suppression of tomato Fusarium—wilt by
amendment of chicken manure to soil. (AbstrJ Bull.
Shikoku Agric. Expt. Sta. 34:103-121.

 

35

Hornby, D. 1979. Take-all decline: A theorist's paradise.
in Soil-borne plant pathogens. Edited By B. Schippers
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Hornby, D. 1983. Suppressive soils. Ann. Rev.
Phytopathol. 21:65-85.

Hsu, SJL, and J.L. Lockwood. 1971. Responses of fungal
hyphae to soil fungistasis. Phytopathology 61 :1355-
13620

Hwang, S.F., R.J. Cook, and W.A. Haglund. 1982. Mechanisms
of suppression of chlamydospore germination for

Fusarium ox S orum f. Sp. pisi in soils. (AbstrJ
Phytopathology 72:948.

 

Hwang, S.F., R.J. Cook, and W.A. Haglund. 1983. More
intense microbial competition for emphemeral carbon

sources may operate in Fusarium-suppressive soils.
(Abstr.) Phytopathology 733812.

 

Jackson, R.M. 1957. Fungistasis as a factor in the
rhizosphere phenomenon. Nature 180:96-97.

Jones, PnR. 1923. Stem and root rot of peas in the United
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Jordan, V.W.L., B. Sneh, and B.P. Eddy. 1972. Influence of
organic amendments on Verticillium dahliae and on the
microbial composition of the strawberry rhizosphere.
Ann. Appl. Biol. 70:139-148.

 

 

 

Kao, CJW. 1982. Nature of suppression of £ythium splendens
in a soil from South Kohala. M. Sc. t esis, University
of Hawaii, Hilo. 56 pp.

Kao, C.W., and W.H. Ko. 1982. A Hawaiian soil suppressive
to Pythium splendens. (Abstr.) Phytopathology 72:985.

 

Kao,CLW., and WQH. Ko. 1983. Nature of suppression of
P thium s lendens in a pasture soil in South Kohala,
Hawaii. hytopathology 73:1284-1289.

Kinsbursky, ILS., and A.R. Weinhold. 1983. Influence of
soil on opulation-disease relationships of Rhizoctonia
solani. Abstr.) Phytopathology 73:812.

 

36

Kloepper, J3W., J. Leong, M. Teintze, and M.N. Schroth.
1980. Pseudomonas siderophores: A mechanism explaining
disease-suppressive soils. Current Microbiol. 4:317-
320.

 

Ko, W.H., and W.C. Ho. 1983. Screening soils for
suppressiveness to Rhizoctonia solani and Pythium
gsplendens. Ann. Phytopath. Soc. Japan 49:1-9.

 

 

Komada, H. 1976. Studies on the evaluation of activity of
Fusarium oxysporum, Fusarium wilt athogen of vegetable
crOpS, in the soil. (In Japanese. Bull. Tokai-Kinki
Natl. Agr. Exp. Sta. 29:132-269.

 

Lin, YkS., and R.J. Cook. 1979. Suppression of Fusarium
rp_S_e__1_1_m 'Avenaceum' by soil microorganisms.
Phytopathology 69:384—388.

 

Liu, S., and R. Baker. 1980. Mechanism of biological
control in soil suppressive to Rhizoctonia solani.
Phytopathology 70:404-412.

 

Lockwood, J.L. 1977. Fungistasis in soils. Biol. Rev.

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relation to its infestation with Ophiobolus graminis
Sacc. Can. J. ReS., C. 21:343-350.

 

Malajczuk, N. 1979. The microflora of unsuberized roots of
Eucalyptus calophylla R. Br. and Eucalyptus marginata
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conducive to Phytophthora cinnamomi Rands. II.
Mycorrhizal rootS and associated microflorad.Aust.éL
Bot. 27:255-272.

 

 

 

 

37

Malajczuk, N., and A.J. McComb. 1979. The microflora of
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soil suppressive and conducive to Phytophthora
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Mantylahti, V., and T. Ylaranta. 1981 . The effect of heat
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Mayfield, (LI., S.T. Williams, S.M. Ruddick, and H.L.
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McCain, ILH., IHE. Pyeatt, T.G. Byne, and D.S. Farnham.
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Menzies, J.D. 1959. Occurrence and transfer of a
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Phytopathology 49:648-652.

Nash, S.M., T.Ghristou, and W.C. Snyder. 1961. Existence of
Fusarium solani f. haseoli as chlamydospores in soil.
Phytopathology 51:3 - .

 

Opgenorth, D.C., and R.M. Endo. 1983. Evidence that
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7080

Rombouts, J.E. 1953. The microorganisms in the rhizosphere
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Academic Press, Lon on. pp. 385-415.

38

Scher, F.M., and R. Baker. 1980. Mechanism of biological
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Scher, F. M., and R. Baker. 1982. Effect of Pseudomonas
utida and a synthetic iron chelator on induction of
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Phytopathology 72:1567-1573.

 

 

Scher, FKM., and R. Baker. 1983. Fluorescent microscope
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Fusarium from coniferous forest soils. Can. J.
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Schneider, ILW., ed. 1982. Suppressive soils and plant
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Schroth, M.N., and J.G. Hancock. 1982. Disease-suppressive
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1381.

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62:273-277. .

 

 

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39

Tramier, R.,¢LC. Pionnat, A. Bettachini, and C. Antonini.
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Walker, J.C., and W.C. Synder. 1934. Pea wilt much less
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INTRODUCTION

In recent years, attempts have been made to identify
the active components of "disease-suppressive" soils, with
the ultimate objective of understanding how to manipulate
"disease-conducive" soils so that disease is reduced in
them (Cook and Baker, 1983; Schneider, 1982; Schroth and
Hancock, 1982). Many workers studying suppressive soils
have found that they impose a greater degree of
fungistasis than disease-conducive soils, as evidenced by
the higher quantity of nutrients required to annul
germination inhibition in the disease-suppressive soils
(Alabouvette, 1983; Arjunarao, 1971; Furuya, 1982; Hwang,
1982, 1983; Kao, 1982; Smith, 1977). With this possible
relationship of elevated fungistasis and increased
disease-suppressiveness in mind, Ko and Ho (1983) have
proposed using a quantitative assay of soil fungistasis as
a Simple, rapid method for identifying suppressive soils.

The objective of this research was to determine the
relationship between the degree of fungistasis imposed by
three Michigan soils and the incidence or severity of
soil-borne diseases occurring in them. Relative disease
suppressiveness of these soils had not been previously
determined, although they differed in their degree of
fungistasis against Bipolaris victoriae and Mortierella

4O

 

 

41

Sp., as determined by adding different quantities of a
glucose and peptone mixture to the soils (Lee and
Lockwood, unpublished data). The hypothesis was that
highly fungistatic soils should have lower disease
incidence or severity than less fungistatic soils.
Relative disease suppressiveness was determined by
adding propagules of four pathogens in increments to these
soils and comparing disease incidence or severity of a
susceptible host planted in them. Fungistasis imposed by
the soils on these pathogens was quantified by measuring
the degree of germination promotion by adding different
quantities of a glucose and peptone mixture to the soils.
Because fungistasis is microbial in origin, the
qualitative microbial composition and the total microbial
biomass were compared among the three soils. .Also, the
ability of the microflora of these soils to utilize added

glucose was compared.

MATERIALS AND METHODS

Characterization of Soils

 

Three different agricultural soils from Michigan were
used (Table 1). Soils were taken from the field,
partially air-dried, passed through a 2 mm-mesh Sieve and
stored in large plastic bags at 4°C for the duration of
all work. Elemental analyses, cation exchange capacity,
and organic matter content were performed by the Soil
Testing Laboratory, Michigan State University. Textural
analyses were done by the hydrometer method (Day, 1965).
The pH was measured by immersing;a.glass electrode into
the supernatant of a 2:1 (v/w) mixture of 0.01 M
Ca012:soil (Peech, 1965). Moisture characteristic curves
for the soils were determined using a pressure-plate
apparatus (Richards, 1965).

Soils were planted to radish (Raphanus sativus L. cv.

 

'Cherny Belle'), wheat (Triticum aestivum 1L cv.

 

'Waldron'), tomato (Lycopersicum esculentum Mill. cv.

 

'Bonny Best'), and pea (Pisum sativum L. cv. 'Wandofl) to

 

detect the presence of indigenous soil-borne pathogens.

No indigenous pathogens were detected in any of the soils.

Microbial Characteristics of Soils

 

Microbial populations in soils were estimated by
dilution plate counts, using serial dilutions made in 0:1%
agar water blanks. The following media were used:

trypticase soy broth agar (Martin, 1975). containing 50
42

hvfioagmo omcmsoxo acuvmou.o.m.o+

Haom cupcamt

 

 

£13

 

 

n w_ .nu 44 eon comm ~.n cm. a m.m mm.o 04.4 cm mm be m.m. smog eoosaoo

S o_ 4, we Sow Papa eon sew S. ¢.m ~_.o S~.m 4m mm as o... smog ooooo

F as A en mar oorm he. .m. o m.o 40.0 o~.~ m. S SS S.n aooa
hvcsm Sohom

a
so a: as on m: mu m m oo_\om: ma somoapfiz copaoa soflo aflnm econ .oaaooaoz Hfiom
+.o.m.o Hoooa oncomoo
2mm u

 

mafiom mo mofiumfiaovoapazo .v manna

44

ppm PCNB to inhibit actinomycetes (Farley and Lockwood,
1968), for aerobic bacteria; colloidal chitin-mineral
salts agar (Hsu and Lockwood, 1975), for actinomycetes;
and peptone-dextrose-rose bengal agar (Martin, 1950), for
fungi. A bent glass rod was used to spread Ou2 ml of soil
suspensions over the surface of the agar. Plates were
incubated at 25° 11°C for 5—7 days for bacteria and fungi
and 10 days for actinomycetes. Anaerobic bacteria were
estimated using the most-probable number method (Koch,
1981). One-ml aliquots from a two-fold dilution series
were dispensed into 10, 15 cm X1 cm screw cap test tubes
containing 11 ml fluid thioglycollate medium (Difco,
Detroit, MI 48226). Determinations were repeated at least
once.

Populations of form species of f; oxysporum added to
soil were determined by Spreading 0.2 m1 aliquots of soil
, dilutions onto Komada's medium (Komada, 1975). Plates
were incubated under fluorescent lights (63uEm‘2S'1) at
25°C :1°C for one week.’ Plates inoculated with pure
cultures of E; oxysporum inocula were included for
comparison of colony types to aid identification and
dilutions from noninfested soils were included as
controls.

Total soil biomass was determined using the
chloroform fumigation method (Jenkinsqn and Powlson,

1976), with certain modifications. Soils were adjusted to

45

a matric potential. of -30 kPa (-0.3 bars) before
fumigation by Spraying water onto soils with an atomizer.
Soils were continuously agitated to ensure even wetting.
Soils were incubated in closed 3.75 l (1 gallon) jars with
(L01 M NaOH to trap carbon dioxide. After incubation, 5
ml saturated BaC12 and(12 ml 1% phenolphthalein in 95%
ethanol were added to the trap before titration with OJM
M HCl. The weight of 002-C was calculated from the
formula, mg 002-C = M HCl X (standard - sample) X 12,
where "standard" represents the volume of acid in ml
required to titrate base from incubation jars without
soil, and "sample" represents the volume of acid required
to titrate base from incubation jars containing fumigated
or unfumigated soil. These values were used to calculate
total carbon biomass according to the formula of Jenkinson
and Powlson (1976), except that 0.411 was used for K, the
proportion of biomass carbon mineralized (Anderson and

Domsch, 1978). Soil biomass was determined twice.

Maintenance of Fungi

 

Cochliobolus victoriae Meehan and Murphy isolate CV3

 

and L sativus (Ho and Kurib.) Drechsl. ex Dastur isolate
T2T1 were maintained on carrot agar containing per liter:
250 ml of carrot broth (30 g carrots in 250 ml water,

autoclaved 10 min and filtered) and 20 g agar. Fusarium

 

oxysporum Schlecht.:f.eqn lypopersici (Sacc.) Snyd. and

 

 

46

Hansen isolate R5-6, F; oxysporum Schlecht. f. sp.

 

conglutinans (wr.) Snyd. and Hansen, and'Fl solani (Mart.)

 

App. and Wr. f. Sp. REE} CFJL Jones) Snyd. and Hansen
isolate F116 were stored in sterilized soil (Toussoun and
Nelson, 1976). Single—spored isolates were grown on
potato-dextrose broth containing per liter: 500 ml potato
broth (200g potatoes in 500 ml water, autoclaved 30 min
and strained through cheesecloth), 20g dextrose and 20g

agar.

Production of Inoculum

 

Cochliobolus sativus: Inoculated carrot agar plates

 

were incubated in the dark at 20°C for four weeks. Plates
were flooded with distilled water and conidia were
dislodged using;a glass rod. The suspension was passed
through a 153 mm sieve and concentrated to a volume of 30
ml by centrifugation. Viability was determined by
counting germination of conidia incubated 6 hours on
potato-dextrose agar.

Fusarium spp.: Chlamydospores. were produced

 

aseptically by growing mycelia in flasks containing 100 ml
potato-dextrose broth. Mycelia were ground 30 sec using
an Omni-Mixer (Ivan Sorvall, Norwalk, CT 06856) set at
9,600 rpm and washed by centrifugation three times.
Mycelial fragments were placed in flasks containing 50 ml
of salt solution (Quereshi and Page, 1970) and incubated

at 25° t1°C and ambient light on a rotary Shaker for 4

47

weeks. Aggregates were then washed, ground with an Omni-
Mixer for 3 minutes and chlamydospores in the suspension
were enumerated with a haemocytometer.

Chlamydospores were also induced in the presence of
soil. Mycelial fragments were prepared as previously
described and incorporated into approximately 1 kg Boyer
sandy loam (5% moisture content). The infested soil was
placed:h1a.shallow tray covered with aluminum foil and
incubated 10 days. After this time, chlamydospores in
mycelial fragments were seen in soil smears and the soil
was allowed to air-dry. Population density was determined
by dilution-plating the soil on KomadaHs agar. Inoculum
produced in this manner was used only for disease assays

in soils.

Assays of Disease

 

Fusarium oxysporum f. Sp. lycopersici: Chlamydospores

 

produced A. 13352 were added to 3 kg aliquots of
sterilized and non-sterilized soil to give densities of
10,000, 25,000 and 50,000 propagules per gram (ppg).
Soils were sterilized by autoclaving for 2 hours. Each
aliquot of infested soil was distributed into 20, 6 cm
diameter X 7 cm waxed paper cups, into which were

transplanted 9-day-old 'Bonny Best' tomato seedlings.

Seedlings were grown in a greenhouse at 18° to 24°C and

were fertilized weekly with 255 gl’1 20-20-20 Peters Soil

 

48

Test Fertilizer (Robert B. Peters 00., Allentown, PA
18105). Wilt incidence was recorded weekly. Plants were
harvested after six weeks. Fresh shoot weight and shoot
length were measured. Stems were cut longitudinally and
the proportion of each stem length with vascular necrosis
was rated as follows: 1=no necrosis; 2=necrosis confined
to the taproot, below the soil line; 3=up to 50% necrosis
of the stem length; 4=50-75% necrosis of the stem length;
5=more than 75% necrosis; 6=dead plant. Segments from
symptomless plants, cut from the cotyledonary node to 1.5
cm above it, were surface-sterilized and plated on
acidified potato-dextrose agar (10 ml 25% lactic acid
added to one liter of medium after autoclaving). The
experiment was repeated using 5.8 X 103, 2.9 X 104 and
1.45 X 105 ppg of chlamydospores induced in Boyer sandy
loam, with 27 plants per treatment.

Fusarium solani f. Sp. pis': Chlamydospores produced

 

1 yitro were incorporated into 10 kg aliquots of each

 

soil to obtain densities of 500 and 1000 ppg. Aliquots
were distributed into five, 11 cm diameter X 15 cm waxed
paper cups. Four, 5-day-old 'Wando' pea seedlings, grown
in vermiculite, were transplanted into each container.
Plants were grown in a growth chamber at 25°C with a 14
hour photoperiod andziquantum flux denisty (400-700 nm)
of 240 uEm’28‘1. After four weeks, root, Shoot and

lesion lengths were measured and shoots were weighed.

49

Disease was rated, based on the sum of foliage, cotyledon
and root ratings of each plant as follows: Foliage:
0=healthy, 1=wilt/some dead leaves, 2=dead; Cotyledon:
0=healthy, 1=trace necrosis, 2=extensive necrosis; Roots:
0=healthy, 1=less than 25% necrosis, 2=25-50% necrosis,
3=more than 50% necrosis. A similar experiment was
performed with transplants grown in 2.5 cm diameter X 12.5
cm plastic containers containing soil infested with 500 or
5000 ppg. These containers were watered from below via
holes in the bottom, by periodically setting them in a
water bath.

Fusarium oxysporum f.£uh conglutinans: Chlamydo-

 

 

spores produced in Boyer sandy loam were used to infest 1
kg soil aliquots to obtain densities of 160, 800, 4000,
20,000, and 100,000 ppg. Each aliquot was distributed
into four, 11.5 cm diameter X 5 cm waxed paper cups.
Fifteen 'Cherry Belle' radish seeds, covered by 40 cc
infested soil, were thinned to 12 seedlings after 5 days.
Plants were grown in a greenhouse at 18° to 24°C. Plants
dying from wilt were counted and removed from pots.
Plants surviving after three weeks were harvested and
upper stem segments were surface-sterilized and plated on
acidified potato-dextrose agar. A similar experiment was
performed using chlamydospores produced 13 13132, then

added to soils to give a density of 100,000 ppg.

The effect of a constant, -10 kPa (-0.1 bars) soil

50

matric potential on radish wilt incidence in the three
soils was determined using tension plates (Duniway, 1976).
Soil was infested with 20,000 and 100,000 ppg of L

oxysporum f. Sp. conglutinans and 125 g (oven-dry weight)

 

 

was placed in each 97 mm internal diameter (ID) Buchner
funnel with a fritted glass plate of fine porosity, while
60 g was placed in each 67 mm (ID) funnel. Five
replications were used with small funnels and three with
large funnels. Soil was saturated with water and
equilibrated overnight before planting radish seeds.
Funnels were kept covered with plastic film. After four
days, seedlings were thinned to 10 in small funnels and to
20 in large funnels. Plants were grown in a growth
chamber at 28°C with a 14 hour photoperiod and a quantum
flux density (4oo-7oo nm) of 240 uEm'ZS'1. The number of
plants dying was recorded every second day for three
weeks. The experiment was repeated several times.

Cochliobolus sativus: Conidia were applied to 250g

 

aliquots of soil by spraying;a conidial suspension into
the soil in a plastic bag, using an atomizer. Sufficient
volume was applied to obtain 750, 3750 and 18,750 conidia
per g (oven-dry weight}. Soils Sprayed with water only
served as controls. Spring wheat seeds (cv. 'Waldron')
were surface-disinfested 5 min in.CL1% AgNOB. rinsed twice
in 0.5% NaCl, rinsed with distilled water and soaked in

water three hours. Five cc sand was placed in a 1.5 cm

51

diameter X 10 cm plastic tube with a bottom drainage hole
and wetted. One seed was placed on top of the sand and
was covered to the top of the tube with soil. Tubes were
placed in plastic bags for five days, which was the time
required for coleoptile emergence. Tubes were watered
from the top with a squeeze bottle to compact the soil at
emergence, the Sides were covered with aluminum foil to
keep light away from the sub-crown internode, and each
inoculum density for each soil type used was divided into
four blocks of six seedlings each. Different blocks
contained the same number of seedlings which were of
similar size. Blocks were randomized in racks, which were
kept in trays. Plants were grown at 25° t1°C under
fluorescent light with a 14 hour photoperiod and a quantum
flux density (400-700 nm) of 63 uEm’ZS'1, and were watered
from the bottom as needed, by periodically setting trays
in a.water baths After four weeks, the necrotic lesions
on leaves were counted and the sub-crown internodes were
rated as follows: 1=no symptoms; 2=Slight disease (some
necrotic flecks); 3=light disease (some necrotic
portionS); 4=moderate disease (necrosis with some healthy
portions); 5=severe disease (extensive necrosis). The

experiment was repeated once.

52

Quantitative Determination of Fungistasis

Propagules borne on 1 cm2, (L4 :un pore Size
polycarbonate membrane filters (Nuclepore Corp.,
Pleasanton, CA 94566) were placed on sterilized soil and
natural soil amended with increasing amounts of glucose
and peptone. Five-g samples of soils in 6-cm-diameter
plastic dishes were wetted to -50 kPa (-O.5 bars) matric
potential and were equilibrated for 16 to 24 hours before
adding nutrients. Glucose concentrations ranged from 75
to 4800 g per gram of soil and peptone concentrations
were 0.2 (w/w) that of glucose. The nutrients were added
in a volume of water that brought all soils to -10 kPa (-
0.1 bars). The soil was stirred and smoothed with a
spatula and four membrane filters per species of fungus
were placed (M1 the surface. In order to detect
fungistasis of abiotic orgin, filters bearing propagules
were placed on wetted soils in glass dishes, which had
been sterilized by autoclaving one hour. After 12 hours

incubation for Fusarium Spp. and 6 hours incubation for

 

Cochliobolus spp., filters bearing these propagules were

 

stained with phenolic rose bengal, destained in water and

mounted on glass slides. Filters bearing Fusarium spp.

 

were completely dried, cleared with a drop of clove oil,
covered with a glass coverslip, and viewed with
transmitted light at 150 X magnification. Conidia of

Cochliobolus spp. were viewed on a white background with

 

53

incident illumination at 150 X magnification. Assays were

repeated at least once.

Soil Solution Assay

 

The procedure of Filonow and Lockwood (1983) was
used. Soils were wetted to -5 kPa (-0.05 bars) matric
potential and equilibrated 24 hours before centrifugation
at 5°C and 3000 X g for 40 min. Soil solutions were
filter-sterilized (0.2 m pore diameter Nuclepore
filters) and OJ Iml aliquots were placed into double-
welled microscope Slides enclosed in Petri dishes.
Nuclepore filters bearing fungal propagules were floated
on the soil solutions. Incubation and examination of
propagules were the same as previously described. Two

experiments were performed.

Bioassay for Volatile Inhibitors

 

Twenty g (oven-dry weight) of soil was equilibrated
24 hours in a glass Petri dish at -5 kPa (-0.05 bars)
matric potential. One-tenth ml aliquots of sterile, 1:100
solution (Bristow and Lockwood, 1975) were placed in
depressions on a plastic plate suspended 2 mm above the
soil surface. Nuclepore membrane filters bearing fungal
propagules were floated on the Pfeffer's solution and the
dishes were sealed with Parafilm (American Can 00.,
Greenwich, CT 06830). Incubation and examination of

propagules were the same as previously described. To

54

ensure that this procedure was effective for detecting
volatile spore germination inhibitors, filters bearing
propagules were incubated over soil on which one drop of
concentrated ammonium hydroxide (as a source of ammonia)

was placed. The experiment was repeated once.

Soil Respiration of Glucose:

 

At -1 kPa (-0.01 bars) matric potential: The
procedure of Filonow and Lockwood (1979) was used. Four-g
aliquots of soil were pulsed with.CLO5 uCir(1uCi=37MBq) of
uniformly labeled [14C]-glucose (specific activity, 200
mCi/mM) and respired [14C]-002, collected in an
ethanolamine scintillation cocktail at two-hour intervals
for 8 hours, was measured using a liquid scintillation
spectrometer. Counting was corrected using a [140]-
toluene internal standard. Three replicates per soil were
used and the experiment was repeated four times.

At -30 kPa (-O.3 bars) matric potential: One hundred
g (oven-dry weight) soil samples in 400 ml beakers were
equilibrated overnight at -30 kPa before adding 1% (w/w)
glucose. Soils were incubated 48 hours in sealed 3.75 l
(1 gallon) jars containing a beaker with 100 ml 0.25 M
NaOH. Carbon dioxide production was determined by
titration as previously described. The experiment was
repeated using 100 ml 0.1 M NaOH titrated after 6, 12, 24

and 48 hours.

RESULTS

Disease Incidence in Three Soils in Relation to Their Level
of Fungistasis

 

 

Fusarium oxysporum f. Sp. gppglptipgpp: In a

 

preliminary experiment, radish mortality 25 days after
planting was 78% in Boyer sandy loam, which was
significantly greater (3=OJED than that in Capac loam (44%)
(Table 2). Colwood loam was intermediate (62%), but did not
differ significantly (3:0.05) from . the other two. The
addition of the same quantity of inoculum to these soils
which had previously been sterilized by autoclaving resulted
in Significantly (3:0.05) higher mortality than in the
unsterilized soils, but mortality among the sterilized soils
dhd not differ significantly (§;0.05) (Table 2). This
preliminary experiment thus showed that the natural
microflora of these soils suppress disease and the degree of
this biologically-mediated suppression differed among the
soils.

These results were confirmed in another experiment, in
which development of radish wilt, first occurring 13 days
after seeding” differed among the soils over the course of
the experiment, 21 days (Figure 1). Mortality was
Significantly greater (P=0.05) in Boyer sandy loam than in
Capac loam or Colwood loam after 16 days and remained so for

the duration of the experiment.

55

56

TableEL Incidence of Fusarium wilt of radish in three
non-sterilized'and sterilized soils, after 25

 

 

 

 

daysy.
Disease Incidence, %Z
Soil Non-sterilized Sterilized
soil soil
Boyer sandy loam 78 b 100 a
Capac loam 44 a 84 a
Colwood loam 62 ab 100 a

 

y Mean values, based on 4 replicates of 12 plants each per
treatment.

Z Based on mortality. Numbers within a column followed b
the same letter are not significantly different (£=0.05§
using Duncanfs multiple range test.

57

.mawom .858 on» :H :3:- Samoa macaw cohom :H 30.0qu nopmoam

hapcmofimficmfim mm: mama mp poems mocmcfiocfi pa“:
mpcmam N— mo mmpwofiamon e no woman .mvsam> cams pcomosmmu Spam

.vcmspmoap com some
.mHfiom

 

mouse cw mmfiwws We Ahpwfiwpuoav oozmcfiozfl paws so mzmcwpsamzoo .mm .m

 

asuommhxo ssfiuwmsm Mo Aafiom Ho swam pom mo? N NV accommochawano mo pommmm

 

 

 

m><n
—N ON a— up up 0— m— V— n—
] . ildllllq . a 3} O
14 a
1 \ .. mu m
u<a<u 4 n.
l\.\. m
.4
.\\\\z\ . t. m
D Dooga8\0 ”-
a
O u!
.c o\\ ’35- .. mu %

 

 

 

.P maswwm

58

Mortality differed among soils when different inoculum
densities were used. In one experiment, mortality was
significantly greater (3:0.001) in Boyer sandy loam than in
Capac loam at 100,000 propagules per gram (ppg) (Figure 2).
Boyer sandy loam also had the greatest mortality among the
soils at 20,000 ppg, 4,000 ppg, and 800 ppg, but the
differences were not statistically significant (3:0.05).
Populations of the pathogen, monitored over the course of
the experiment in soils in which 100,000 ppg and 20,000 ppg
had been added, either increased or remained constant (Table
3). Although initial recovered populations of the pathogen
were similar in Capac loam and Colwood loam, the initial
recovered population in Boyer sandy loam was approximately
one-half that of the other soils. In all three soils, the
populations of the pathogen enumerated by dilution-plating
at the start of the experiment ranged from 10-58% of that
estimated to have been originally added.

In.greenhouse>and growth chamber experiments, pots of
radish seedlings were watered as needed. Since pots
containing Boyer sandy loam tended to dry more quickly than
other soils, experiments were performed in which all soils
were maintained at -10 kPa L4L1 bar) matric potential to
determine if the greater likelihood of’moisture stress in
Boyer sandy loam could have accounted for the greater

mortality in this soil than in the other two soils. In

Figure

2.

\l
(n
.11
fi
0.

C”

O
I

l

DISEASE INCIDENCE, %
ha
tn
ill

59

 

BOY! It.) A

R

CAPAC

.A
o a «celwooo

oWfiL/d 1 1..
0.8 4 20 3 100
PROPAGULES / g , X10

 

 

Effect of five inoculum densities of Fusarium
oxysporum f. Sp. conglutinans on wilt ihcidence
Tmortality) of radish in three soils, 24 days
after planting. Data represent mean values,
based on 4 replicates of 12 plants each per
treatment. Wilt incidence at 100,000
propagules per gram of soil was Significantly

greater (P=OXKM) in Boyer sandy loam than in
he other-Soils.

 

60

Table 3. Recovered populations of Fusarium oxysporum f. sp.
con lutinans from three sdils infested with 20,000
and 100,000 propagules per gram (ppg), at the time
of seeding and 24 days after seeding to radishy.

 

 

 

Colony forming units ' g'1 soil, X 103

 

 

 

 

20,000 ppg 100,000 ppg
Soil 0 days 24 days 0 days 24 days
Boyer sandy loam 2.6 b 5.1 a 27.2 b 59.9 a
Capac loam 5.7 a 9.2 a 57.6 a 48.4 a
Colwood loam 4.1 ab 8.2 a 55.0 a 65.3 a

 

y Mean values, based on 5 plates per treatment.

2 Numbers within a column followed by the same letter are
not significantly different (2:0.05) using Duncan's
multiple range test.

61

these experiments, radish mortality was approximately twice
as great (P=OJEU in Boyer sandy loam as in Capac loam at
inoculum densities of 100,000 ppg and 20,000 ppg (Table 4).
Populations of the pathogen, monitored in Boyer sandy loam
and Capac loam to which 20,000 ppg had been added, had
increased in both soils after 20 days, but there was no
significant (_P_=0.05) difference between the final
populations in these soils. Thus, these experiments
demonstrated a Significantly'greater disease incidence in
Boyer sandy loam than in Capac loam when.matric potential
was kept constant at -10 kPa (-0.1 bars).

In several experiments, the level of fungistasis was
consistently highest in Colwood loam and lowest in Capac
loam, as determined by the nutrient titration technique.
For example, with the addition of 300 ug glucose and 60 ug
peptone per gram of soil, germination of chlamydospores was
Significantly (2:0.05) higher in Capac loam than in Boyer
sandy loam (Figure 3). Colwood loam had the lowest (13:0.05)
germination of the soils. Thus, relative levels of
fungistasis differed among the soils, Inrt were not
correlated with relative incidences of radish wilt.

Fusarium oxysporum f. sp. lycopersici: Disease

 

 

incidence, based on wilting and vascular discoloration, was

highest in Capac loam, followed by Boyer sandy loam, and

62

Table 4. Incidence of radish wilt in two Soils infested
with two concentrations of Fusarium ox S orum f.
Sp. conglutinans and maintained at -10 EPa.(-O.1
barST_matric pdtential, and recovered populations
of the pathogen, both three weeks after planting.

 

 

Colony forming units of
-1

 

 

Fusarium,’ g soil,
Disease incidence, %y X 1032
Soil 2 X 104 ppg 105 ppg 0 days 21 days
Boyer sandy
loam 83 97 5-5 8-3
Capac loam 48 47 8.1 12.2

 

y Based on mortality. Me n values, based on 5 replicates of
6 plants ea h at 2 X10 ppg and 3 replicates of 20 plants
each at 10 ppg. Mean values within each column are
Significantly different (2:0.05) using Student's t-test.

2 Mean values based on 5 replicates per treatment.

GERMINATION,%

Figure 3.

63

 

 

 

loo—+ 1 . _
A
75 *
CAPAC\ OYER
o
50F
25 -
o a COLWOOD
z’Il/llgz’I/I/l'l J

 

75 150 300 600
ug GLUCOSE / 9 son

Germination of chlamydospores of Fusarium
oxysporum f. Sp. conglutinans on three soils
amended with different concentrations of 5:1
(w/w) glucose:peptone, after 12 hours. Data
represent mean values, based on 4 replicates per
treatment; 100 spores were counted per replicate.
Duncanfls multiple range test showed the followng
differences amo means: 150 ug glucose/soil,
Boyerb Capac , and Colwooda; 300 ue
glucose/soil, Boyer , Capacc, and Colwooda. Mean
values of soils followed by the same letter did
not differ significantly (§;CL05).

 

 

 

64

then Colwood loam (Figure 4). This trend was observed at
all three inoculum densities used, but disease incidence was
Significantly (2:0.01) lower in Colwood loam than in the
other soils only at an inoculum density of 25,000
chlamydospores per gram. Plants grown in Capac loam showed
greater reduction of Shoot height and weight in pathogen-
infested soil, in relation to that in non-infested soils,
than those’in Colwood loam and Boyer sandy loam (Table 5).
The proportions of vascular discoloration in stem lengths
did not differ among the soils, indicating that this
parameter was not useful for disease evaluation (Table 5).
In a repeated experiment using 5,800, 29,000 and 145,000
ppg, no wilt symptoms were evident. The mean incidence of
vascular discoloration was 1% in Colwood loam and Boyer
sandy loam, as compared with 6% in Capac loam, but because
of the low frequency of disease, these differences were not
statistically significant. Thus, the ranking of disease
incidence in these soils, with Capac loam the highest and
Colwood loam the lowest, must be considered tentative. In
other experiments, disease incidence in sterilized soils
containing only 1,000 chlamydospores per gram was 100%.
Thus, the presence of the natural microflora in these soils
results in disease suppression and accounts for differences

in disease incidence among the soils.

65

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pom mpcmam ON .5 was—350.: Haves. pcmmmpmou Spam .mcfipcwamwzwpw

 

 

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«2x .o\ 33303255

 

 

 

 

 

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henna cohom

 

 

 

 

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so— N m e or N m.N sop 0 v0— M m vow N m.m VOF 0 so. N m e0? N m.w eow o HHom
umfimOpoo: awasoms> w .pgmwmz poonm smash So .spmcma poosm

 

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poonm opmaov so Ammgv mofipfimcmc azasoocfi moan» pm Hofimammooma .mm .m asnmmnwxo sswummsm mo vommmm .m wanna

 

 

67

Colwood loam was Significantly (2:0.05) more

fungistatic to chlamydospores of F; oxysporum f. sp.

 

lyc0persici than Boyer sandy loam, which was significantly

 

(§;CLO1) more fungistatic than Capac loam, as determined in
soils amended with 150 ug glucose and 30 ug peptone per gram
of soil (Figure 5%. Thus, the levels of fungistasis to F;

oxysporum f. Sp. lycgpersici were inversely correlated with

 

 

observed incidences of disease. Chlamydospores of L

oxysporum f. Sp. lycopersici germinated more readily in soil

 

 

in the presence of nutrients than those of F; oxysporum f.

 

Sp. lycopersici.

 

Cochliobolus sativus: Incidence of sub-crown internode

 

lesions was compared among the three soils in two
experiments. Leaf lesions also were enumerated in one
experiment, but did not occur in the other experiment.
Results of the two experiments were contradictory. In the
first experiment, sub-crown internode lesions were
Significantly (3:0.05) more severe in Boyer sandy loam than
in Capac loam at inoculum densities of 3,750 and 750 conidia
per gram (Table 6). At both inoculum densities, the ranking
of Colwood loam was intermediate, but did not differ
significantly from either of the other soils. There was no
significant difference in sub-crown internode lesion
severity among soils at an inoculum density of 18,750

conidia per gram. There was no significant difference in

GERMINATION, %

Figure 5.

68

 

 

 

 

100 u 1 1
A.
75 __ o 110an
CAPAC‘
50 - -
ficmwooo
25 b «-
OLA—L—J—‘_

75 150
ug GLUC053EO/ g Sglol.

Germination of chlamydospores of Fusarium
oxysporum f. sp. lycopersici on three soils
amended with different concentrations of 5:1
(w/w) glucose:peptone, after 122 hours. Data
represent mean values, based on 4 replicates per
treatment; 100 spores were counted per replicate.

Duncan '8 multiple range test showed the following
diff rences among means: 150 ug glucose/soil,

Boyer , Capacc and Colwooda; 300 ug glucose/sci,
Boyer a, Capac , ColwoodC . Mean values of soils
followed by the same letter did not differ
significantly (3:0.05).

 

 

69

 

 

 

 

 

Table 6. Effect of Cochliobolus sativus at three inoculum
densities on severity H sub-crown internode
disease and numbers of leaf lesions on wheat in
three soils, four weeks after seedingx.

Disease indexy Leaf lesions, no.z

Soil 750 3750 18,750 750 3750 18.750

PPS PPS PPS PPS PPS PPS

Boyer

sandy 3.1 a 3.7 a 3.4 a 0.4 0.7 3.1
loam

Capac

loam 2.4 b 2.7 b 3.1 a 0.1 0.6 2.6

Colwood

loam 2.9 ab 3.3 ab 3.3 a 0.2 0.3 2.5

 

treatment.

y Severity of necrosis of sub-crown internode:
necrosis, 2=Slight (some flecks),
portions),

portions),

4=moderate

Mean values, based on 4 replicates of 6 plants each per

1=no
3=light (some necrotic

(necrosis with some healthy
5=severe (extensive necrosis).

Numbers within

a column followed by the same letter are not significantly
different (3:0.05) using Duncan's multiple range test.

(13:0.05) among treatments .

Analysis of variance showed no significant differences

70

leaf lesion numbers among soils at all three inoculum
densities.

In the second experiment, sub-crown internode lesions
were significantly (2:0.01) more severe in Capac loam than
in Colwood loam at an inoculum density of 8,000 conidia per
gram (Table 7). Boyer sandy loam was intermediate, but did
not differ Significantly from either of the other soils.
There was no Significant difference in lesion severity among
soils at an inoculum density of 80 or 800 conidia per gram
of soil. The ranking of severity of _C_._ sativus root rot in
these soils based on these experiments is therefore
inconclusive.

As determined by the nutrient titration technique, the
level of fungistasis against _C_. sativus was significantly
(3:0.05) lower in Capac loam than in Colwood loam or in
Boyer sandy loam (Figure 6). Nutrient titration of these

soils using conidia of Q; victoriae gave Similar results.

 

 

Fusarium solani f.Eflh pig}: Results of disease
incidence determinations for two experiments were
contradictory. In the first experiment, root lengths,
lesion lengths, and disease ratings were not Significantly
(§;Ou05) different among the soils at 500 and 1000
chlamydospores per gram (Table 8). However, shoot length

and fresh weight were significantly (3:0.01) lower in Boyer

71

Table 7. Effect of Cochliobolus sativus at three inoculum
densities on severity oFsub-crown internode
disease on wheat in three soils, four weeks after
seedingx.

 

 

Disease indexy

 

 

Soil 80 ppg 800 ppg 8000 ppg
Boyer sandy loam 2.1 a 2.5 a 3.3 ab
Capac loam 1.7 a 3.1 a 3.7 a
Colwood loam 1.7 a 2.4 a 2.7 b

 

x Mean values, based on 4 replicates of 6 plants each per
treatment.

y Severity of necrosis of sub-crown internode: 1=no
necrosis, 2=Slight (some flecks), 3=light (some necrotic
portions), 4=moderate (necrosis with some healthy
portions), 5=severe (extensive necrosis). Numbers within
a column followed by the same letter are not significantly
different (2:0.05) using Duncan's multiple range test.

Figure 6.

100

GERMINATION, 9;

72

cancg D
D ..
0'
R

IOYER

D
“HCOHNVCHDD

C) I 4} n I
600 1200 2400 4800
ng GLUCOSE / g SOIL

Germination of conidia of Cgchliobolus sativus on
three soils amended with different concentrations
of 5:1 (w/w) glucose:peptone, after 6 hours.
Data represent mean values, based on 4 replicates
per treatment; 100 spores were counted per
replicate. Duncan' S multiple range test showed
the following differences among means: 60
1200, and 2400 pg glucose/soil, Boyera, Capac
and Colwooda. Mean values of soils followed by
the same latter did not differ significantly
(2:0.05).

‘4
Ln

tn
(3

BD

C"
V
11.

 

 

 

73

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74

sandy loam than in Capac loam or Colwood loam. In the
second experiment, lesion length was significantly (3:0.05)
less in Capac loam than in Boyer sandy loam or Colwood loam
at an inoculum density of 500 chlamydospores per gram (Table
9). However, differences were not significant at an
inoculum density of 5000 chlamydospores per gram. The
ranking of severity of'j; solani f3 sp. pig; root rot in
these soils based on these experiments is therefore
inconclusive. Disease incidence in sterilized soils did not
differ at either 500 or 5000 chlamydospores per gram of
soil.

The level of fungistasis against}:L solani f: sp. pig;
in Capac loam was significantly (_P=0.05) lower than that in
Colwood loam and Boyer sandy loam, which did not differ

significantly (3:0.05) (Figure 7).

Germination of Propagules on Natural and Sterilized Soils

 

PrOpaguleS of all the fungi used in this work failed to
germinate on all three, unamended soils. However, when
these same soils were autoclaved for 1 1/2 hours, propagules

germinated 99-100%.

Assay of Soil Solutions for Germination Inhibitors

 

Germination of chlamydospores of F; oxysporum f. sp.

 

congultinans was 99-100% in sterile aqueous extracts of the

 

three soils, as compared with 99% germination in 1:100

75

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76

 

Ioo . H 4 , l,

\l
0'!
I
C!
|_l

h)
tn
I
la

Figure 7.

GERMINATION, %
CD
CD

“neg ‘4

CO [WOOD

0
“DOVER

 

 

 

75 150 300 600
ug GLUCOSE/ g SOIL

Germination of chlamydospores of Fusarium solani
f. Sp. isi on three soils amended with different
concen rations txf 5:1 (w/w) glucose:peptone,
after 12 hours. Data represent mean values,
based on 4 replicates per treatment; 100 spores
were counted per replicate. Duncan's multiple
range test showed the following differences among
means% 150 and 300 ug glucose/soil, Boyera,
Capac h and Colwooda; 600 ug glucose/soil,
Boyera' , Capacb, and Colwood a. Mean values of
soils followed by the same letter did not differ
significantly (£=0.05).

77

Pfeffer's solution. Conidia of g; sativus germinated 98% in
sterile extracts of the three soils, as compared with 66%

germination in 1:100 Pfeffer's solution.

Assay of Soils for Volatile Inhibitors

 

Chlamydospores of f; oxysporum f. Sp. conglutinans and

 

conidia of g; sativus germinated 99% and 60%, respectively,
in Pfeffer‘s solution incubated over soil. In both cases,
spore germination over soil did not differ from spore
germination over Pfeffer's solution. The technique used was
effective at trapping inhibitory volatile compounds, as
Shown by nearly 100% inhibition of spore germination of
these fungi incubated over soil to which 1500 ppm ammonia

was added.

Microbial Biomass and Microbial Composition of the Soils

 

The biomass of Colwood loam, as determined by the
chloroform fumigation method, was 107.8 mg C/100g soil -
twice that of Capac loam and Boyer sandy loam (Table 10).
Colwood loam had significantly'(§=0.05) more aerobic
bacteria than Capac loam, while Boyer sandy loam ranked
intermediate (Table 10). Populations of actinomycetes were
significantly'(§;CL05) greater in Colwood loam and Boyer
sandy loam than in Capac loam (Table 10). Populations of

anaerobic bacteria were Significantly'(£=0xfifl greater in

.nowo mOHmoHHmmH OH HO

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.HHow Hon

mmpmoHHmoa m no woman .mOSHm> :80: .chm8 o>HHOOHmm so mcHHmHmIcoHHSHHw an cmzHEHOHOQ h

.HHom pom mmeoHHmoa e no woman .mmsam> new: .coHHmeSSH EHOHOHOHno an cmcHanopoo x

HSOCOHHHS thcmoHHHcmHm we: was COHHOH mamm map an cmSOHHOH casHoo w :HSHHS mampasz

.Hmmp awash OHQHHHSS m.:wozsa mchs Amo.oumv

 

 

 

3
O m.vv Q >.vm p N.HN p >.v> n m.>oH ESOH ooozHoo
p a.mm o m.ml o m.m_ o n.0m s m.mm ssoH oesno
n
m o.mH m o.oH pm m.o— n m.H> m m.mm ESOH
hogan Homom
No. x O— N moH x o— x HHom
.h.m:sm .SHH pomp .st pomp .hmmpmo SOSHHO< HHom m
uoHnouoms< hoHpoao< ooH\o we
.xmmmaoHn
Hmpoe
3HHom HIm . wHHss maHsSom ASOHOO

 

.mHHom Swamp :H mcoHHmquom HSHQOHOHS can mmmSon .oH OHQSB

79

Capac loam and Colwood loam than in Boyer sandy loam (Table
10). Populations of fungi were significantly (2=Olfifl
greater in Colwood loam than in Capac loam, while
populations in Boyer sandy loam were least (P=0.05) (Table
10).

Nutrient Sinks of Three Soils

 

Usingzniincubation.chamber continually flushed with
air, there was 1K) significant (£;0.05) difference in
utilization of 14C-labelled glucose among soils adjusted to
-1 kPa (-0.01 bars) (Figure 8). Most of the respiration
occurred within four hours after the addition of label. In
contrast, using a closed, large-volume system, significant
differences (_P=0.05) in utilization of glucose among soils
adjusted to -1 kPa (-0u01 bars) were shown after 24 hours of
incubation, but not after only 12 hours (Figure 9). Between
12 and 24 hours, significantly (_P=0.05) more carbon dioxide
was respired by Colwood loam and Capac loam than by Boyer
sandy loam. When the closed, large-volume system was used
to measure respiration of soils adjusted to a matric
potential of -30 kPa (-0.3 bars), there was 2- to 4-fold
greater respiration in all soils after 12 hours, than in
soils adjusted to -1 kPa (-0.01 bars). Respiration in
Colwood loam after 24 hours was 5 times greater (3=OXEH

than that in Boyer sandy loam and 3 times greater than that

0

J5

"CO, RESPIRED, dpm x102
0 01
f

00
O
I

Figure 8.

80

O
I

O
I

 

 

 

4
HOURS

Cumulative 14CO respiration by three soils at -1
kPa (-0&H bars? matric pote tial, pulsed with
0.05 1101 (1.85 MBq) of 1 C-glucose. Data
represent mean values, based on 3 replicates per
soil. No significant differences (fi=0.05)1were
observed among the soils during 2-8 hours.

Figure 9.

mg co,— C RESPIRED /100 9 SOIL

I“
[j CAPAC
Em...

 

12 24
HOURS

002 respiration by three soils at -1 kPa (-0.01
bars) matric potential measured 12 and 24 hours
after the addition of 1% (w/w) glucose. Data
represent mean values, based on 4 replicates per
soil. DuncanIS'multiple range test showed the
folloging differences among means after 24 hours:
Boyer , Capaca, Colwooda. Mean values of soils
followed by the same letter did not differ
significantly (2:0.05).

82

in Capac loam (§?0.05) (Figure 10).

Oll.

Figure 10.

FIRED / 100 g 5

83

 

 
   

 

 

 

301
- BOYER E
25 " Z: d
D CAPAC E
20- E -
E COLwooo E
15 - E .I
10 - E .L
5 - E -
_ I E
6 12 24
HOURS

002 respiration by three soils at -30 kPa (-O.3
bars) matric potential measured 6,12, and 24
hours after the addition of 1% (w/w) glucose.
Data represent mean values, based on 4
replicates per soil. Duncan's multiple range
test showed the following difoference among
means: 12 hours, Boyerab Capac , Colwooda ; 24
hours, Boyer a, Capac and Colwoodc. Means
values of soils followed by the same letter did
not differ significantly.

DISCUSSION

The objective of this work was to determine if a
relationship exists between levels of fungistasis in soils
and disease incidence or severity occurring in them. This
question had not been explicitly addressed previously.

Results with Fusarium oxysporum f. Sp. lycopersici supported

 

this hypothesis in that highly fungistatic soils had lower
disease incidence than less fungistatic soils. Other
workers have observed that disease-suppressive soils are
apparently more fungistatic than disease-conducive soils
(Alabouvette, 1983; Arjunarao, 1971; Furuya, 1982; Hwang et
al., 1983; Kao and Ko, 1983; Smith, 1977). However, results
with Fusarium wilt of radish did not show this relationship:
the lowest incidence of wilt, caused by L oxysporum f. sp.

conglutinans, was in Capac loam, which had the lowest level

 

of fungistasis to the pathogen, suggesting that factors
other than fungistasis are involved in disease expression in
the case of this pathogen (Table 11). Possibly, differences
in fungistasis in the rhizosphere may be correlated with
differences in disease in soils, or other mechanisms of
antagonism may be involved. However, the low incidence of
radish wilt in Capac loam cannot be explained by
hyperparasitism, or other lethal antagonisms, as the

population of the pathogen did not decrease during the

84

85

.SOHHSHHHH HSOHHHSS esp an cocHaampoo

.mpmcprcoo
eOmOHo .mmamH :H mwoosHm_?wHHoancs Scum :oHHoscoamnmoo_Ho HSOSOHSmmoa co commm a

:oprmHSdH SHOHOHOHno mp cmcHapmHmm
pcosHammxm cmEHHHSOOSS one no woman .m>HHwH:ma
mpcmsHHmnxm Hmpm>mm so cmmmm

snoaoH n m .oansosnoHsH u m .HnosmHs u H s

 

 

H H H m m SSOH ooosHoo

m m N H n asoH oases

m m m m H SSOH henna Cohom

anmemHmSSH stmmmnaoHp zoocoeHozH >mozmoHozH HHom
Ho Ho>oH pcoHapsz HnHoa HHH3 HHH3
OHSSOH anowm

 

.SHHHS OpmsOH and :chma Ho mmocmoHocH can .mHmmHmeSSH Ho mHm>mH .mxch
HcoHpHSS .mommmaoHn HprOHOHs pHosH OH Hoommmu 5H6: mHHom Ho wSchmm .HHmHnme

86

course Ixf the experiments. Results obtained with

Cochliobolus sativus causing root rot of wheat and with

 

F; £32221 f. Sp. Rig} causing root rot of pea where
inconsistent and so conclusions cannot be drawn from these
experiments.

Where competition for nutrients is the mechanism of
disease suppression in a soil, nutrient titration, or an
analogous technique for determining differences in
fungistasis, should be effective in identifying suppressive
soils. Such an approach was used by K0 and Ho (1983), who
found that soils highly fungistatic to R; solani and 2;
splgppgpg, suppressed disease caused by these fungi.
However, in my work, radish wilt development was least in

Capac loam, which was not as fungistatic to L oxysporum f.

Sp. conglutinans as the other soils. Therefore, the method

 

of Ko and Ho may not be applicable in all cases, since other
mechanisms may be involved in suppression.

All pathogens used in this work caused greater
incidences of disease in soils which had been sterilized,
than in non-sterile soils, showing that the natural
microflora played an important role in suppressing disease
development. The Similar levels of disease observed in the
sterilized soils suggest that abiotic factors were not
responsible for disease differences in non—sterile soils.

However, abiotic factors, such as fertility and soil texture

87

as it affects water availability, may indirectly affect the
ability to detect differences in disease among soils,
because they can affect host susceptibility. Lack of
consistent results when comparing pea and wheat root rot
severity among soils could be ascribed to variability in
water potential, since this factor was not controlled.
However, the order of severity of radish wilt among the
three soils remained consistent, whether plants were grown
at constant or fluctuating matric potentials or
temperatures. Thus, some plant/pathogen systems, like

Fusarium wilt of radish, are more suitable for detecting

 

differences in disease among soils than others. Alabouvette
et al. (1982) also found that differences in suppressiveness
among soils could be more readily shown using Fusarium wilt

of flax, than Fusarium wilt of muskmelon because of less

 

variation in results with flax wilt. Additional advantages
to the flax wilt system, which were also seen with the
radish wilt system, included rapid development of wilt and a
small plant size, which made increased replication possible.
Other workers have used damping-off fungi such as

Rhizoctonia solani on radish (Kinsbursky and Weinhold,

 

1983). and Pythium splendens on cucumber (Kao and Ko, 1983),

 

 

as rapid, repeatable systems for identifying differences in

disease development in soils.

88

Soil fertility is another important factor that can
affect disease comparisons among different soil types. As
shown in Table 1, the concentrations of'mineral nutrients
among the three soils were different. Since differences in
fertility may affect disease development, supplementary
fertilization was used. However, supplementary
fertilization could not fully compensate for differences
among soils, as shown by tomato shoot lengths and weights
that were Significantly lower (3;0.05) in non-pathogen-
infested Boyer sandy loam than plants grown in the other two
non-infested soils (Table 5). Thus, comparisons of these
growth parameters could not be directly made among pathogen-
infested soils, but rather, had to be made from proportional
growth differences in relation to the non-infested controls.
In contrast, disease incidence of tomato was a parameter
less influenced by soil fertility than by biotic factors, as
shown by differences among non-sterilized, but not among
sterilized soils.

The importance of using several inoculum densities to
measure disease incidence in soils was demonstrated in this
work. My results with radish and tomato wilts, and the
results of Alabouvette et al. (1982) with flax and muskmelon
wilt, demonstrated that statistically significant
differences in disease can be demonstrated only at certain

inoculum concentrations.

89

Total microbial biomass was greatest in Colwood loam,

which had the lowest incidence of Fusarium wilt of tomato

 

and the highest degree of fungistasis of the three soils
(Table 11). Biomass and fungistasis were less in Boyer
sandy loam and Capac loam, which had greater incidences of
tomato wilt. These results suggested a possible
relationship between biomass and the suppression of the
pathogen. However, the order of the incidence of radish
wilt in these soils differed from that of tomato, suggesting
that other factors have a greater effect on development of
this disease than microbial biomass. Counts of specific
microbial components of soils were imperfectly correlated
with total biomass. The ranking of soils with respect to
counts of fungi was similar to the ranking of total biomass
values, which reflects the fact that fungi account for 75%
of the total microbial biomass (Anderson and Domsch, 1978).
Populations of actinomycetes and bacteria also were greatest
in Colwood loam. However, Capac loam had a higher
population of anaerobic bacteria and a lower population of
actinomycetes, than Boyer sandy loam, demonstrating a lack
of correlation of these microbial components with total
biomass.

One objective of dilution plate enumeration of the

microflora was to detect the presence of specific components

90

of the microflora which may be important determinants of
disease suppression. In other work (Chet and Baker, 1981),
dilution plating revealed the presence of high numbers of

Trichoderma viride, which, although representing only'(h8%

 

of the total number of fungi, accounted for disease
suppression. However, there were no obvious differences in
the composition of the microflora of the three soils
examined.

The nutrient sinks of the soils, as determined by
measuring the respiration of added glucose, showed some
correlation with total biomass (Table 11). The nutrient
Sinks and biomasses were greatest in Colwood loam, but the
nutrient sink of Capac loam was greater than that of Boyer
sandy loam,.although their mass values were identical.
From the work of Filonow and Lockwood (1983), Boyer sandy
loam had a greater nutrient sink than several other soils
whose composition was similar to that of Capac loam. By
contrast, Boyer sandy loam had a relatively low level of
fungistasis as compared with these soils. The reasons for
the discrepancies between their results and the present
results are not known, although there may be differences in
the microflora between samples of soils used in the two
studies.

Results of nutrient sink determinations using a closed,

large volume system and a continuous collection system

91

differed. The continuous collection system, using 140-
glucose, permits a high degree of sensitivity, so that
responses to added glucose could be measured in the same
time frame required to evaluate spore germination. However,
differences between the nutrient sinks of the soils were not
shown by this method. Respiration reached a maximum in all
soils within four hours after the addition of label, and
decreased thereafter at similar rates. In contrast,
differences in respiration among soils were shown only after
12 hours using the closed, large volume system. Further
evaluation of these techniques is necessary to account for
the lack of correlation of results between them.

It is noteworthy that the order of fungistasis among
the soils was similar for all the fungi tested. Colwood
loam was the most fungistatic of the soils, while Capac loam
was the least fungistatic (Table 11). Germination of all
fungi was inhibited on natural, unamended soils, whereas
they germinated unimpeded on sterilized soils. No volatile
compounds or inhibitors from sterile soils solutions were
detected.in any of the soils. These results suggest that
nutrient deprivation is the primary mechanism of fungistasis
in these soils (Lockwood, 1977).

Conidia of g; victoriae and Q; satiyps required an

 

amendment of 4800 pg glucose and 960 ug peptone per gram of

92

soil for approximately 98% germination, while chlamydospores

of E; oxysporum f. sp. lycopersici, F; oxysporum f. Sp.

 

 

conglutinans and L solani f. Sp. pisi required only 600 ug

 

glucose and 120 ug peptone per gram of soil to germinate to

the same extent. The greater sensitivity'of’Cochliobolus

 

Spp. conidia is an exception to the general relationship of
decreasing sensitivity to fungistasis with increasing Spore
‘volume (Steiner and Lockwood, 1969). .Also, the conidia of

Cochliobolus spp. required less than half the time required

 

for germination of chlamydospores of Fusarium spp., whereas
propagules more sensitive to fungistasis generally required
a longer germination time (Steiner and Lockwood, 1969). It

is not known why Cochliobolus spp. was more sensitive to

 

fungistasis than Fusarium Spp. The sensitivity of Fusarium
Spp. in this work was similar to that reported by others
(Alabouvette, 1983; Smith, 1977). However, previous

workers, using the same isolate of 9; victoriae and the same

 

soil types, found that an amendment of only 1000 ug glucose
and 200 ug peptone per gram of soil resulted in 60-90%
germination (Lee and Lockwood, unpublished). Differences in
the production of conidia or mutation of the culture may
account for the high sensitivity to fungistasis of the

Cochliobolus Spp. used in this work.

 

Chlamydospores of L oxysporum f. sp. lchpersici and

 

.3; oxysporum f. Sp. conglutinans were nutrient-independent,

 

93

as shown by their high germination (80% and 99%,
respectively) on 1:100 PfefferHs solution, which contrasts
with previous reports of 0-1% germination of chlamydospores

of L oxysporum f. Spp. in the absence of nutrients (Smith

 

and Snyder, 1972). However, chlamydospores of g; solani f.
Sp. pisi germinated only 1% on 1:100 Pfeffer's solution,
which is similar to the results of Griffin (1970). Conidia

of g; victoriae and g; sativus were partially nutrient

 

independent, germinating 30% and 50%, respectively. This

is in contrast to other workers reporting 97% germination of
these fungi in the absence of nutrients (Hsu and Lockwood,
1973).

In conclusion, this work did not Show a consistent
relationship between the level of fungistasis in soils and
disease incidence or severity occurring in them. However,
because of the inherent difficulties in ruling out factors
other than the soil microflora as an explanation for disease
differences, the hypothesis that highly fungistatic soils
have lower incidence or severity of diseases cannot yet be
rejected. There may be differences in fungistasis occurring
within the rhizospheres of these soils that could account
for differences between radish and tomato wilt incidences.
Instead of comparing disease in different soils with

different levels of fungistasis, another approach might have

94

been to alter the fungistasis of one soil and observe
changes in disease. The problem with this approach is
finding a treatment that would affect fungistasis, but not

other properties of the soil. Prolonged storage could be

such a treatment.

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fusarioses vasculaires. Role de la competition
nutritive entre microorganisms. PhJL.dissertation,
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Alabouvette, C., Y. Couteaudier, and J. Louvet. 1982.
Comparaison de la réceptivité de différents sols et
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Anderson, J.P.E., and K.H. Domsch. 1978. Mineralization of
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Arjunarao, V. 1971. Biological control of cotton wilt. I.
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Chet, I., and R. Baker. 1981. Isolation and biocontrol
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Cook, R.J., and K.F. Baker. 1983. The nature and practice
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Day, PJL 1965. IParticle fractionation and particle-Size
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Duniway, JJM. 1976. Movement of zoospores of Phytgphthora
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Farley, J.D., and J.L. Lockwood. 1968. The suppression of
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96

Filonow, AJL, and JuL. Lockwood. 1979. Conidial exudation
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Filonow, AHB., and J.L. Lockwood. 1983. Mycostasis in
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Furuya, H. 1982. Studies on Fusarium root rot of bean.
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Hwang, S.F., R.J. Cook, and W.A. Haglund. 1983. More
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Schroth, M.N., and J.G. Hancock. 1982. Disease-suppressive
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Smith, S.N., and W.C. Snyder. 1972. Germination of
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Steiner, G.W., and J.L. Lockwood. 1969. Soil fungistasis:
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.I1lllll“ 91(“I .4 I