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Q Michigan State
University

   

I 1
This is to certfi' . at the ‘ 3 ‘
thesisgtitled

 

Soil Fungistasis: Nature of the Inhibition of Nutrient-
Independent Propaqules

presented by
Peter R. Bristow

has been accepted towards fulfillment
of the requirements for

Ph.D. dPM?ph1 Botany and Piant
U' Pathology

\ . J-/ x
C\: % (K, K/tiflryjki/

Major professor

in

     

 

 

 

m..—

ABSTRACT

SOIL FUNGISTASIS: NATURE OF THE INHIBITION
OF NUTRIENT-INDEPENDENT PROPAGULES

BY

Peter R. Bristow

Certain fungal propagules germinate to varying
degrees in glass distilled water, but fail to do so on
natural soil. Germination of these nutrient-independent
yet soil fungistasis—sensitive propagules was also
suppressed in a model system designed to imitate the
microbial removal of exuded spore nutrients in soil.
The model system imposed a nutrient stress by the
continuous aqueous leaching of sand or glass microbeads
upon which membrane filters bearing the propagules were
incubated. Experiments were designed to gain a further
understanding of the mechanism of inhibition of these
propagules by the microbial nutrient sink in soil, and
of the sink's significance in relation to possible
volatile fungistatic factors.

Germination of Helminthosporium victoriae conidia

 

in the model system with mineral salts solutions or sub-
soil extract as the leaching media was suppressed as

effectively as with water. Conidia of this fungus and

In

Peter R. Bristow

Curvularia lunata, and sclerotia of Sclerotium cepivorum

 

collected from cultures containing 1"C-glucose,
initially lost about twice as much radioactivity on
leached sand as on unleached sand. Over 90 percent

of the radioactivity exuded by g. victoriae conidia

 

was detected in the leachings after only 12 hr.

Conidia of g. sativum and g. victoriae lost

 

the capability to germinate in buffer solution after

7 days of prior continuous incubation on Conover loam
soil or in the model system. Viability of g. sativum
decreased rapidly 1—2 days following loss of nutrient-
independence, whereas significant decreases in viability

of g. victoriae occurred only after another 21 days of

 

nutrient deprivation.
Glucose and amino acids were among the substances

present in conidial exudates. Conidia of g. victoriae,

 

first converted to nutrient dependency by extended

incubation on Conover loam soil or in the model system,
accumulated radioactivity and germinated when incubated
in exudate collected from 11+C-labelled spores. Germi-
nation was stimulated, over that occurring with buffer,

when conidia of g. victoriae and g. lunata were leached

 

with their own exudate or the exudate of the other
fungus. A similar stimulation of germination occurred
when a solution of glucose and casein hydrolysate was

used as the leaching medium: the concentration of each

Peter R. Bristow

being equal to that in spore exudates.

Term tubes began to emerge from g. victoriae

 

conidia after only 1.5 hr incubation on water-saturated

sand. Germ tube formation was prevented by transferring

conidia to conover loam soil or to the model system

before they had completed 1.0 hr incubation on water-

saturated sand. However, after 1.0 hr the conidia

were irreversibly committed to producing germ tubes.

In contrast, transfer of already committed spores to

a clay loam soil from Colorado, which is known to

produce a volatile fungistatic factor, prevented germ

tube formation in about 50% of the committed spores.
Radioactive carbon dioxide was detected within

3 minutes after the addition of l"C-glucose to either

Conover loam or the Colorado clay loam soil. This

demonstrates the very early establishment of a microbial

nutrient sink in the soils. Exudates from l"C-labelled

g. victoriae conidia were also readily and quickly

 

metabolized in both soils. Similarly, glucose loss

from glucose-containing paper discs incubated on either
soil was more rapid than when incubated on their
respective sterile soil controls. The rates of 1"C02
evolution and glucose loss were greater with Conover
loam soil than with the Colorado clay loam soil. More-
over, bacteria isolated from these two soils accumulated

1"C--labelled materials exuded from spores.

Peter R. Bristow

metabolites of spore exudates. In the Colorado clay
loam soil, where the microbial nutrient sink appeared
to be less than in Conover loam soil, the volatile
fungistatic factor may have played a major role in

the inhibition of germination.

SOIL FUNGISTASIS: NATURE OF THE INHIBITION

OF NUTRIENT-INDEPENDENT PROPAGULES

BY

Peter R. Bristow

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Department of Botany and Plant Pathology

1974

 

 

To Libbie

ii

ACKNOWLEDGMENTS

To Dr. John L. Lockwood, I express deep
appreciation for his friendship and guidance during
my tenure as a graduate student, and for his encour—
agement and assistance in the preparation of this
manuscript.

To the members of my guidance committee:
Drs.N. E. Good, D. J. deZeeuw, M. L. Lacy, J. M.
Vargas, Jr., and A. R. Wolcott, I extend my thanks
for their suggestions and critical evaluation of the
manuscript.

Thanks are also due to the numerous people
in this and other Departments for the use of equipment.

Funds received from the Ernst A. Bessey Graduate
Student Award for 1972 were applied toward publication
of this thesis.

Without the understanding and encouragement of

my wife Libbie, this study would never have been possible.

LIST OF

LIST OF

TABLE OF CONTENTS

TABLES I O I I O O I l I O O O D O 9 O I

FIGURES I I I I I I I I I I I I I I I I

INTRODUCTION I I I I I I I I I I I I I I I I I I

LITERATURE REVIEW 0 O O O O O I I O C I O l C O

METHODS

RESULTS

AND MATERIALS D O I O I O I O I O O O 0
Maintenance of fungi and bacteria . . .

Production, collection, and preparation
of propagules . . . . . . . . . . . . .

Spore exudates . . . . . . . . . . . . .
Source and preparation of soils . . . .

Model system for imitating the
microbial nutrient sink of soil . . . .

Metabolism of glucose and spore
exudates by soil microorganisms . . . .

Paper disc technique for assaying the
microbial nutrient sink of soil . . . .

Fungistasis assays . . . . . . . . . . .

Radiochemical purity of glucose
in agar discs incubated on soil . . . .

Enumeration of soil microorganisms . . .

Effect of washing conidia, and of
different leaching media on
germination in the model system . . . .

iv

20
20

20
22
24

25

27

29
30

33
35
37

37

Exudation from spores incubated for
alternate periods when leached and
not leached . . . . . . . . . . . . . . . . 38

Exudation from g. victoriae conidia
incubated continuously in the
model system . . . . . . . . . . . . . . . 43

 

Effect of extended incubation on
Conover loam soil or in the model
system . . . . . . . . . . . . . . . . . . 47

Accumulation of exudates by spores . . . . 48

Stimulation of germination by
spore exudates . . . . . . . . . . . . . . 52

The point in the germination of
g. victoriae at which fungistasis
becomes ineffective . . . . . . . . . . . . 58

 

Establishment of the microbial
nutrient sink in soil . . . . . . . . . . . 62

Role of the microbial nutrient

sink of soil in fungistasis . . . . . . . . 72
Fungistatic substances in soil . . . . . . 79
DISCUSSION . . . . . . . . . . . . . . . . . . . . 94
APPENDIX . . . . . . . . . . . . . . . . . . . . . 112

Modification of the paper disc
technique for assaying the

microbial nutrient sink of soil . . . . . . 112

Characteristics of the model system . . . . 116

LIST OF REFERENCES . . . . . . . . . . . . . . . . 120
v

LIST OF TABLES

Table

1.

Germination of Helminthbsporium victoriae
conidia (unwashed) incubated in the model
system leached with various media, in the
corresponding non-leached controls, on
natural soils, and on PDA . . . . . . . . . .

 

Germination of washed and unwashed
Helminthosporium victoriae conidia

incubated for 12 hr in the model

system, on saturated sand, on

natural Conover loam soil, and on PDA . . . .

Uptake of radioactivity by nutrient-dependent
conidia of Helminthosporium victoriae
incubated 12 and 24 hr on 1.0 ml of
concentrated 1"C-labelled spore exudate . . .

 

Quantities of nutrients exuded from

conidia of Thielaviopsis basicola,

Curvularia lunata, and Helminthosporium
victoriae incubated in sterile glass
distilled water for periods less than

those required for germination . . . . . . .

 

 

 

Populations of fungi, bacteria, and
actinomycetes in Conover loam and

the clay loam soil 24 hr after

remoistening and air-dried soils . . . . . .

Carbohydrates and amino compounds in

natural and sterile Conover loam and

the clay loam soil 1 day after

remoistening the air-dried soils . . . . . .

vi

Page

39

40

52

54

71

77

LIST OF FIGURES
Figure Page

1. The model system used to impose a diffusion
stress on fungal propagules and discs . . . . . 26

2. Exudation from 1"C—labelled conidia of
A) Helminthosporium victoriae,
B) Curvularia lunata, and
C) Thielaviopsis basicola incubated
for alternate periods in the model
system (leaching) and on buffer—saturated
glass beads (no leaching) . . . . . . . . . . . 42

 

 

 

3. Exudation from 1“C-labelled conidia of
figlminthosporium victoriae (unwashed)
incubated in the model system for 96 hr . . . . 45

 

4. Radioactivity in each of ten successive
10 m1 aqueous washings of ll‘C-labelled,
initially unwashed, Helminthosporium
victoriae conidia . . . . . . . . . . . . . . . 46

5. Conidial germination of A) Helminthosporium
victoriae and B) Helminthosporium sativum
incubated on natural Conover loam soil or
in the model system (45 m1 buffer/hr) for
up to 56 days before transfer to either
buffer solution or PDA for an additional
12 hr incubation . . . . . . . . . . . . . . . 49

 

6. Diagram of the model system, modified to
measure the uptake of l*C-labelled spore
exudates by non-radioactive spores . . . . . . 50

7. Germination of Helminthosporium victoriae
(A and B) and Curvularia lunata (C and D7
conidia incubated in the model system with
either water, natural exudate from both
fungi, or their corresponding synthetic
exudates, and on sand saturated with each
of the media . . . . . . . . . . . . . . . . . 57

 

 

Figure

8.

10.

ll.

12.

13.

14.

15.

Germination of Helminthosporium victoriae
conidia (unwashed) incubated on water-
saturated sand for 0-6 hr prior to transfer
to the following for 4 hr additional
incubation: a) Conover loam soil,

b) the clay loam soil, c) the model

system, or d) water-saturated sand . . . . .

Loss of 1"C-glucose from amended paper discs
incubated on sterile and natural Conover
loam soil. Discs were amended with 90 ul

0.05 (°-°), 0.005 (---), and 0.0605% (———)
glucose solutions each containing 0.05
“Ci/ml I I I I I I I I I I I I I I I I I I I

Loss of ll'C-glucose from paper discs
amended with 90 ul 0.0005% glucose

solution (0.05 uCi/ml) when incubated

on the sterile and natural clay loam soil .

Evolution of 1"C02 from natural and sterile
Conover loam and the clay loam soil plates
following application of 2 uCi ll‘C-glucose
(72 ug) to the soil surface . . . . . . . .

Evolution of 1"C02 from Conover loam and the
clay loam soil suspensions (soil:water, 1:2,
w/v), amended with concentrated exudate from
1|‘C—labelled conidia of Helminthosporium

victoriae . . . . . . . . . . . . . . . . .

Evolution of ll‘COZ from Conover loam and the
clay loam soil suspensions (soil:water, 1:2,
w/v), amended with 0.05 uCi 1“C-glucose . .

Accumulation of radioactive exudates from
1"‘C—labelled Helminthosporium victoriae
conidia by bacterial isolates from Conover
loam soil (A and B) and the clay loam

soil (C and D) . . . . . . . . . . . . . . .

Loss of 1“C-glucose from amended agar discs
incubated on water-saturated sand, in the
model system (10 m1 water/hr), or on sterile
and natural Conover loam and the clay loam
soil . . . . . . . . . . . . . . . . . . .

viii

Page

61

64

65

66

68

69

70

73

Figure

16.

17.

18.

19.

Conidial germination of Thielaviopsis

‘basicola on discs, initially amended
to contain 0.1% glucose, after incubation for

up to 24 hr A) in the model system

(10 ml water/hr), and on natural

Conover loam and the clay loam soil,

and B) on water—saturated sand and
sterile soils. Germination is

expressed as the percent of that on
untreated glucose-amended discs.

Amended agar discs containing 1"C--glucose
(ca. 3,000 cpm) were incubated exactly as
non-radioactive discs to determine the
percent glucose remaining . . . . . . . .

Germination of Helminthosporium victoriae

 

conidia on purified agar discs that were
incubated on (———) or over ("') the
natural clay loam soil for up to 24 hr
before inoculation, and then either

left on soil (on soil) or transferred

to a petri dish (empty dish) for an
additional 8 hr . . . . . . . . . . .

Germination of ThielaviOpsis basicola
conidia on purified agar discs incubated
in the model system (10 m1 water/hr), on
Conover loam soil, and on the clay loam
soil, then removed and amended to contain
A) 0.1% or B) 0.005% glucose before
inoculation . . . . . . . . . . . .

 

Germination of A) Neurospora tetrasperma

 

ascospores and B) Helminthosporium victoriae

 

conidia on polyacrylamide gel discs that were

incubated on (———) or over (~--) natural clay

loam soil for up to 24 hr before inoculation,

and then either left on soil (on soil), placed
on a glass microscope slide over soil (over

soil), or transferred to a petri dish
(empty dish) for an additional 8 hr . .

ix

Page

76

81

83

85

Figure

20.

21.

22.

23.

24.

25.

26.

Germination of Helminthosporium victoriae
conidia on polyacrylamide gel discs that

were incubated on natural Conover loam

soil (-—-) or in the model system (---)

for up to 24 hr before inoculation, and

then either left in place (on) or

transferred to a petri dish (empty

dish) for an additional 8 hr . . . . . . . . .

 

Determination of pH, with pH indicator

paper, of 50 ul glass distilled water
incubated for 1 hr on purified agar

discs which were previously incubated

for 24 hr on Conover loam soil and

the clay loam soil . . . . . . . . . . . . . .

Germination of Helminthosporium victoriae
conidia in solutions and on purified agar
discs containing 0.1 M potassium

phosphate buffers at various pH values . . .

 

Germination of Thielaviopsis basicola

conidia in solutions and on purified

agar discs containing glucose and 0.1 M
potassium phosphate buffers at various

pH values . . . . . . . . . . . . . . . . . .

 

Germination of Thielaviopsis basicola
conidia on glucose amended agar discs
incubated over Conover loam soil, the
clay loam soil, and water-saturated sand . . .

 

Loss of glucose from Whatman and Schleicher
and Schuell (S & S) paper discs incubated

on natural and sterile Conover loam soil.

Each disc initially contained 90 ul of a

0.05% glucose solution, with the amount
remaining determined by the Glucostat

reagent . . . . . . . . . . . . . . . . . . .

Loss of glucose from Millipore membrane
filters (13 mm diam, 0.22 n) to which 15 ul
of a 0.005% l“C«-glucose solution (5.0 uCi/ml)
had been added prior to incubation on
natural and sterile Conover loam soil.

Discs were dissolved in scintillation

fluid and counted . . . . . . . . . . . . .

Page

87

89

91

91

115

117

INTRODUCTION

Previous work from this laboratory has shown a
highly significant direct correlation between the ability
of fungal spores to germinate in glass distilled water
and on soil (54). Conidia of four fungi, however, were
exceptions in that they germinated to various degrees
in water, but were completely inhibited on soil. Eleven
other nutrient-independent yet fungistasis-sensitive
fungal propagules were later identified (43). Germi—
nation of these propagules was suppressed when gently
leached with water or dilute buffer solution in a
model system thought to imitate the microbial nutrient
sink of soil. Other work from this laboratory demon-
strated that nutrients exuded from this type of
propagule were rapidly utilized by soil microorganisms
(59). Moreover, incubating conidia of Helminthosporium

victoriae or Glomerella cingulata with washed cells of

 

bacteria or actinomycetes inhibited germination (59).
The reduction in germination was not accompanied by
the production of detectable inhibitory substances.
All these results supported the hypothesis that the

removal of nutrients due to the microbial nutrient

sink prevented germination of nutrient—independent
propagules.

My research was undertaken with two primary
objectives, to gain a further understanding: a) of
the mechanism of the inhibition by the microbial
nutrient sink in soil, and b) of its significance
in relation to recently reported volatile fungistatic
factors (36). A clay loam soil from Colorado known
to produce a volatile inhibitor was used to compare
results with Conover loam soil, the soil used in all

previous fungistasis studies from this laboratory.

LITERATURE REVIEW

Fungistasis and mycostasis are the descriptive
terms given to the phenomenon whereby fungal propagules
fail to germinate in soil under conditions considered
favorable for germination. The phenomenon was first
described just over twenty years ago by Dobbs and
Hinson (22), and since then considerable research
effort has been directed toward understanding its
nature, not only because of its importance in soil
ecology, but because of the implication for control
of soil-borne plant pathogens. Fungistasis occurs in
most soils throughout the world, and can be annulled
by the following: i) sterilization of the soil, ii) the
addition of nutrients, iii) the proximity of fresh
organic matter, iv) the proximity of the rhizosphere or
spermosphere of plants, or v) long periods of air drying
the soil (63). There is agreement among researchers
that widespread fungistasis is microbial in origin, and
Dobbs termed the general phenomenon "microbial"
fungistasis to differentiate it from the few soils
where a permanent thermostable inhibition, that cannot

be overcome by nutrients, exists: the latter being

called “residual" fungistasis (18). The subject was
comprehensively reviewed by Lockwood in 1964 (63), while
a recent interpretive, but less thorough review was made
by Watson and Ford (91). The review presented here will
consider literature concerning the mechanism of
fungistasis, especially those papers directly related

to research reported herein.

Nutrient Requirements for

Eermination and Their
Availability in Soil

 

 

 

Before reviewing the various mechanisms proposed
for the inhibition in soil, a brief consideration of the
nutrient requirements for spore germination and the
availability of such nutrients in soil might provide a
framework upon which further discussion can be developed.

From the outset it must be assumed that any
constituitive dormancy requirements have been fulfilled.
Germination of fungal propagules is an active metabolic
process which requires energy, and two extremes are
found in fungi with respect to exogenous nutrients:

a) those completely dependent upon, and b) those
completely independent of them (54). A borderline
character is exhibited by some species, in that within
a population of spores a low percentage will germinate
without an exogenous substrate. For many of the

nutrient-dependent spores, glucose alone may satisfy

the energy requirements for germination, while others
may need some specific carbon source or form of nitrogen
(24,28,34,49,55,59).

Soil is characterized by an abundant and diverse
microflora (4,28,32), which is normally inactive as
indicated by the minimal respiration rates measured for
fallow unamended soil (27,59). Even the addition of
the fungicide pentachloronitrobenzene to unamended soil
did not further reduce respiratory activity (27). In
addition, the soil microflora may be responsible for
only a portion of the total respiration measured, the
balance owing to microfaunal activity. Gray and
Williams (31) conclude, based on calculations of the
yearly input of energy substrates to soil and the
biomass which must be supported, that most microbes
existing in soil are present as resting or survival
forms. They and Babiuk and Paul (5) also point out
that the amount of energy available in soil on an annual
basis, taking into account that required for maintenance
of existing populations, would allow cells to divide
only a few times a year. Thus respiration in unamended
soil likely represents maintenance metabolism, not
growth and reproduction. The soil is thus, an envi-
ronment in which energy yielding substances are in

chronically short supply. This idea is further supported

by the observation that no nutrient-independent fungal
propagules, which are also fungistasis—sensitive, have
ever been identified. Propagules representing the
other three possible combinations of these characters

are abundant.

Nature of Soil Fungistasis

As early as 1949 it was suggested that the
inability of most human and animal pathogens to survive
for more than a few days in soil was due to their
failure: a) to obtain proper nutrients, b) to compete
with indigenous microorganisms, or c) to withstand
antibiotics produced by soil microbes (72). Since
the original report by Dobbs and Hinson numerous
mechanisms have been proposed to explain the nature of
fungistasis. Physical factors of the soil environment
such as hydrogen ion concentration and oxidation-
reduction potential have been ruled out as playing a
significant role. There are at present two hypotheses
regarding the nature of the phenomenon: i) the nutrient
hypothesis where nutrients needed as an energy source
for germination are deficient due to microbial activity
in the soil, and ii) the inhibitor hypothesis which
postulates the existence of inhibitory substances of
microbial origin. The latter includes recently reported

volatile fungistatic factors.

The inhibitor hypothesis has been championed by
Dobbs and co-workers who proposed that the observed
inhibition was caused by a soluble yet unstable substance
present in the soil solution (22,23). However, Jackson
(44) demonstrated that soils remained fungistatic even
after extensive aqueous leaching, and believed that
fungistasis was caused by resistant organic matter that
leaching could not remove. Lingappa and Lockwood (58)
found that decomposition products of lignin could
inhibit spore germination, but they did not suggest
that these compounds were responsible for the widespread
inhibition. Lockwood (62) initially proposed that

antibiotics produced by Streptomyces might be the

 

inhibitory factor, but later discounted the idea since
such substances could not be extracted from soil (57).
Brian (12) after a thorough review of the literature on
antibiotics in soil concluded, that they are not produced
in unamended soil because of insufficient energy, and
therefore probably are not responsible for fungistasis.
Both degradation of resistant organic matter and
synthesis of antibiotics assume active metabolism by
appropriate microorganisms, but as already indicated the
energy for such activity is not available. Moreover,
Lingappa and Lockwood (57) point out that sites in soil

where antibiotics are most likely to be synthesized

I r:

(i.e. adjacent to fresh organic debris or the rhizosphere)
are microsites favorable for spore germination. Even
sand without organic matter was found to be fungistatic
(63).

Evidence has since been obtained to show that
fungal propagules, notably those which do not require
exogenous nutrients for germination, themselves provide
energy for microbial activity in soil (29,59). Spores
and spore washings markedly increase microbial popu-
lations, as well as oxygen uptake in soil (29). Gilbert
and Linderman (29) report an increase in bacteria only,

in the "mycosphere" of Sclerotium rolfsii sclerotia.

 

However, there could have been a corresponding increase
in the biomass of fungi and actinomycetes adjacent to
sclerotia, as the result of germination, but before
sporulation. They also reported a shift toward greater
numbers of streptomycin-tolerant bacteria, and suggest
that such a shift represents a response to a selection
pressure because of elevated antibiotic concentrations
in the "mycosphere" soil. No attempt, however, was
made to directly demonstrate the presence of antibiotics;
moreover, streptomycin is readily degraded and adsorbed
in soil (12).

The similarity between some of the morphological
changes in staled fungal cultures and on soil led Park

(70) to suggest that staling products may cause

fungistasis. Staling products do exist in culture, but
their presence in soil has not been shown (16,17,75).
Moreover, the morphological changes observed by Park,
also occurred when the fungi were deprived of nutrients
(34,42). If staling products or antibiotics play a
role in fungistasis, then reinfested sterilized soil
should be an ideal environment for their production.

It is well documented that reinoculation of
sterilized soil with a pinch of natural soil or any one
of a number of microorganisms restores inhibitory
properties to the sterile soil (33,64,84). Griffin (33)
found that both microorganisms antagonistic and non-
antagonistic in culture restored fungistasis, and
concluded that the accumulation of staling products was
responsible for the inhibition by the non-antagonists.
Each of sixty-seven microorganisms tested by Lingappa
and Lockwood (64), some of which produced antibiotics
in culture, returned some level of inhibition to auto—
claved soil, even when reinoculated into sterilized soil
which was first thoroughly washed to remove nutrients
which would be used for antibiotic or staling product
synthesis. Filter—sterilized aqueous extracts from
reinfested topsoil were not inhibitory, but some
contained carbohydrates which can annul fungistasis;
thus it was not until after extracts from reinfested

subsoil were shown to be both non-toxic and nutrient-free

10

were Steiner and Lockwood (83) able to conclude that
fungistatic substances were not involved. Their
extensive reexamination of the mechanism of fungistasis
in reinfested soil provided no evidence that inhibition
was due to anything other than the depletion of
nutrients released upon sterilization, and the main-
tenance of a nutrient-deprived environment by the
reinoculated organism. They further pointed out that

if inhibitors of microbial origin accumulated in
reinoculated soils, then the rate of glucose utilization
in soil should decrease with time of incubation. Their
data and that of others (3) clearly showed that the rate
actually increased.

Both volatile (17,75) and non-volatile (l6)
inhibitors from fungal cultures have been isolated and
identified. However, if the concentrations necessary
to inhibit fungal spore germination in vitro are the
same in soil, then such compounds should have been easily
extracted from reinfested sterilized soil, if not from
natural soil. The same considerations apply to the work
of Chinn (14), who found that sand amended with only
0.005% natural soil was still inhibitory, and stated
that the fungistatic principle was over ZOO-times more
concentrated than needed.

There are numerous reports of inhibition of spore

germination in non—sterile extracts and diffusates (23,

11

34,76,86). However, the effect is negated partially or
completely when those extracts are sterilized by heat

or filtration (23,34,76,86). Brown (13), providing the
first evidence of soil bacteriostasis, also showed that
natural soil and non-sterile extracts retarded growth of
bacteria, and that the inhibition was removed by filter
sterilization. Despite this evidence to the contrary,
Brown still proposed that bacteriostasis was caused by
bacteriostatic factors of biological origin. By
extracting garden and forest soils and bioassaying the
extracts under nitrogen, Dobbs and Carter (20) were

able to retain some of the inhibition in sterile extracts.
Attempts to obtain inhibitory sterile extracts from
other soils were unsuccessful (54,57,63,83). However,
in a recent preliminary report, Basmith and Vaartaja

(8) found that both sterile and non-sterile extracts of

natural soil restricted growth of Pythium ultimum. The

 

inhibition was overcome by heating or adding nutrients
to the soil, or passing the extracts through filter
paper. Interestingly, those extracts, inhibitory to

P. ultimum, stimulated Fusarium oxysporum. When

 

Vaartaja (90) fractionated aqueous soil extracts by
molecular sieve chromatography, certain fractions
inhibited while other fractions stimulated or had no
effect on mycelial growth. Estimates of inhibitor and

stimulator molecular weights ranged from 100 to 80,000

12

and 2,000 to 50,000, respectively. If inhibitory
fractions were not contaminated and further research
demonstrates the inhibition of germination of a wide
range of fungal spores, it will be the first strong
evidence for a non-volatile inhibitor in soils.

Agar discs incubated on soil often fail to
support spore germination. This inhibition has been
taken to represent the diffusion of a soluble inhibitor
from soil into the agar. Using gel electrophoresis
techniques on fungistatic agar, Weltzein found an
inhibitory band migrating toward the anode (92). Such
results may instead represent the migration of nutrients
establishing a zone depleted of energy sources or the
accumulation of ions directly or indirectly preventing
germination. Griffin (34) incubated agar in contact
with soil at 3 C to lower microbial activity and to
reduce the loss of essential nutrients. However, Ko
and Lockwood (54) clearly showed that nutrients diffuse
into soil from agar rapidly at either 24 and l C. More-
over, inhibitors could not be extracted from agar
previously in contact with soil (57).

The occurrence of residual fungistasis has been
reported in a few soils. Dobbs and Gash (21) attributed
the residual inhibition in calcareous dune sands and
limestone subsoils to inorganic iron and CaCOB, respec-

tively. Ko and Hora (50,51) recently reported that an

13

aluminum ion was the fungitoxin in certain cultivated
acidic soils in Hawaii. Liming these soils to a neutral
pH annulled the fungicidal effect. Moreover, aqueous
solution of Al from AlCl3 or A12(SO4)3 at concentrations

equal to that in soil inhibited germination of Neurospora

 

tetrasperma ascospores, which are sensitive to the
residual fungistasis but not to the more common microbial
fungistasis.

Volatile substances have also been suggested as
the cause of soil fungistasis (l9): Lingappa and Lockwood
(57) could not demonstrate their presence in soil.

More recently, Kouyeas and Balis (7,48) provided indirect
evidence that volatile fungistatic factors may be
involved in the phenomenon. Remoistened air-dried soil
was fungistatic when suspended over water, but only
partially so when incubated over either silver nitrate
or mercuric perchlorate solutions, which were thought

to inactivate the volatile substance by binding with it
at points of unsaturation. Toxic vapors from these
solutions may have partially sterilized the surface of
the soil, releasing nutrients stimulatory to spores of
the test fungus. Balis (6) tentatively identified allyl
alcohol as the volatile inhibitory factor found in water
condensates collected from the head space over soil.
This conforms to earlier assumptions that the volatile

was a water soluble unsaturated carbon compound (7).

_14

Allyl alcohol at 10 ppm inhibited conidial germination

of Penicillium chrysogenum in vitro, and the inhibitory

 

effect was overcome by nutrients.

Hora and Baker (36) provided the first direct
evidence for the existence of a volatile factor in
certain remoistened air-dried alkaline soils. The
inhibitory effect of the volatile generally increased
with increasing soil pH. When the sensitivity of the
bioassay technique was improved, through exhaustive
leaching of the test agar discs, significant volatile
inhibition was detected from acidic soils as well, even
25 days after remoistening (77). Furthermore, the
inhibition of Helminthosporium sativum conidia by the
volatile factor was nearly equal to that when membrane
filters bearing conidia were incubated directly on the
soils.

The most inhibitory soil, one particular alkaline
clay loam, was used in more extensive studies. Auto-
claving did not eliminate the volatile factor (38,52),
and spores incubated directly on the surface of either
sterile or nutrient-amended soil failed to germinate,
fulfilling the criterion for residual fungistasis (52).
When this soil was treated with hydrochloric acid, it no
longer released the volatile inhibitor (52). The
volatile was inactiviated by passage through water and

absorbed by activiated charcoal (76), however, the

 

 

15

presence of conidia were required in the water traps
during volatile extraction to demonstrate the inhibition
of conidial germination (37). This suggests that spores
have a strong affinity for the inhibitor. Moreover,
Hora and Baker (39) found that a volatile fungistatic
factor can be generated non-biologically in soil,
showing that its release was dependent upon alkaline
pH's. Reinoculating the sterilized clay loam soil with
actinomycetes increased volatile production, while
bacteria and fungi removed its effect; when assayed 2
weeks after reinfestation (38). Furthermore, when this
soil was amended with chitin, glucose, cellulose, or
lignin, only chitin increased inhibition by a volatile
factor. Interestingly, reinoculation of sterilized

soil by microorganisms or the addition of an organic
amendment increased the level of fungistasis compared
with that in the natural clay loam soil, as determined
by the direct method. This suggests that the microbial
component of fungistasis was increased by all the
treatments, even though their affect on volatile
production differed. The stimulation of volatile
generation by chitin amendment and the observation that
they were only detected above those Streptomyces cultures
with earthy odors, suggests that actinomycetes were
involved in its production (38). They may be involved

in reinoculated sterile soil and in chitin-amended soil,

16

but their involvement in natural unamended soil seems
unlikely because of nutrient deficiency. Germination
and growth of streptomycetes, like that of fungi and
bacteria, is also suppressed on natural soil (61). To
this reviewer these results suggest that the volatile
inhibitor in the clay loam soil from Colorado is not
of biological origin, but that its generation may in
part be mediated by actinomycetes when sufficient
energy for their activity is available.

Smith (79) recently reported that microbially
synthesized ethylene was the main inhibitor of fungal
propagule germination in soil. This volatile was the
only material detected in fifty Australian soils at
concentrations high enough to be biologically active.
Such soils were, however, oven dried at 40 C for 24 hr
prior to rewetting and subsequent sampling for ethylene.
Ethylene concentrations equal to that detected in some
of the rewetted soils inhibited germination of

Sclerotium rolfsii sclerotia and Helminthosporium

 

 

sativum conidia. Fungistasis was reduced when a water-
saturated air stream was swept over the surface of these
soils. He proposes that nutrients are the main stimu-
lators in soil, and that the balance between them and
ethylene determines whether soil is fungistatic at any

given time.

17

If volatile inhibitors are as universal as Baker
and co-workers, and Smith suggest, it is a wonder that
Dobbs and Hinson did not detect them in their "gap
traps" or on the margins of cellophane folds extending
out beyond soil, which they used for control germination.
In reference to ethylene, it has been demonstrated that
this compound is only produced in soil by microbes when
oxygen concentrations are less than 2 percent (1), and
is rapidly degraded under aerobic conditions (80).

Lockwood (63) proposed a nutrient deficiency
hypothesis to explain the widespread microbial fun-
gistasis. Some of the evidence presented by K0 and
Lockwood (54) to support the nutrient hypothesis
appeared previously in this review. Analysis of sterile
soil showed nutrients were present in sufficient quan-
tities to support germination of nutrient-dependent
spores, whereas those in natural soil were not. More
importantly, they demonstrated that soil microorganisms
can rapidly remove nutrients around spores, not only
creating, but maintaining an environment deprived of
energy substrates. Spores of nutrient-dependent fungi
require an exposure to an energy source throughout the
germination process in order to produce a germ tube
(82,93). Thus, unless there is enough energy in the
vicinity of the spore to satisfy both its germination

requirements until germ tube emergence, as well as that

18

of the surrounding soil microbes, the spore fails to
germinate. Hsu and Lockwood (41) also demonstrated
that the inhibition of fungi by some streptomycetes
in culture was due to nutrient deprivation.

The extension of the nutrient deprivation
hypothesis to explain inhibition of nutrient-independent
yet fungistasis-sensitive spores (54), has been confirmed

by others. Germination of Verticillium albo-atrum micro-

 

sclerotia was suppressed when the propagules were contin-
uously leached with water on a modified leaching system,
but not when leaching was discontinued (25). Similar
results were reported by Adams et a1. (3) for chlamydo-

spores of Fusarium solani f. sp. phaseoli. These

 

propagules germinated freely when leached with glucose
or asparagine solutions. Germination of the nutrient-

independent conidia of Botrytis cinerea was inhibited

 

in the phyllosphere (10), and also in a model system
(87) similar to that used by K0 and Lockwood (54).
Suspensions of phyllosphere bacteria were inhibitory
to conidia, while sterile filtrates of such mixtures
were stimulatory.

Fungistasis can be thought of as a survival
mechanism, in that it prevents the spontaneous germi-
nation of fungal propagules in the absence of a suitable
substrate. Therefore, it seems unreasonable that a

mechanism for survival based on the continuous

19

expenditure of energy for production of inhibitory
substances by soil microorganisms, would have evolved
in a highly competitive environment where energy-
producing substrates are in chronically short supply.
In an environment where conservation of limited energy
reserves is necessary for survival, a passive rather
than an active mechanism for dormancy seems more

reasonable and consistent with reported observations.

METHODS AND MATERIALS

Maintenance of Fungi
and Bacteria

 

 

The following fungi were maintained on potato-

dextrose agar (PDA): Helminthosporium sativum Pam.,

 

King and Bakke, Helminthosporium victoriae Meehan and

 

Murphy, Neurospora tetrasperma Shear and Dodge,

 

Sclerotium cepivorum Berk., and Thielaviopsis basicola

 

 

(Berk. and Br.) Ferr. Curvularia lunata (Wakker)

 

Boedijn was kept on V-8 juice agar (per liter: 200 m1
V-8 juice [Campbell Soup Co.], 2.0 g CaCOB, 20 g agar).

Bacteria isolated from soil dilution plates on
soil extract agar (71) were cultured on a nutrient agar
(per liter: 5 g peptone, 5 g yeast extract, 1 g glucose,
5 g K2HPO4, 20 g agar).

Production, Collection, and
Preparation of Propagules

 

 

Conidia of the helminthosporia were produced by
Lukens‘ method (65). Shake cultures were grown in 25
ml modified Fries No. 3 basal medium (74) with glucose
substituted for sucrose for 5-7 days. The mycelium
produced was comminuted in a Servall Omnimixer for 2

minutes. The resulting hyphal fragments were washed

20

21

by suspending them in sterile 0.02 M phosphate buffer
(pH 6.4) after each of three refrigerated centrifu-
gations. A 2.5 m1 aliquot of the final suspension was
Spread over sterile filter paper in a 9.0 cm diam petri
dish. Sporulation commenced within several days under
continuous fluorescent light (four, 40 watt, cool white,
tubes). Conidia were not used until the filter paper
was dry, and kept for up to 6 months.

Neurospora tetrasperma was cultured on plates
of PDA and ascospores were obtained by washing the
ejected spores from the inside surface of petri dish
lids with sterile glass distilled water. Ascospores
were separated from any conidia of the fungus by
sedimentation in sterile water. After washing three
times by centrifugation the spores were stored in
sterile glass distilled water at 4 C. Ascospores
were heat activated for use at 58 C for 20 min in a
water bath (30).

Conidial suspensions of T. basicola were prepared
by adding glass distilled water to PDA-slant cultures
and agitating on a Vortex Genie mixer. Conidia of
Q. lunata and of the helminthosporia produced on petri
plates of V-8 juice agar and filter paper, respectively,
were rubbed free with a glass rod after irrigating the
plates with glass-distilled water. 9. lunata suspensions

were then passed through a 325-mesh sieve (44 u) to

22

remove hyphal fragments. The suspensions were washed 3
times by refrigerated centrifugation.

Sclerotia of S. cepivorum were collected from

 

PDA plates covered with non-coated cellophane. The
sclerotia were treated in 1% sodium hypochlorite for
1 min to kill attached hyphae and then washed as
previously described.' I

When radioactive spores were required the culture
medium was amended with the following quantities of ll'C-
glucose (D-glucose-U-C-l4, 20 hour grade, specific
activity 50 mCi/mM, Calatomic, La Jolla, Calif.): a) H.
victoriae, l uCi/ml Fries basal medium; b) T. basicola,
10 uCi/ml PDA or 50 uCi/slant; c) g, lunata, 25 uCi/plate;
and d) g. cepivorum, 25 uCi/plate. The same procedures
outlined above for collection, etc., were followed.

Unwashed conidia of H. victoriae were usually

 

collected by rubbing a Millipore membrane filter or

the dull side, not shiny, of a Nuclepore membrane filter
on the surface of dry filter paper cultures of the
fungus. When large quantities of unwashed conidia were
required, they were collected with a sterile Tervet

cyclone spore collector.

Spore Exudates

 

Conidia (5-70 X 103/ml) were suspended in 0.5-1

liter sterile glass distilled water and incubated at

23

25 i 2 C with stirring for a period of time short of
germination (3 hr for Q. lunata, 1.5 hr for H. victoriae).
Conidia of the nutrient-dependent fungus T. basicola

were incubated similarly for 20 hr. Exudate was
collected and sterilized by passage through a 0.22 u
Millipore membrane filter. One-half (ca. 1 liter) of

the collected exudate was taken to dryness in a flash
evaporator at 45 C and then redissolved in 10 ml sterile
glass distilled water. The other half was used in
germination experiments.

Carbohydrates in the concentrated exudates were
determined using the anthrone reagent (69), while glucose
was measured by the Glucostat reagent (Worthington
Biochemicals Corp., Freehold, New Jersey). The ninhydrin
method (68) was used to determine amino compounds.

Synthetic exudates were prepared from a mixture
of glucose and casein hydrolysate (acid hydrolysed,
vitamin free, Nutritional Biochemicals Corp., Cleveland,
Ohio). The concentration of each was based on the
respective glucose and glycine equivalents of total
carbohydrates and amino acids in natural exudates.

Exudates collected from 1"C-labelled conidia
were reduced to approximately 1/80 their original volume
in a flash evaporator at 45 C before being filter—

sterilized again, and stored until use at 4 C.

24
Source and Preparation
of Soils

Conover loam soil collected from the Michigan
State University Botany and Plant Pathology Farm
possessed the following characteristics: pH 6.7,
organic matter 3.8%, water holding capacity (WHC)
42.7%, clay 7.5%, silt 42.8%, and sand 49.7%. A clay
loam soil from Colorado, reported to produce a volatile
fungistatic factor (36), was supplied courtesy of Dr.
Ralph Baker, Colorado State University. Hereafter, this
soil will be referred to as the clay loam soil. The
clay loam soil was collected from a low lying swale
where water frequently collected then evaporated. Its
characteristics are as follows: pH 8.6, organic matter
1.1%, lime 5.9%, NHZ-N 54.5 ppm, Nog—N 34.7 ppm, 13205
140 lbs/acre, K20 865 lbs/acre, and conductivity (salts)
48.0 mhos/cm. All soils were screened through a 10-
mesh sieve and stored air dry in plastic bags in the
laboratory. Subsoil collected from beneath Conover
loam soil was similarly screened but stored at ca. 8%
moisture in plastic bags.

For use, the Conover loam and the clay loam
soils were brought to 27 and 33% moisture levels,
respectively, by thorough mixing with distilled water.
These levels represented an equivalent moisture tension

of 0.06 bars in each soil as measured with a tensiometer

25

(Soilmoisture Probe, Cat. No. 2100, Soilmoisture
Equipment Corp., Santa Barbara, Calif.). In most
experiments the moist soil was incubated for at least
24 hr prior to use. Soil plates were prepared by
placing moist soil in petri dishes and smoothing the
surface with a stainless steel spatula. Plates of
sterile soil were made in glass petri dishes and
autoclaved at 121 C (15 psi) for at least 30 min.
Moisture content of sterile soil was returned to the
level prior to autoclaving by the addition of sterile
glass-distilled water, and then the surface was
smoothed again with a sterile spatula.

Model System for Imitating the
Microbial Nutrient Sink of Soil

 

 

A model system previously described (43) was
used to impose a diffusion stress upon spores or agar
and polyacrylamide gel discs incubated on an acid-
washed silica sand or glass microbead bed (Figure 1).
Discs and spores were separated from the bed by either
a Nuclepore (0.4 u) or Millipore (0.22 u) membrane
filter. For most experiments the model system was
modified by removing the needle valve and connecting
a 1/8 in O.D., l/16 in I.D. plastic (Tygon) tube between
the stem of the separatory funnel and the inlet tube of
the leaching dish, then accurately metering the flow

rate with a precision peristaltic pump (Manostat

26

      
 
  

  

STERILE
MEDIUM

'STOPCOCK

 

J
or -NEEDLE VALVE

l—a' ......

 

 

 

' ..... l
SAND on GLASS 1 r
MICROBEADS
MEMBRANE FILTER a
BEARING SPORES
LEACHINGS
~1-

Figure l. The model system used to impose a diffusion
stress on fungal propagules and discs.

27

Cassette Pump, Manostat, New York, N.Y.). Sterile
glass—distilled water or a dilute buffer solution
(0.025 M potassium phosphate buffer, pH 7.0) were
generally used as the leaching media. The term
‘buffer solution‘ henceforth will refer to this buffer
unless otherwise specified. Both natural and synthetic
spore exudates were also used. In experiments designed
to evaluate the effect of mineral nutrients on germi-
nation in the model system Pfeffer solution (87) without
trace element supplement (per liter: 0.8 g Ca(NO3)2,
0.2 g MgSO4, 0.2 g KNO3, 0.1 9 KCl, and 0.2 g KZHPO4)
and subsoil extract were used. Subsoil extract was
prepared as follows: 1.0 Kg natural subsoil was
vigorously stirred with 1.0 liter distilled water for
30 min then filtered through Whatman No. 1 filter paper
and finally filtered sterilized using a 0.22 u Millipore
membrane filter.

When non-leaching conditions were required as
a control, silica sand or glass microbeads in a petri
dish were saturated with the same aqueous medium that
was used in the model system.

Metabolism of Glucose and Sppre
Exudates by Soil Microorgafiisms

 

Metabolism of 1"C-glucose was measured by two
methods: a) moist soil (50.0 g) with a smooth surface

was prepared in the bottom of a ground-glass weighing

28

dish (90 X 15 mm). The lid was fitted with inlet and
outlet tubes, and moist air was passed over the soil
surface at a rate of 3 liters per hr. Two uCi of
1"C-glucose was placed on the soil in ten 2 ul drops
with a microsyringe. Air from the soil chamber passed
through a drying tube of anhydrous calcium sulfate
before respired carbon dioxide was trapped by bubbling,
via a capillary pipette, into 2.0 ml ethanolamine
solution (methyl cellosolve: ethanolamine, 7:3, v/v)
contained in a scintillation vial. At predetermined
intervals the pipette was transferred to additional
vials containing ethanolamine solution. Fifteen ml
modified Bray‘s liquid scintillation fluid (11) was
added to each vial and the samples counted in a Packard
Tricarb Spectrometer. b) Soil suspensions (1.0 g air-
dried soil plus 1.0 ml H20) were incubated for 1 hr at
25 i 2 C before 2.0 uCi ll’C-glucose and an additional
1.0 ml of water were added. Air was bubbled through
the suspension using a capillary pipette, and the
respired 1"C02 was trapped and measured as described
above. To measure respiration of concentrated radio-

active exudate of H. victoriae conidia, 1.0 m1 of the

 

exudate was added to the soil suspensions after the 1 hr
incubation. Application of 1"C-glucose to sterile soils

or soil suspensions served as controls for both methods.

29

In experiments measuring the uptake of radio-
active spore exudate by bacteria, the bacteria were
grown in nutrient broth (nutrient agar minus the agar)
for 3—4 days on a shaker. Before use the cells were
washed by centrifugation in buffer solution and the
concentration of cells in the final suspension adjusted
to A500==0.40 in a B & L Spect 20. One ml of concen-
trated exudate from 1"C—labelled conidia of H. victoriae
was added to an equal volume of the adjusted cell
suspension. At zero time and specified time intervals,
0.30 ml was removed and placed on a Nuclepore membrane
filter. The excess exudate was removed by suction and
the cells were washed with 10 ml cold 0.01% Tergitol
TMN (non-ionic surfactant) followed by 10 m1 glass
distilled water. Solutions without bacteria but
similarly handled served as controls. Membranes and
cells were placed in vials containing scintillation
fluid and counted.

Paper Disc Technique for

Assaying thgiMicrobial
Nutrient Sink of Soil

 

 

Paper antibiotic assay discs (4 in diam, Carl
Schleicher and Schuell Co., No. 470-E) were amended with
90 ul 0.05, 0.005, and 0.0005% glucose solutions each
containing 0.05 uCi 1“C—glucose/ml using a microsyringe

fitted with a Chaney adapter. Amended discs were then

30

placed directly on the soil surface and incubated at
24 C for various periods of time. Upon removal, discs
were immediately dried in an oven (100 C), and then
combusted in a Nuclear-Chicago Semi-Automatic Sample
Combustor (Model 3152, Nuclear-Chicago, Chicago, I11.).
To facilitate combusion discs were first wrapped in
small pieces of Kimwipes tissue. Radioactive carbon
dioxide was trapped in 15 m1 ethanolamine solution
(ethanol:ethanolamine, 2:1, v/V) and an aliquot was
transferred to a scintillation vial containing 15 m1
liquid scintillation fluid and counted. The loss of
glucose on soil, expressed as the percent of radio-
activity initially present in the discs, was plotted

as a function of incubation time on the soil.

Fungistasis Assays

 

Various assays of fungal spore germination
required the use of agar discs. These were prepared
from 18 m1 of 2% purified agar (Difco, Detroit, Mich.)
poured into a 9.0 cm diam sterile plastic petri dish.
Discs were cut from the 2.5 mm thick agar with a 9.0
mm diam sterile cork borer. Nutrient amendments to
the agar were made prior to autoclaving, but sterile
1"C—glucose (usually 0.1 uCi) was added to the sterile
molten agar using a microsyringe. When discs of various

hydrogen ion concentrations were needed, 9.0 ml 4% molten

31

purified agar was mixed with 9.0 ml hot sterile 0.2 M
potassium phosphate buffer solutions and after cooling
discs were cut from plates of these mixtures.

In some experiments discs of polyacrylamide gel
were used. A modified procedure of BishOp et al. (9)
was used to prepare polyacrylamide gels. Eight ml of
an aqueous solution of 15% acrylamide and 0.75%
bisacrylamide were mixed with 8.45 ml glass distilled
water and 8.33 ml 3E buffer (0.12 M Tris, 0.06 M
sodium acetate, 0.003 M sodium EDTA adjusted to pH
7.8 with glacial acetic acid). Dissolved air was
removed from this mixture by vacuum before 0.02 ml of
N,N,N',N' tetramethylethylenediamine followed by 0.2 ml
of a freshly prepared 10% ammonium persulfate solution
were added. The final mixture was stirred quickly and
18.0 ml pipetted into a 9.0 cm diam plastic petri dish.
Once polymerized 9.0 mm diam discs were cut from the
gel with a cork borer and transferred to buffer solution
in which they were soaked for at least 7 days with
daily changes of buffer solution to completely remove
toxic materials. Before use the gel discs were auto-
claved and then collected in a sterile Buchner funnel.

To assay for fungistatic materials in the soil
agar or gel discs were incubated on the soil surface,
and for volatile inhibitors they were placed on sterile

glass microscope slides separated from soil by glass rods

32

or suspended over soil on the inside surface of petri
dish lids. After incubation the discs were bioassayed
in situ or after transfer to an empty petri dish.

Test fungi included conidia of H. victoriae and asco—

 

spores of N. tetrasperma. When conidia of T. basicola

 

were used, 50 ul sterile glucose solution was added to
each agar disc with a microsyringe 12 hr prior to
inoculation with the test organism. Once inoculated,
all discs were incubated for 12 hr at 24 C unless
otherwise stated. Discs incubated on the model system,
or on or above water-saturated sand served as controls.
An assay, henceforth called the radiochemical-
biological (R-B) assay, was developed to separate that
portion of the fungistasis phenomenon due to the
microbially-induced nutrient sink in soil from any
other fungistatic factor(s). Agar discs containing
0.1% glucose with and without 1“C-glucose label (ca.
3,000 cpm per disc) were incubated in the model system
(10 m1 water/hr) or on soil for periods of 0, 0.5, l,
2, 4, 8, 12, 16, and 24 hr. When they were removed
from the model system and soil radioactive discs were
dropped immediately into scintillation fluid and counted
to determine the amount of glucose remaining. Non—
radioactive discs were placed in a petri dish and each
was inoculated with one drop (ca. 0.04 ml) of a washed

conidial suspension of T. basicola. Inoculated discs

33

were incubated at 24 C for 12 hr before the conidia
were strained and killed with phenolic rose bengal

(per 100 ml distilled water: 1 g rose bengal, 5 g
phenol, 0.01 g CaCl2 and the solution clarified by
passage through filter paper). Standard discs of

known glucose concentrations were prepared from
solutions ranging from 0.1 to 0.00001% glucose. Both
germination and radioactivity were expressed as percent
of controls without incubation on soil or in the model
system.

In all fungistasis assay experiments discs
incubated on soil or in the model system were kept
sterile by a Nuclepore membrane filter (0.4 u) placed
between discs and the soil, or sand or glass bead bed.
Maintenance of sterility was routinely checked by
transferring some discs to PDA.

Radiochemical Purity of Glucose
in Agar Discs Incubated on Soil

In the R-B assay it was important to know
whether the radioactivity remaining in the discs
represented ll’C-glucose or some labelled metabolite
of the sugar. Radioactive agar discs containing 0.1%
glucose were incubated on soil for the intervals of
time used in the R-B assay. Each disc, initially,
contained ca. 200,000 cpm instead of the 3,000 cpm

per disc normally used in the R-B assay. After

34

removal from soil, 2 discs for each time interval were
shaken in 1.5 ml sterile glass—distilled water at 45 C
for 1 hr. Duplicate discs were also dissolved by adding
0.5 ml 4N HCl to the eluting water and incubating at
100 C for 20 min. One-tenth ml of each aqueous eluate
was streaked in a narrow band across the center 1 in
of a 2 in wide strip of Whatman No. 1 chromatography
paper or spotted on Silica Gel GF thin layer chroma-
tography (TLC) plates (250 u, Analtech Inc., Newark,
Delaware). The same volume of acid-dissolved discs
were spotted on TLC plates only. Paper chromatograms
Were developed by ascending chromatography in
n-butanolzn-propanol:water (4:1:1, v/v/v) with the
radioactive bands being detected on a Packard Radio-
chromatograph Scanner (Model 7201) with a 5 mm aperture.
Triplicate TLC plates were run in three different
solvent systems: a) t-butanol:n—propanol:water (4:1:1,
v/v/v), b) n-butanol:acetone:water (4:5:1, v/v/v), and
c) methyl ethyl ketone:acetic acid:water (3:1:1, v/v/v).
Radioactive spots were detected by exposing the plates
to No-screen Medical X-ray film (NS-2T, Eastman Kodak
Corp., Rochester, New York) for 6 wks in the dark at
6 C. Exposed film was developed in total darkness
for 15 min in the following developer at 21 C (per
liter: 1 g p-methylaminophenol sulfate, 75 g anhydr.

sodium sulfite, 9 g hydroquinone, 58 g sodium

35

carbonate-10 HOH, and 5 g potassium bromide). Developed
films were rinsed in water, fixed in commercial fixer,
for 15 min, and rinsed again before drying. Prior to
fixing, the films were previewed under a 25 watt No. 2
dark red safety light to identify any spots. Radio-
active spots were then compared with spots on the TLC
plates that were developed by spraying with 50% H2504

and then charring at 120 C.

Enumeration of Soil

"‘1' .
Microorganisms

Microbial populations in Conover loam and the

 

clay loam soil were estimated by the soil dilution plate
technique. Ten grams of air dried soil were remoistened
to previously mentioned soil moistures and incubated at
24 C for 1 day. Soil suspensions were prepared by
adding 95 ml sterile 0.85% NaCl solution to the soil

and shaking vigorously for 30 min on a Burrell wrist
action shaker. All further dilutions were made in
saline solution. One ml of the diluted soil suspensions
was swirled in a petri dish with ca. 15 m1 of molten

(43 C) soil extract agar containing 25 ppm PCNB (26)

and chitin agar (60) to determine numbers of bacteria
and actinomycetes, respectively. For fungi, 0.5 m1 of
the diluted suspensions were spread on solidified plates

of acidified NPX—PDA (84). A11 plates were incubated at

36

24 C for 2 wks before counts were made with a Quebec
colony counter.
All experiments were run in duplicate and

repeated at least once.

RESULTS

Effect of Washing Conidia,
and of Different Leaching
Media on Germination in
the Model System

 

 

 

Nutrient-independent spores germinate readily
in glass distilled water, while few do so when incu-
bated on soil, or when leached with water or buffer
solution in the model system. To test whether media
more like the natural soil solution could also

suppress germination, conidia of H. victoriae were

 

leached in the model system with Pfeffer's solution
and subsoil extract, as well as water and buffer
solution. An extract of natural subsoil was used

to approximate the mineral environment of the soil
solution. It contained only a small fraction of

the energy-yielding compounds present in extracts

of topsoil. For comparison, conidia were also
incubated on natural subsoil, Conover loam soil, and
PDA. The various mineral salt solutions were neither
more nor less effective in reducing germination than

water; likewise the solutions stimulated no greater

37

38

germination than water in the non-leached controls
(Table 1). Thus it appears that water alone and
buffer impose stresses on the spore like those
imposed by the soil solution.

Two common practices are to wash spores before
use and to incubate them on membrane filters. Three
membrane filters bearing both washed and unwashed

conidia of H. victoriae were incubated under several

 

conditions to determine the effects of each on
germination. In the model system and on soil the
unwashed conidia germinated better than the unwashed
spores (Table 2). Germination on Nuclepore membranes
and nylon mesh (28 u) on soil was not unlike that of
spores placed directly on the soil. Millipore
membranes were somewhat stimulatory under the
nutrient deprived conditions of soil and the model
system. Where possible, unwashed conidia of H.

victoriae were used in this work.

 

Exudation fromtSppres Incubated
for Alternate Periods when
Leached and not Leached

 

 

Materials are lost from fungal propagules,
via exudation, prior to the formation of a germ tube
(9,15,46,59). Ko and Lockwood (54) proposed that

the inhibition of spores requiring no exogenous

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w .cOHumcflEuoO

 

 

 

 

 

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.cOAusaom oouooaocw nvwz ooumuavwm ocmm

 

40

 

 

 

 

 

 

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41

energy for germination may be due to the rapid
utilization of exuded endogenous materials by micro-
organisms in the soil, or their physical removal
from the spore in the model system. Alternatively,
spores may be able to modify their pattern of
exudation under nutrient stress conditions so as to
conserve endogenous energy reserves.

To determine whether less materials are lost
from spores under diffusion stress conditions, 1"Cu
labelled conidia of H. victoriae, g. lunata, and T.
basicola on membrane filters were incubated alter-
nately on glass beads leached with buffer solution
and on buffer—saturated glass beads without leaching.
Conidia of the latter fungus requires exogenous
nutrients for germination. At various times the
exudates and the washings of the glass bead beds
were collected and taken to dryness, and the radio-
activity measured. One ml of a 0.3% thimerosal
solution was added to the exudates during drying
to prevent microbial activity.

More radioactivity was lost from the spores
when they were leached than when they were not
leached. This indicates that increased loss occurs
under diffusion stress conditions (Figure 2). Most

of the radioactivity was collected during the early

42

 

  
  
   

§ 2 '

 

    
 

V/////_

O

 

   
 
 

 

 

"° '%////////////j

    

////////

 

0 I

     
 

 

 

 

z? . 8 éfi .
: //
I72 W:

 

 

 

*— 1
O

was ’Sonava'I

IOWF
O

.1

I
\

FE

TMAE,

THHE,IH"I

Figure 2. Exudation from l“C-labelled conidia of A) Helminthosporium victoriae, B)

 

ching) and on buffer-saturated glass beads

 

Curvularia lunata, and C) Thielaviopsis basicola incubated for alternate

periods in the model system (lea

 

(no leaching).

43

incubation periods. A similar pattern of exudation

was obtained when sclerotia of H. cepivorum were

 

incubated for alternate periods. Interestingly, the
nutrient—dependent conidia of g. basicola exhibited
the same type of exudation pattern as the three
nutrient-independent propagules.

Attempts to measure exudation from spores
on soil were unsuccessful as the variation in the
amount of radioactivity in labelled spores applied
to several replicate membrane filters was greater
than the difference between untreated spores and
those incubated on soil.

Exudation from H. victoriae

Conidia Incubated Continuously
in the Model System

 

 

 

To determine the pattern of exudation under
continuous diffusion stress, 11+C-labelled conidia

(unwashed) of H. victoriae were incubated in the

 

model system leached with buffer solution for 96 hr.
The leachings were collected in 10 ml fractions
with a fraction collector (LKB Instruments Inc.,
Chicago, I11.). Each fraction was transferred to

a scintillation vial containing 1.0 ml 0.3%
thimerosal solution and taken to dryness on a hot

plate at 60 C, before the addition of scintillation

44

fluid, and then counted. After 12 hr several
fractions were combined to obtain enough radio-
activity to count.

Approximately 10% of the initial 65,558
cpm in the spores was lost after 96 hr. More than
90% of the 6,233 cpm lost was leached from the
spores within the first 12 hr, and over 99% by 24
hr (Figure 3). Most of the exuded material was
readily leached from the conidia and after its
removal exudation continued at a lower, almost
constant rate.

Similar results were obtained by passing
ten 10 m1 volumes of sterile glass distilled water,
in rapid succession, through 1"C-labelled (unwashed)

conidia of H. victoriae on a Millipore membrane

 

filter (0.22 u), and then determining radioactivity
in each of the ten dried filtrates (Figure 4).

Such treatment removed ca. 2.5% of the radio-
activity from the conidia. Unwashed conidia of

H. victoriae germinated ca. 10% when incubated

 

in the model system (50 ml water/hr) for 12 hr,
whereas conidia washed by centrifugation failed
to germinate. Both washed and unwashed conidia
germinated greater than 85% on water-saturated

sand, and were completely inhibited directly on

45

.Hmimm How EmumMm Hoooa may cw poumnsosw Aconmm3c5v

 

 

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62.2055: .59
am— om— ow. m
«1.52:.
co as me vs 0
”1F L1 iilq - —

 

 

 

WdD

 

46

 

 

‘0 .I l i I I I 1 I I 1
H.VICTORIAE
. TOTAL CPM' IN UNWASHED -
SPORES= 2.3 x105

CPM/WASHING
8

 

2!- '5

IC) 1 I 1 l on I J 1 I 1

I 2 3 4 5 6 7 8 9 IO
SEPARATE 10ml WASHINGS,N0.

Figure 4. Radioactivity in each of ten successive
10 ml aqueous washings of l"C—labelled,
initially unwashed, Helminthosporium
victoriae conidia.

 

 

 

 

 

 

47

natural Conover loam soil. The ability of washed
spores to germinate in water suggests that spores
still contain sufficient reserves.

Effect of Extended Incubation
on Conover Loam Soil or in

the Model System

Data presented in Figure 3 suggest that loss

 

of materials from conidia of g. victoriae after 24 hr
on the model system proceeds at a very low, but almost
continuous level. This raised the question whether
the endogenous energy reserves of the spore eventually
become depleted to the extent that an exogenous
source of nutrients is required for germination.
Hsu and Lockwood (43) recently reported that the
nutritionally independent propagules of five test
fungi did not lose the ability to germinate in
buffer solution after 24 hr exposure to either
the model system or soil.

To further examine the possibility of such
a conversion, conidia of E. victoriae and g. sativum
were incubated for up to 56 days in the model
system or on soil. At predetermined intervals
membrane filters bearing the conidia were either
stained and killed, or were transferred to sterile

buffer solution or PDA for an additional incubation

48

of 12 hr to assess nutrient—independence and
viability, respectively.

Germination of g. victoriae conidia in buffer
decreased from 87 to 58% after only 12 hr incubation
in the model system or on soil (Figure 5—A). By 7
days almost all the spores had become nutrient-
dependent. Extending the incubation period in the .
buffer solution to 24 hr did not increase germination
over that at 12 hr. Viability of conidia incubated
in the model system began to decrease after 21 days
and declined to 30% by 56 days. Viability declined
more slowly for conidia incubated on soil. The
loss of nutrient-independence by E. sativum conidia
was as rapid as that by g. victoriae, but viability
of g. sativum declined much more rapidly both on

soil and in the model system (Figure S-B).

Accumulation of Exudates by Spores

To test whether spore exudates may be utilized
in the process of germination, radioactive conidia of
g. lunata were incubated in the model system assembled
so that their leachings passed directly into a second
leaching dish containing non—radioactive ("cold")
conidia (Figure 6). After 12 hr ca. 2% of the radio-

activity (2,080 cpm) was leached from the 1“C—labelled

49

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leIMNV Emumwm Hmwoe may ca Ho Hwom EmoH Hm>o

Edfiuommonucflfiawm Am cam mwauouofl> Eswuommosua

 

   

.coHuMQSUCH us NH Hoseauflopm am How

 

 

 

   

 

 

 

 

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STERILE
WATER
OR
BUFFER

 

 

 

50

   

I4c LABELLED spoaes

 

Ir

.L

 

Figure 6.

com spones

at l

ir-

 

LEACHINGS

 

Diagram of the model system, modified to
measure the uptake of I‘C--1abelled spore
exudates by non-radioactive spores.

51

conidia. Membrane filters with the “cold" conidia
from the lower dish gave 38 cpm over membrane filters
similarly incubated in the lower dish, but without
spores. In absolute amounts the radioactivity taken
up was low, but it should be considered that: a) the
“cold“ conidia covered only a small portion of the
glass bead bed, b) the leachings entering the lower
dish became diluted, and c) the "cold" spores were
undergoing leaching at the same time they were taking
up the labelled leachings from the upper dish.

In other experiments conidia of g, victoriae

 

converted to a nutrient—dependent condition by extended
incubation on soil or in the model system were tested
for their ability to take up materials in spore
exudates, and for stimulation of germination by the
exudates. Membrane filters hearing such spores were
floated for 12 hr on 1.0 m1 concentrated 1“C-labelled

exudate from g, victoriae conidia. Following

 

incubation the conidia and membranes were washed
with 30 ml glass distilled water on a Millipore
filter holder. Membranes were scanned under the
microscope to estimate germingtion before being

placed into scintillation fluid for counting. Over

50% of the radioactivity available was found in the

52

spores after only 12 hr (Table 3), and more than

one-half of the spores had germinated.

Table 3. Uptake of radioactivity by nutrient-dependent
conidia of“gelminthosporium-victoriae
incubated 12 and 24 hr on 1.0 m1 of
concentrated 1"C-labelled spore exudate.

 

 

Radioactivity, % of
Previous incubation of conidia total availablea

 

Treatment Time 12 hr 24 hr
days
Model system 14 72.8 87.0
21 51.5 90.0
Conover loam soil 14 70.6 86.2
21 64.1 67.5

 

aTotal available

1050 cpm.

Stimulation of Germination
by Spore Exudates

If the removal of exudates by leaching is
important in the inhibition of germination of nutrient—
independent spores, then the exuded materials should
contain substances which when resupplied to Spores
stimulates germination. Exudates collected from

conidia of g, victoriae, g. lunata, and T. basicola

 

each contained amino compounds and carbohydrates,

53

including glucose (Table 4). Conidia of T. basicola
exuded only 0.16 ug glucose equivalents of carbohydrate
per million spores during 20 hours of incubation,

while g. victoriae lost over 95 pg and Q, lunata

 

ca. 4.3 ug in 1.5 and 3.0 hr, respectively. Approx-
imately 30% of the carbohydrates detected in the
exuded materials of g. lunata and T, basicola were
accounted for as glucose, while greater than 90% was

measured for g, victoriae. Exudation of amino compounds

 

was one-sixth to one-half that of the carbohydrates
for each of the three fungi.

In order to determine directly whether
substances present in conidial exudates would stimulate
germination, conidia were leached in the model system

with exudate from Q. lunata and g. victoriae. To

 

establish the role of nutrients present in natural
spore exudate, conidia were also leached with a
synthetic exudate, based on nutrient concentrations
found in natural exudate. Synthetic exudate for

g. victoriae contained 523.5 ug glucose and 83.5 ug

 

glycine equivalents of casein hydrolysate per liter
of glass distilled water, and that for g, lunata
319.0 and 124.0 mg per liter of the two nutrients,

respectively.

54

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.ucmmmou wcou£9c¢m

 

 

 

 

 

 

 

 

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an
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mapwmmm woa\m1 coaumnsocH sofiumummmum

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.maoowmwn mamm0fl>mamwn9 mo «amazon Eoum wowsxw muqmfiuus: mo mofiuwucmua .v magma

55

Exudate from conidia of g, victoriae increased
the germination of its own conidia in the model system
from 18% under leaching conditions with water to 45%
with natural exudate and over 50% with synthetic
exudate (Figure 7-A). The apparent decrease in
germination after 12 hr leaching with natural and
synthetic exudates (Figure 7-A and B) was the result
of either germ tube lysis or the retraction of germ
tube protoplasm into the spore proper. This obser-
vation suggests that the nutrients provided in the
natural and synthetic exudates were sufficient to
stimulate germination but not to maintain germ tube
growth under the diffusion stress of the model system.
Germination of g. lunata conidia was stimulated by its
own natural and corresponding synthetic exudate and
by that of g. victoriae (Figure 7-C and D). Similar
trends were observed when conidia of g. sativum were
leached with natural and synthetic exudates of g.
lunata. Germination on sand saturated with the
various exudates was never greater than with water.
Germination of g. victoriae was never as great as in
non-leached controls (75%), indicating that even
though materials were being resupplied, the effect

of the diffusion sink was still evident.

Figure 7.

56

Germination of Helminthosporium victoriae
(A and B) and Curvularia lunata (C and D)
conidia incubated in the model system with
either water, natural exudate from both
fungi, or their corresponding synthetic
exudates, and on sand saturated with each
of the media.

 

 

GERMINATION,%

GERMINATION, %

57

Figure 7.

 

 

 

  

 

 

 

 

 

 

 

 

I” __l l T I - I I I I -
— WATER H.VICTORIAE ... WATER , o |A£i
------- MALEXUDAIHHM) A NAT. EXUDATE(C.|..) l3
_- SYN-EXUDATE "- SYN.EXUDAIE
7s - NNO lEACHING ' 3.5 - - "0 ““"mG -
..§..\.
.1.
so- .~~ - .
// %“~‘
// .0...” \
/ ' ~~ . \
( .o‘. “5.. \
./ sf '3} \\
I lEACHING ‘
25 I- I 3.. l 3.... \‘u q
I I. .5
I.’ '~.
’3'. .3
I...“
4’
p son.

0 " I 4 I I l I I J "
‘OOLI l l I q I I . I u
— WATER C lUNAT _ WATER LUNATA

""" " NAT- EXUDAWHW -----NAT.EXUDATE(C.I..)
-- SYN.EXUDATE ,x‘ —-SYN.EXUDATE 1...-
L No ”:.‘ .......... NO ”/"’
7s , ---- «- - lEACHING -
LEACHING f’ / .
/ I." l .................. ..
I .9. I, .........
/ I , ..........
/ j ,
50L ,/ , - ~ ¥lEACHINGN
, LEACHING\
I ’
I
I d
y
25... - - -
SOIL so“
0 I l 1 - l I L l-
6 I2 24 48 o 6 I2 24 30
TIME,HR "ME HR

58

The Point in the Germination
SETH. victoriae at which

Fin istasis Becomes
Inegfective

Previous results have shown that conidia of

 

 

g, victoriae germinate well over 50% in several hours
when incubated on water-saturated sand. The question
arose as to whether there was a point in the spore‘s
metabolism, prior to the formation of a germ tube,
beyond which the spore was no longer sensitive to
fungistasis.

To answer this question Nuclepore membrane

filters bearing unwashed conidia of g, victoriae were

 

incubated on water-saturated sand for intervals up to
6 hr before transfer to one of the following for 4 hr
additional incubation: a) Conover loam soil, b) the
clay-loam soil, c) model system [83 m1 water/hr], or
d) returned to water-saturated sand. The following

diagram illustrates the treatments:

I Killed immediately

 

Time on water-saturated I Conover loam soil I
sand prior to transfer to:
0-6 hr 3 I Clay-loam soil I

 

 

I Model system (83 ml/hr) I

 

 

I.‘ Water saturated sand I

 

I 4 n:-——————»

 

 

59

During the initial incubation on water-
saturated sand germ tubes began to emerge by 1.5 hr
(Figure 8). After only 1.0 hr some of the conidia
were no longer sensitive to fungistasis: they had
become irreversibly committed to the formation of
a germ tube. Transfer of these spores to either
the model system or natural Conover loam soil
failed to stOp their germination. All the conidia
capable of germinating independent of exogenous
nutrients were irreversibly committed to germinate
after 3 hr incubation on water saturated sand with
the exception of some of those transferred to the
clay loam soil. This suggests that the clay loam
soil is more fungistatic than Conover loam soil
or the model system.

Germination of spores placed directly in
the model system, without prior exposure to water—
saturated sand was 30% compared with 18% for those
incubated on water-saturated sand for 0.5 hr before
transfer to the model system. It is not known why
these conidia, having had initiated germination
but not yet produced a germ tube, were more
sensitive to the diffusion stress of the model

system.

60

Germination of Helminthosporium victoriae
conidia (unwashed) incubated on water-
saturated sand for 0—6 hr prior to
transfer to the following for 4 hr
additional incubation: a) Conover loam
soil, b) the clay loam soil, c) the

model system, or d) water—saturated sand.

61

Figure 8.

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9‘ ‘Nouvmwuas

62

This experiment emphasizes the importance
of time in the inhibition of nutrient—independent
propagules. Whatever factor(s) is responsible for
inhibition in soil, it must act on the spore before
the point of irreversible committment to germination.
Thus, if microbial utilization of spore exudates is
a major cause of fungistasis, then the activity of
microorganisms in the vicinity of the spore must be
such to remove essential materials within a relatively
short period of time.

Establishment of the Microbial
Nutrient Sink in soil

 

Rapid decreases in carbohydrate levels in
amended soil have been reported by several researchers
(27,40,83). More recently Lockwood (unpublished
results) developed an assay to obtain a quanti-
tative estimate of the energy sink caused by
microbial activity in soil, and the diffusion
sink in the model system by measuring the rate of
glucose loss from amended paper discs incubated on
soil and in the model system.

The microbial nutrient sink in Conover loam
and the clay loam soil was assessed using a modifi—

cation of the paper disc technique. Very small

63

quantities of ll‘C—glucose were added to paper discs
(90 p1 of 0.05, 0.005, and 0.0005% glucose solutions,
each containing 0.05 pCi/ml) that were incubated on
the two soils. Loss of glucose was detected within
15 minutes (Figures 9 and 10). Moreover, less time
was required for 50% of the radioactivity to be
lost from discs the lower the initial concentration
of glucose in the discs. This may be of particular
importance in the region of the spore where very
minute quantities of substrate are exuded. Utili—
zation of such materials probably occurs quite
rapidly, if not immediately.

To better determine the minimum time
required for the expression of the microbial
sink, evolution of radioactive carbon dioxide
from metabolism of 1"C-glucose applied to the soil
surface was monitored. Radioactive CO2 was
detected within 3 minutes from both Conover loam
and the clay loam soils, with ca. 7 and 3% of the
2.0 pCi (72 pg glucose) applied to each soil,
respectively, respired after just 4 hr (Figure 11).
No 1"C02 was respired when radioactive glucose was
applied to sterile soils, nor when non-radioactive

glucose was used.

64

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67

When 1"C—labelled exudates from g. victoriae

 

conidia were mixed with suspensions of either soil,
ll’C02 was respired within 15 minutes (Figure 12).
After 4 hr 3% of the radioactivity added to the
Conover loam soil suspension was respired, while
only 1.4% was trapped from the clay loam soil.
However during the first two hours, the rate of
exudate metabolism in both soils was similar.
Addition of ll‘C-—glucose to soil suspensions resulted
in respiration of 20 and 11%, respectively, of the
added radioactivity in 4 hr (Figure 13). The slower
rate at which exudates were metabolized, compared
with glucose, indicates that only a fraction of
the exuded materials can be readily utilized by
soil microorganisms.

Bacterial isolates obtained from soil
dilution plates were tested directly for their
ability to take up radioactive exudates collected

from g, victoriae conidia. All four isolates, two

 

each from Conover loam and the clay loam soil,
accumulated radioactivity within 15 minutes and

two of the isolates contained ca. 6% of the initial
amount of radioactivity after 60 minutes incubation

(Figure 14).

68

 

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.A>\3 .mua .kumzuaflOmv mcowmcmmmsm HHOm Emoa

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69

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71

The difference in the two soils used might
be due to differences in microbial populations
and/or their activities. The number of bacteria
and actinomycetes in the two soils was quite
similar. However, the fungal population in the
clay loam was less than 0.03 that in Conover loam
soil (Table 5). This was not surprising as a
volatile fungistatic factor has been reported for
the clay loam soil (36). The lower fungal populations
and reduced microbial activity, possibly due to the
volatile inhibitor may account for the less active
sink in the clay loam soil.

Table 5. Populations of fungi, bacteria, and
actinomycetes in Conover loam and

the clay loam soil 24 hr after
remoistening the air-dried soils.

 

 

 

 

xenm‘ Propagules per giof air-dried soila

Soil Fungi Bacteria Actinomycetes
Conover loam 6.96 x 10” 5.75 x 106 8.60 x 106
Clay loam 2.10 x 103 7.89 x 106 6.21 x 106

 

aFungi were enumerated on acidified PDA
plus detergent (84), bacteria on soil extract
agar (26), and actinomycetes on chitin agar (60).

72

Role ofEhe Microbial Nutrient
Sink of Soil in Fungistasis

In previous experiments more g, victoriae

 

conidia germinated in the model system than on soil.
Also the rate of glucose loss from paper discs on
the model system, operated at flow rates capable of
inhibiting spore germination, was greater than that
on soil (Lockwood, unpublished results). This was
confirmed by incubating agar discs amended with
glucose on Conover loam and the clay loam soils,

and the model system, even though agar discs were
far less sensitive than the paper discs in measuring
the loss of glucose (Figure 15).

A radiochemical-biological (R-B) assay was
developed to determine whether the microbial nutrient
sink accounted for all the inhibition observed in
soil. The assay was designed to experimentally
separate the role of the microbial nutrient sink from
any other mechanism of inhibition in soil. Purified
agar discs amended with 0.1% glucose were incubated
on the two soils, in the model system, and on water—
saturated sand.

Germination of T. basicola on discs removed
at intervals from either the model system or water-

saturated sand corresponded with that on standard

73

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momwo Homo poocoeo Eoum omoosHmIoz mo moon

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74

discs prepared with known glucose concentrations
(Figure 16A and B). In contrast, germination on
discs which had been incubated on natural Conover
loam soil or the clay loam soil was markedly reduced.
For example, conidial germination was ca. 15 and 5%
of the controls (percent germination in the controls
was ca. 80%) on discs previously incubated for 8 hr
on Conover loam and the clay loam soil, respectively,
whereas discs from the model system containing the
same amount of glucose supported over 90%. Inhibition
was recorded for discs incubated for only 0.5 hr on
either soil. Discs assayed after removal from the
clay loam soil were more inhibitory than those from
Conover loam soil. Thus in the R-B assay, removal
of glucose from amended discs through microbial
activity in the two soils did not account for all
the inhibition.

A stimulatory effect was recorded when the
R-B assay was performed using sterile Conover loam
soil, whereas fungistasis in the clay loam soil was
only partially annulled by sterilization (Figure 163),
even though the nutrients released during autoclaving
were approximately equal to or greater than those

released in Conover loam soil (Table 6). Autoclaving

Figure 16.

75

Conidial germination of Thielaviopsis
basicola on discs, initially amended

to contain 0.1% glucose, after incubation
for up to 24 hr A) in the model system
(10 ml water/hr), and on natural Conover
loam and the clay loam soil, and B) on
water—saturated sand and sterile soils.
Germination is expressed as the percent
of that on untreated glucose—amended
discs. Amended agar discs containing
l"C--glucose (ca. 3,000 cpm) were
incubated exactly as non radioactive
discs to determine the percent glucose
remaining.

 

 

GERMINATION, x or CONTROL

76
Figure 16.

 

 

 

 

 

 

 

 

 

 

Iool' ' ' ' K.
\ STANDARD
75 - .
MODEL
SYSTEM
5° _ I coucven I I
10AM
I
25 ' ClAY— I '
lOAM ‘
o P l l I v
125 7 ' ' ' BJ
couovea
LOAM
‘00 ’ WATER- '
\ SATURATED
SAND
\
75 r- .
STANDARD
CLAY- I _.
LOAM r ' '
50+ 4
1‘ I;
o l I I I L -
100 75 50 25 0

‘4c-6Iucoss,% or CONTROL

77

may have reduced the concentration of the heat-stable

volatile inhibitor from the clay loam soil. These

observations agree with those of Ko and Hora (52)

who demonstrated the inhibitory nature of this

particular clay loam soil following autoclaving

or amendment of nutrients.

Table 6. Carbohydrates and amino compounds in
natural and sterile Conover loam and

the clay loam soil 1 day after
remoistening the air dried soil.

 

 

pg/g air dried soil

Soil Condition Carbohydratea Amino

Compoundsb

 

glucose eq glycine eq

Natural 8.5 1.9
Conover loam

Sterilec 352.9 45.8

Natural 44.0 85.6
Clay-loam

Sterile 297.4 107.3

 

aAnthrone reagent.
bNinhydrin reagent.

cAutoclaved at 121 C (15 psi) for 60 min.

Radiochemical purity of the 11+C-glucose
remaining in amended agar discs incubated in the
various treatments was determined to make certain

that all radioactivity measured represented glucose.

78

No radioactive peaks or spots other than glucose
were identified on paper or thin layer chromatograms,
respectively. Moreover, no additional spots other
than those present from untreated discs, were
observed on TLC plates sprayed with sulfuric acid.

In the R-B assay, discs were inoculated
immediately after removal from the various treatments.
To test whether the inhibition was due to unstable
or volatile materials, as reported for the clay loam
soil, discs were incubated in a petri dish for 12 hr
at 24 C prior to inoculation. The inhibition was
not diminished by delaying inoculation for any of
the treatments.

Cellulose-amended soil, 1-2 weeks after
amendment, required addition of greater amounts of
glucose than unamended soil in order to achieve the

same level of germination of Fusarium solani f. sp.

 

pig} chlamydospores (2). The elevated level of
fungistasis was related to an increased rate of
microbial utilization of glucose, but the possibility
of greater amounts of inhibitory materials in the
amended soil was not ruled out (3). Conover loam
soil was amended with 2% (w/w) alfalfa hay particles
(<40 mesh, 420 p), incubated for two weeks, then

assayed for the rate of glucose utilization and the

79

presence of inhibitory compounds using agar discs.
Glucose was lost more rapidly from agar discs
incubated on amended than unamended soil. On
amended soil 4.0 hr were required for 50% (half-
life) of the glucose to be lost, compared with 7.0
hr on unamended soil. The half-life of glucose was
ca. 12.5 hr on sterile soil with and without
amendment. The R-B assay was used to test whether
the level of inhibitory compounds increased in

soil amended with the plant residue. There was

no significant difference between the levels of
inhibition in amended and non-amended soils,
indicating that alfalfa hay amendment did not
stimulate production of greater amounts of inhib-
itory substances. This suggests that increased
microbial activity was the major cause for elevated
fungistasis in cellulose-amended soil reported by

Adams et a1. (3).

Fungistatic Substances in Soil

 

The metabolism of glucose lost from amended
agar discs in the R-B assay may lead to microbial
synthesis of fungistatic materials in soil, even
though the radiochemical purity tests did not

indicate the involvement of glucose metabolites in

88

the observed inhibition. To test for the existence
of a preformed fungistatic substances in unamended
soil, purified agar discs, first leached on the
model system with glass distilled water (flow rate
60 ml/hr) for 24 hr to render them as nutrient-free
as possible, were used. The discs were incubated
on or over the surface of soil to trap non-volatile
and volatile materials, respectively.

No substances, volatile or non—volatile,
were detected in discs from Conover loam soil assayed
with g. victoriae conidia or N. tetrasperma asco-
spores. Germination was reduced when discs were
left on soil following inoculation with g. victoriae,
but not when transferred to an empty petri dish.
This reduction appeared to be caused by the microbial
nutrient sink below the disc, as germination on discs
similarly treated on the model system (10 ml water/hr)
was correspondingly reduced. Conidia of g. victoriae
germinated ca. 90% on discs removed after 12 hr
incubation on sterile soil, compared with 75% on
the untreated discs.

Agar discs incubated on or over the natural
clay loam soil, then removed, inhibited germination
of E. victoriae (Figure 17). Further decreases in

germination occurred when the discs were left in situ

GERMINATION , %

81

 

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I

 

W

mm msu

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Figure 17.

8 12 24
TIME,HR

Germination of Helminthosporium
victoriae conidia on purified
agar—discs that were incubated
on (-——) or over (°--) the
natural clay loam soil for up
to 24 hr before inoculation,
and then either left on soil
(on soil) or transferred to a
petri dish (empty dish) for an
additional 8 hr.

 

 

 

82

after inoculation. A volatile fungistatic factor
was detected in the sterile as well as the natural
clay loam soil. As in the R-B assay, discs
incubated on the natural clay loam soil were more
inhibitory than those from sterile soil. Similar
results were obtained with discs inoculated with
ascospores of N. tetrasperma.

For germination of T. basicola, the test
organism in the R-B assay, glucose had to be added
to the discs following their removal from soil or
the model system. Discs were finally inoculated
12 hr after the addition of glucose, to test for
the presence and stability of any inhibitor. Discs
incubated on the clay loam soil then amended to
contain 0.1 or 0.005% glucose inhibited germination,
while discs on Conover loam soil or in the model
system did not (Figure 18A), except that at 0.005%
glucose a marked decrease in the germination of
T. basicola conidia was noted at 24 but not 12 hr
or earlier (Figure 18B). There was about a 50%
reduction in germ tube lengths on discs incubated
for 24 hr on Conover loam soil then assayed at 0.1%

glucose.

\

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83

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%'Nouvmwaao

84

It was still possible that the reduction in
germination and germ tube growth on Conover loam
soil was due to microbially synthesized inhibitors
in the agar, as the leached discs may contain
traces of nutrients and/or the soil microorganisms
may be able to utilize the agar itself. To avoid
any microbial stimulation from materials in agar
discs, nutritionally inert discs, prepared from
polyacrylamide gel (PAG), were used in similar
experiments. Germination of N. tetrasperma asco-
spores, which were completely insensitive to fungi-
stasis in Conover loam soil, steadily decreased the
longer PAG discs were incubated on the natural clay
loam soil (Figure 19A). Further reductions in
germination were recorded on discs first incubated
on soil, then left in situ or placed over soil on
a glass microscope slide after inoculation. Similar
reductions occurred on discs bioassayed with N.
victoriae conidia (Figure 19B). No inhibition of
either test fungus occurred when PAG discs were
treated in the same manner in sterile clay loam
soil. As with agar discs, germination of N.
victoriae conidia was not reduced by any treatment
on Conover loam soil, except where discs were

incubated on natural soil after inoculation (Figure

85

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86

20). Results using both agar and PAG discs
demonstrated the presence of volatile and non-
volatile fungistatic substances, which may be one
in the same, in the clay loam soil, but provided
little evidence for such in Conover loam soil.

The inhibition of germination and germ tube growth
of T. basicola on agar discs only after 24 hr
incubation on Conover loam soil suggests that the
fungistatic factor was synthesized to late, or

if in a preformed state may be in too low a concen-
tration to be effective in retarding germination of
nutrient-independent spores.

There were, however, several differences in
the results obtained with the two kinds of discs.
For example, PAG discs were unable to trap the
volatile inhibitor from the clay loam and they did
not show the stimulatory effect of sterile Conover
loam soil. Because of these differences in results,
it seemed possible that the PAG discs were less
permeable than the agar discs. Therefore, diffusion
of glucose through discs of each type was compared.
Two PAG discs were stacked one on top of the other,
with purified agar discs handled similarly. A
purified agar disc containing 0.1% ll’C-glucose

(ca. 3,000 cpm), the donor disc, was placed atop

GERMINATION , %

87

 

 

  
 
    

 

 

 

 

 

'00 * H. VICTORIAE "
a'.‘~. . _{ETET:PE?L'::1=

75 M ”"fi” ‘vr' "
ON 4—0

50 " \w’/ \\\\ON "'
--"

25 ' -— CONOVER LOAM -

-- MODEL SYSTEM

()Illl I I I I I;

O 2 4 8 I2 24
TIME,HR
Figure 20. Germination of Helminthosporium

 

victoriae conidia on poly—
acrylamide gel discs that were
incubated on natural Conover
loam soil (--) or in the model
system (~——) for up to 24 hr
before inoculation, and then
either left in place (on) or
transferred to a petri dish
(empty dish) for an additional
8 hr.

 

88

each stack. Each disc was separated one from the
other by a Nuclepore membrane filter (0.4 p). At
predetermined intervals, the radioactivity in each
disc was measured by liquid scintillation. Glucose
was completely excluded from the PAG discs, even
that disc adjacent to the donor. By contrast, an
equal amount of radioactivity was found in all

three agar discs after only 12 hr. This may explain
the lack of germination stimulation from sterile
Conover loam soil.

The rapid reduction in germination on agar
and PAG discs incubated on the clay loam soil may
reflect a direct effect of the alkaline pH of that
soil. The pH of purified agar discs incubated on
the surface of both soils was measured indirectly.
After removing the discs from the soils, 50 pl of
glass distilled water was incubated on the surface
of each disc for 1 hr, then the pH of the water was
determined with pH indicator paper. The pH of discs
previously incubated on Conover loam soil did not
change, whereas that of discs from the clay loam
soil increased the longer the discs remained on
that soil (Figure 21). Romine and Baker (77)

reported only a slight decrease in the germination

89

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touonsosw hamsow>oum who: gown: momwc Homo cowmwusm
so u: a How wouonnucw Houok voaawumwo woman an om
mo tummom Houoowocw mm auw3 .mm mo sowuoswaumuoa

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90

of N. sativum conidia on buffered agar discs and
in solutions with pH values similar to the pH 8.6
of the clay loam soil. Similarly, buffered agar
discs and solutions adjusted to various pH values
from 5.0-8.0 did not affect germination of either

N. victoriae (Figure 22) and N. basicola (Figure 23)

 

 

conidia. This lack of inhibition strongly suggests
that hydrogen ion concentration per se, within this
range, does not account for the inhibition observed
on discs incubated on the clay loam soil. It is
possible that metallic cations from this soil, which
has a high salt content (77), could cause the high
pH and be the inhibitory factor.

The presence of glucose did not completely
annul either the non-volatile (Figure 16) or the
volatile (52) fungistatic substances from the clay
loam soil. Thus, to test the ability of nutrients
to overcome the effect of the volatile inhibitor,
purified agar discs amended with up to 0.1% glucose
were incubated over both soils (200 g) and water-
saturated sand for 24 hr before being inoculated

in situ with conidia of N. basicola. Inoculated

 

discs were incubated over the treatments for an
additional 12 hr. The volatile inhibitor emanated

from the clay loam soil reduced germination on discs

91

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92

at all nutrient concentrations (Figure 24), whereas
germination on discs from over Conover loam soil
corresponded with that on discs incubated over
water-saturated sand.

Numerous attempts to negate fungistasis by
the removal of volatile fungistatic substances,
which may have been present, failed. When moist
air was passed over either soil at a rate of ca.

75 ml/min conidia of N. victoriae on the soil

 

surface did not germinate, although those on water-
saturated sand treated identically germinated over
75% in 4 hr. Treating remoistened air-dried Conover
loam and the clay loam soil in such a manner for one
month did not allow any mold growth, and sclerotia

of Sclerotium rolfsii picked from the surface of

 

PDA-slant cultures, remained dormant on the surface
of the aerated soils. Sclerotia incubated on
water-saturated sand controls germinated within 3
days. Passing moist air over the surface of
Australian soils allowed both copious mold growth

and germination of N. rolfsii sclerotia (79).

GERMINATION,%

93

 

1001' IWATER SAT. SAND
oCONOVER LOAM
oClAY LOAM

 

75

(n
C)

25

T. BASICOLA

 

 

l l l l l

  

 

OI-
Id" 162 10'3 10'4 10'

GLUCOSE CONCENTRATION, %

Figure 24. Germination of ThielaviOpsis
basicola conidia on glucosE-
amended agar discs incubated
over Conover loam soil, the
clay loam soil, and water—
saturated sand.

 

O

 

 

DISCUSSION

The research reported in this thesis
confirmed the findings of others in this laboratory
(43,54) and elsewhere (25,45,87), that propagules
which will germinate in distilled water, fail to
germinate when exposed to conditions of greater
diffusion stress. This research also established
the importance of nutrients in spore exudates in
initiating germination, and of their loss in
preventing it. Spore materials, including carbo-
hydrates and amino acids, were exuded more rapidly
during incubation on leached glass microbeads than
without leaching. Losses occurring in non—leaching
conditions were not sufficient to prevent germination,
but when the diffusion gradient away from the spore
was steep enough germination was inhibited. The
bulk of the exuded materials were lost from N.
victoriae conidia within twelve hours when spores
were incubated in the model system, or when washed
with successive aliquots of water in a membrane

filter holder. Similarly, Jackson and Knight (45)

94

95

found that most of the glucose lost from unwashed
conidia of N. sativum occurred within several
minutes when washed with water in a membrane filter
holder: they ruled out the source being traces of
adhering media. When they washed conidia for four
days the pattern of exudation was nearly identical
to that found in the present research for N.
victoriae (Figure 2). Interestingly, the nutrient—
dependent conidia of N. basicola also exuded more
materials under leaching than non—leaching conditions.
This may explain why most conidia of this fungus
remain viable for such a short time in natural soil
(56,78,88). It is thought that continuous washing
or leaching in a membrane filter holder or the
model system, respectively, imitates the diffusion
stress of the nutrient sink created by micro-
organisms around the spore in soil. These findings
support the proposal (54) that elevated microbial
activity in the vicinity of the spore, stimulated
by exuded energy yielding compounds, increases the
diffusion gradient away from the spore, removing
constituents normally available for germ tube
synthesis. Blakeman (personal communication)
suggested that the spore's energy reserves are

insoluble and for germination they must first be

96

transformed into a soluble form, which may occur
at a constant rate, and that it is the loss of
these soluble materials at a rate faster than
replacement that prevents germination.

Germination of N. victoriae and N. sativum
conidia, as well as other nutrient-independent
propagules (43), was inhibited in the model system
when leached for periods up to 24 hr, but germinated
freely when subsequently transferred to water— or
buffer-saturated sand. In contrast, extended
incubation in the model system or on soil converted
conidia of N. victoriae and N. sativum to a nutrient-
dependent condition; some of the conidia in the
population were converted in only 12 hr. Apparently
the endogenous energy supply required for germi-
nation in the absence of exogenous nutrients was
depleted by such treatment. Further incubation
for up to 28 days reduced the viability of N.
sativum but not of N. victoriae. Several days
incubation on either soil or in the model system
were required to convert all the thick—walled spores
of these two fungi, whereas thin—walled conidia may
become nutrient—dependent more quickly. Ko and
Lockwood (54) found that almost all conidia of

Neurospora tetrasperma become dependent after only

97

12 hr exposure on soil or in the model system, and
that the conversion was correlated with the loss

of carbohydrates and amino acids. Viability data,
however, was not provided in their report.

Sztejnberg and Blakeman (87) reported the conversion
of Botrytis cinerea conidia within 24 hr by leaching.
Other nutrient-independent propagules may not undergo
conversion, or are converted at a slower rate in a
diffusion stress environment. For example,
chlamydospores of N. basicola were still capable

of germinating in water after extensive leaching
(35,89).

The bulk of the exuded materials was not
required to maintain the nutrient-independent status
of N. victoriae conidia. This was also true for
conidia of N. sativum (47). The rapidly lost
materials may have been substances exuded during
the formation of the spore and retained in the
spore wall matrix. Exudation after the initial
flush could represent the gradual depletion of
the reserves which confer nutrient—independence,
or the loss of soluble conversion products from
an insoluble reserve, as suggested by Sztejnberg
and Blakeman (87). Continued loss of this energy

reserve, not only through exudation but also by

98

the spores own maintenance metabolism, would result
in the conversion to nutrient dependence and finally
loss of spore viability."Once spores are converted
they behave like other nutrient-dependent propagules
and germinate only upon the addition of an exogenous
energy source. One mechanism which may prevent such

rapid conversion and possible loss of viability

 

would be the formation of chlamydospores within
the conidia (67). Chlamydospore formation, however,
was not observed in this study.

The importance of spore nutrients in
germination was emphasized by the results of several
experiments. The stimulation of germination of
conidia resupplied with their own exudates in the
model system strongly suggests that loss of such
materials is responsible for the lack of germination
where the diffusion gradient is steep enough to
induce a sufficient degree of depletion. Stimu-
lation by a very dilute solution of glucose and
casein hydrolysate, or by exudates from another
nutrient-independent spore implies that the germi-
nation stimulants are non-specific and may well be}
energy yielding compounds. Furthermore, nutrient-
independent conidia took-up and metabolized some of

their own exudates during germination under non-stress

99

conditions. The accumulation of exudates by N.
victoriae conidia and their germination after
conversion to nutrient dependency, clearly demon-
strates the importance of exuded materials in
germination. Likewise, Daly et a1. (15) demon-

strated the ability of Puccinia graminis f. sp.

 

triticiuredospores to utilize exuded carbohydrates
such as trehalose and mannitol during germination.
This research also provides further insight
into the various mechanisms proposed to account for
soil fungistasis. Whatever the mechanism for
inhibition of nutrient-independent spores it must
operate before the spores are irreversibly committed
to producing a germ tube. Conidia of N. victoriae
became insensitive to fungistasis in the model
system and Conover loam soil by prior incubation
for only 0.75—3.0 hr on water-saturated sand. By
contrast, those spores which require an exogenous
source of nutrients do not appear to have a point
beyond which germination is insured (82,93). When
the nutrient supply to nutrient—dependent spores
was interrupted, by transfer to the model system or
soil, progress toward formation of a germ tube
ceased. Extended incubation in the model system

or on soil resulted in reversal of the progress

made during the exposure to nutrients. Yoder and
Lockwood (93) also found that incubation on Conover
loam soil or water-saturated sand allowed nutrient-
independent as well as nutrient-dependent spores to
partially progress toward germination; such progress
required only water and was irreversible. For
instance, conidia of N. sativum required only 1.0
hr on sterile soil to germinate 50 percent after
a one day exposure to natural soil, compared with
1.6 hr without prior incubation on soil.

Evidence obtained from this work and that
of others supports the assumption in quantitative
terms, that the model system imitates the microbial
nutrient sink of soil; the evidence is as follows:
i) The rates at which conidia of N. victoriae and
N. sativum were converted to nutrient dependence
in the model system and on Conover loam soil were
similar. ii) Yoder and Lockwood (93) found that
incubation of ngicillium frequentans conidia on
each reversed to the same extent the progress made
toward germination by previous exposure to nutrients.
iii) They also found that ca. 50 percent of the 1"C-
label in conidia, pulsed for 1 hr with radioactive
glucose, was lost during subsequent incubation on

either Conover loam soil or in the model system for

101

six days. iv) Both systems were unable to prevent

germ tube formation in N. victoriae conidia in which

 

germination had progressed to the point where they
were no longer sensitive to soil fungistasis.

v) Germination of ascospores of N. tetrasperma was

 

not inhibited on either Conover loam soil or in
the model system (54).
Not all results support the quantitative

similarity between Conover loam soil and the model

 

system. Glucose-amended paper (Lockwood, unpublished
results) and agar discs lost glucose more rapidly on
the model system than on Conover loam or the clay
loam soil. In some experiments the diffusion of
glucose from agar discs incubated on water-saturated
sand, where nutrient-independent spores readily
germinate, was greater than on natural soil. This
indicates the loss of materials from spores may
differ in rate from loss from various kinds of
inaminate carriers. The disc assays might be

useful in obtaining comparative estimates of the
microbial sink in various soils, as presently used,
but may not be valid for calibrating the model
system. Data for the latter purpose may best be

acquired by the use of propagules themselves.

 

182

Further work is necessary to more accurately measure
the diffusion stress created by each. Such assays
will have to be qualitative, as well as quantitative,
as the diffusion sink in soil may be more specific
for certain substances than the model system. How-
ever, from the limited biological data available,
the diffusion sinks in soil and the model system
may be more parallel than the disc assays indicate.
If the loss of exudates from spores in the
model system equalled or exceeded that on Conover
loam soil, it is difficult to explain the greater
inhibition occurring on soil solely in terms of
nutrient deprivation. The R-B assay provided
evidence that the microbial nutrient sink may not
be the only fungistatic mechanism operative in
Conover loam soil. Correlations between the
decrease in nutrients because of microbial
utilization and decreasing fungal spore germination
have been reported previously (3,27,54,83), but the
R-B assay was the first attempt to quantitatively
assess germination with respect to the available
nutrients. The assay indicated that conidial

germination of g. basicola on glucose-amended agar

 

discs incubated on soil was much less than expected

for the concentration of glucose present at the

103

time of bioassay. However, results obtained with
the R-B assay are subject to their own interpretive
problems. Inputs of large amounts of glucose to
the soil were unavoidable in the R-B assay: these
amendments could have provided the energy needed
to stimulate microbial synthesis of fungistatic
substances which might not be produced from spore
exudates. However, the inability to detect radio—
active metabolites of glucose in discs from the R-B
assay suggests that such compounds were not produced,
or were present in biologically active but chemically
undetectible concentrations. The radiochemical
purity test may have overlooked volatile inhibitors.
An attractive explanation for fungistasis
of nutrient-independent spores is that their exudates
provide the energy required for the production of
antibiotics adjacent to the spore. Lingappa and
Lockwood (59) were unable to find inhibitory

microbial metabolites of exudates from N. victoriae

 

or Glomerella cingulata conidia. It seems unlikely

 

that soil fungi could contribute to antibiosis in
the sporosphere, in that the propagules of such
producers must themselves germinate rapidly within

the sporosphere of the nutrient-independent spore.

184

Soil bacteria and actinomycetes could synthesize
inhibitors, but even under the most favorable
conditions, these microorganisms have a lag period
before antibiotics can be detected (73). Moreover,
the production of inhibitory substances in any
quantity requires large amounts of energy which
are generally not available in soil, thus their
continuous synthesis in soil is unlikely.
Alternatively, the exudation of readily
metabolized materials may, through "priming action,“
allow soil microbes to degrade more resistant
organic matter such as humus or lignin. Decom-
position products of lignicolous materials have
been shown to inhibit spore germination (58). Even
if spore exudates stimulate microorganisms in the
sporosphere capable of producing antibiotics or of
degrading resistant organic matter to toxic sub-
units, the question still remains, whether such
substances are produced in sufficient quantity to
act before nutrient-independent spores become
committed to germination. However, once conidia
are converted to nutrient—dependency, microbially
synthesized inhibitors would not be necessary, as

dormancy would be maintained by the lack of an energy

105

substrate. Concurrent with conversion, reduced
exudation would markedly lower the energy available
for the synthesis of inhibitors.

The use of exhaustively leached agar discs
and polyacrylamide gel discs on soil was intended to
eliminate the possibility of microbial synthesis of
inhibitors by removing the energy source for their
production. Using these discs, no volatile or non—
volatile substances inhibitory to N. victoriae
conidia or N. tetrasperma ascospores were detected
in Conover loam soil. The inhibition of g. basicola
on unamended discs occurring after 24 hr incubation
on Conover loam soil, but not earlier, may be due
to microbial metabolism of the agar itself to
produce toxic materials in sufficient concentration
to affect this fungus, but not the other two test
organisms.

The clay loam soil was used in this study for
comparative purposes because it contains a known
volatile fungistatic factor (36). In various
experiments this soil was shown to be more inhibitory
than the Conover loam soil. For example, it was able
to prevent germination of N. tetrasperma ascospores,

which are insensitive to microbial fungistasis (54).

 

106

Also, R—B assay discs from this soil were more
inhibitory than those from Conover loam, even
though the microbially induced nutrient sink in
the clay loam soil was less. Inhibition by both
volative and non—volatile substances was obtained
'with the clay loam soil. The inhibitory material,
however, may have been one in the same. Low
hydrogen ion concentration per se in this soil was
not responsible for the non-volatile inhibition,
although various basic cationic species which could
account for the alkalinity of this high salt content
soil may have been involved. Ko and Hora (51)
submitted that an aluminum ion appears to be the
fungitoxin in some acid Hawaiian soils, however,
this seems unlikely for the clay loam soil where
the high pH would precipitate aluminum ions as
aluminum hydroxide. Dobbs and Gash (21) reported
that the cause of residual fungistasis in certain
alkaline dune sands may be iron ions, the effect
of which was partially annulled by lowering the pH
to allow chelation with organic matter.
Explanations for the inhibition based on
alkaline cations would not extend to the volatile
fungistatic factor. It seems possible that ammonia

might be the volatile compound, as it is extremely

107

toxic to fungal spores at very low concentrations
(66), and would be readily emanated from alkaline
soils. Lewis (personal communication) has found
that ammonia liberated from decomposing cr0p

residues in soil deformed Rhizoctonia solani hyphae.

 

Ko and Hora (53, and personal communication) have
identified ammonia produced from ammonium chloride
as the volatile fungistatic substance in an
alkaline Hawaiian soil.

Although no volatile fungistatic factor
was detected in Conover loam soil, using methods
capable of detecting such materials in the clay
loam soil, Baker and his co-workers have found a
volatile inhibitor in the Conover loam soil
(personal communication).

The microbial nutrient sink alone may account
for complete inhibition of nutrient-independent
propagules. The microbially induced nutrient sink
was established almost immediately which points out
that the microorganisms are 'poised' and ready to
metabolize an energy source as soon as it becomes
available. Sztejnberg and Blakeman (85), submit
that bacteria may increase the concentration gradient
away from spores by the accumulation of exuded spore

materials into their polysaccharide sheathes. The

 

108

passive establishment of a diffusion sink by this
mechanism in addition to that provided by microbial
activity might increase the diffusion stress on the
spore. The loss of nitrogenous compounds, in
addition to the exudation of carbohydrates, may
accelerate the latter‘s rate of utilization by
microbes. Glucose was found to be metabolized at

a faster rate in soil when nitrogen and phosphorus
were added (85). Fungistasis of Botrytis cinerea
conidia on leaf surfaces was though to be due to
removal of spore exudates by bacteria (10). Droplets
containing spores previously incubated on leaves were
fungistatic only when the bacteria were present in
the droplet. Furthermore, droplet filtrates were
stimulatory rather than inhibitory, demonstrating

the absence of inhibitors from bacteria or the leaf.
Conidial germination of N. cinerea was likewise
inhibited in a model system similar to that used

in the present research (87). Calculations made

from the data of Blakeman and Fraser (10) indicate
that fewer than one bacterium per conidium prevented
germination. Similar calculations from the data of
Lingappa and Lockwood (59) showed that 1-4 X 103
bacteria per conidium completely inhibited germination

of N. victoriae and g. cingulata, while only one-tenth

109

that number were needed to inhibit some of the conidia
of the two test fungi. Filtrates of the bacteria-
spore suspensions were not inhibitory to either
fungus.

Germination and the production of inhibitors
of microorganisms are controlled by the availability
of nutrients, which in soil are in chronically short
supply. The introduction of a nutrient-independent
spore, or a dependent one for that matter, into soil
may provide a temporary source of energy for microbial
activity around that spore, stimulating an increase
in the nutrient sink, followed by the production of
inhibitors. It seems unlikely that microbially
produced inhibitors, especially volatile inhibitors,
are involved in fungistasis in unamended soil,
because the energy required for synthesis on a
continuous basis to maintain inhibitory concen-
trations is not available (31). Moreover, diffusion
rates of gaseous compounds in aerobic soils are high
enough to prevent their accumulation (Tiedje,
personal communication).

Thus, for germination to occur sufficient
exogenous nutrients must be added to soil, not only
to meet the requirements of the spore but also to

overcome any fungistatic factors. Fungistasis may be

 

110

of significant survival value in that nutrient-
independent propagules would only germinate when
sufficient substrate was available for colonization
or to support production of new propagules.

In summary, the main cause for inhibition
of nutrient-independent propagules in a majority of
soils is most probably due to exudation of propagule
constituents beyond a critical rate. The critical

rate is maintained in those soils by a steep

 

diffusion gradient away from the spore as a result
of intense microbial competition for exuded nutrient
materials. Fungistasis in Conover loam soil seems
to be mainly caused by such a microbial nutrient
sink, however, some microbially produced inhibitors
synthesized from the nutrients in spore exudates
may play a role in some instances. The latter
providing a possible explanation for the less
complete inhibition in the model system compared
with that on soil. In contrast, the residual
fungistatic factor in the clay loam soil from
Colorado, and possibly other alkaline soils,

likely accounts for a significant portion of the
inhibition in such soils. Moreover, the toxic

nature and the volatility of this factor may also

111

reduce the activity of all soil microorganisms,
thus decreasing the contribution to fungistasis
made by the microbial nutrient sink in the clay

loam soil.

 

 

 

APPENDIX

 

 

APPENDIX

A permanent record of the results from
experiments which a) led to the modification of
the paper disc technique used to estimate the
microbialnuufientsink in soil, and b) further
defined parameters of the model system are made
in this appendix.

Modification of the Paper
Disc Technique for Assaying

the Microbial Nutrient Sink
of Soil

 

 

As the assay was originally developed by
Lockwood (unpublished results) Whatman filter paper
antibiotic assay discs (1/2 in diam), each amended
with 90 pl of a 0.05% glucose solution were used.
The glucose remaining in the discs after incubation
on soil was determined with the Glucostat reagent,
and was expressed as the percent of that initially
in the discs. The increased rate of loss on natural
soil as compared with that on sterile soil was

thought to be due to an increased diffusion gradient

112

 

113

due to microbial activity beneath the discs. In an
attempt to increase the proportion of glucose lost
by microbial activity to that lost by diffusion in
the absence of the microbial nutrient sink the
initial amount was decreased. Reducing the initial
amount to 90 pl of a 0.0005% glucose solution
markedly increased the sensitivity of the assay as
shown in Figure 9. Since each disc contained only

0.45 pg glucose, a quantity well below the range of

 

the Glucostat color test, radioactive (1“C) glucose
was used.

Various methods of measuring the radio-
activity remaining in the discs were found useful.
A) To use the gas—flow counter, discs upon removal
from soil, were first dried in a stainless steel
planchet on a hot plate at 60 C, with that side
formerly in contact with the soil down. Acceptable
results were obtained even though counts represented
only a small fraction of the ll’C—glucose remaining
in the discs. Most of the Beta particles were
absorbed by the paper. Considerable variation
occurred when the side originally in contact with
the soil was counted. B) Liquid scintillation

counting in which dried discs were placed into

vials of scintillation fluid was less variable than

 

 

114

gas-flow methods. Over 80 percent of the radio—
activity in the discs was detected. C) The most
accurate method, although the most time consuming,
was the determination of 1“C-glucose indirectly by
measuring the ll'COZ produced upon combustion of
amended discs.

Whatman discs are not available in the United
States. However, Whatman discs, kindly supplied by

S.D. Garrett, were compared with similar discs made

 

by Schleicher and Schuell (S & S). Glucose was
lost more rapidly from the S & S than from Whatman
discs incubated on either natural or sterile Conover
loam soil (Figure 25). In a similar experiment,
after only one minute on soil, approximately 18% of
the glucose had already diffused from the S & S discs,
while essentially all the glucose remained in the
Whatman discs. S & S discs, although the same
dimensions as the Whatman discs, weighed only two-
thirds as much. The greater bulk of the Whatman
discs may account for the better retention of the
glucose solutions. It is important therefore, to
use discs from a single source.

Another carrier for glucose was Millipore

membrane filters (13 mm diam, 0.22 p). Each membrane

 

115

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116

filter was amended with 15 ul of a 0.005% ll’C-glucose
solution (5.0 uCi/ml). The glucose remaining in each
disc was determined by either the gas-flow counter,
or dissolving the membrane filter in scintillation
fluid and counting. Glucose was lost very rapidly
from membranes incubated on the soil, and the
activity of the microbial nutrient sink was detected

within minutes (Figure 26).

Characteristics of the

Model System

The rates of glucose loss from agar discs on
soil and in the model system were compared. Agar,
rather than paper discs, was used as a carrier for
glucose because the former were used in the R—B
assay. The diffusion sink in the model system
exceeded that in natural Conover loam or the clay
loam soil (Figure 15). For example, the half-life
of glucose in the model system was ca. 1.0 hr compared
with 7.5 and 9.0 hr for natural Conover loam and the
clay loam soil, respectively. Thus, several
alterations in the model system were tested in an
attempt to make the rate of loss of glucose more

closely imitate that of natural soil.

 

 

117

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Three model systems were operated simulta-
neously at different flow rates to determine whether
this factor would alter the rate of loss. The half—
life of glucose was about equal for discs leached
with water at flow rates of 8.9, 18.1 and 28.4 ml/hr.
Leaching with buffer solution at 18.1 ml/hr resulted
in a half—life similar to water at the same flow

rate.

 

Reducing the diameter of the glass beads in
the bed of the leaching dish increased the half—life
of glucose only slightly. Sand, or glass beads of
470, 200 and 100 u diam gave half-lives of ca. 70,
110, 100, and 120 min, respectively. Even this
_slight increase in half-life did not approach
that for natural soil.

A change in either the flow rate or the size
of the particles in the bed had little effect on the
leaching characteristics of the model system with
respect to loss of glucose from agar discs. It
appears that any movement of the leaching medium
at flow rates faster than ca. 9 ml/hr must keep
this area directly below the disc devoid of
glucose, thus maintaining a maximum diffusion

gradient against distilled water. Using the paper

119

disc technique, Lockwood (unpublished results)
also found that flow rates had a minimal effect
on the rate of glucose loss as measured by the
slope of the line; calculated for readings made
between 0.5 and 4 hr, but had a greater effect on
half-life. The half—life for natural soil was
approximated when paper discs were leached in the

model system with buffer at ca. 10 ml/hr.

 

 

 

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