ABSTRACT

MECHANISM OF SOIL FUNGISTASIS
by Wen—hsiung Ko

For 18 out of 22 fungi tested, the ability of their
spores to germinate on soil was directly correlated
(r = 0.94; p < 0.5%) with the ability to germinate in the
absence of exogenous nutrients. Fungi which required
exogenous nutrients for germination failed to germinate
on soil, and nutritionally independent fungi germinated
on soil. Four exceptional fungi were nutritionally
independent but did not germinate on soil. Their
sensitivity to soil fungistasis was considered due to a
strong diffusion gradient imposed by microbial activity
in soil, by which nutrients diffused rapidly away from
spores to soil. Evidence for this mechanism was the
failure of these spores to germinate when leached with
dripping sterile water. Nutrients were detected in the
leach. Conversely, ascospores of Neurospora tetrasperma
which germinated on soil also germinated during leaching
with water. In addition, when nutritionally dependent
spores were supplied with nutrients and placed on Millipore
filters on soil, the nutrients were rapidly lost and the

spores failed to germinate.

 

 

Wen—hsiung Ko

Spores requiring exogenous nutrients for germination
failed to germinate in aqueous extracts from natural soil,
but they germinated in extracts from sterilized soil.
Sterilization of natural soil by autoclaving increased
free carbohydrates 27-fold and amino acids 37-fold.

Based on a comparison of synthetic soil extracts with
natural soil extracts, it was shown that extracts of
sterilized soil contained sufficient nutrients for
germination; extracts of natural soil did not.

In tests on alfalfa—supplemented natural soil,
germination of Aspergillus fumigatus conidia occurred only
in close proximity to the added organic matter, and not
farther away. Extracts from alfalfa-supplemented natural
soil, however, supported spore germination, whereas those
of nonsupplemented soil did not. These experiments indicate
that the bulk of soil apart from fresh pieces of organic

matter is deficient in nutrients.

 

Decreased spore germination on agar disks incubated
on soil was correlated with the rapid diffusion of nutrients
from agar disks to soil. A similar decreased germination
also occurred on water agar disks incubated with sterilized
charcoal or washed with sterile glass distilled water.

The results indicate that soil fungistasis is a

consequence of the unavailability in soil, or loss from

spores, of nutrients required for spore germination.

 

 

MECHANISM OF SOIL FUNGISTASIS

By

Wen—hsuing Ko

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1966

 

 

 

ACKNOWLEDGMENTS

I would especially like to thank Dr. J. L. Lockwood,
my major professor, for his encouragement and guidance
during the 3 years work on this problem.

I wish also to thank the other members of my guidance
committee for reading the manuscript and making valuable
suggestions: Drs. J. E. Cantlon, N. E. Good, R. P. Scheffer,
and A. R. Wolcott.

Thanks are also extended to Mr. P. G. Coleman for
photographs and Miss Sharon Marbury for the original

typing of the manuscript.

 

ii

 

 

TABLE OF CONTENTS

ACKNOWLEDGMENTS . . . . . . . . . . .
LIST OF TABLES . . . . . . . . . . . .
LIST OF FIGURES . . . . . . . . .
INTRODUCTION . . . . . . . . . . . . .
LITERATURE REVIEW . . . .

METHODS AND MATERIALS . . . . . . . . . .

Preparation of fungal spores . . . .

Direct assay for fungistasis

Indirect assay for fungistasis

Determination of carbohydrates

Determination of amino acids and related
compounds . . . . . . . . .

RESULTS . . . . . . . . . . . . . .

Relation of nutrient requirements for fungal
spore germination to soil fungistasis

Nutrient status of soil and its relation
to fungistasis

Nutrient status of agar disks and its relation
to fungistasis . . . . . . . . .

DISCUSSION - s a n c a . n I I u n C

LITERATURE CITED . . . . . . . . . .

Page
ii

iv

:0

 

 

 

 

 

Table

LIST OF TABLES

Page

Relation between spore germination on
natural soil and in glass distilled water . l6

Germination of spores carrying exogenous
nutrients on Millipore filters placed on
soil, and subsequent germination of the same
spores in glass distilled water or a
nutrient solution . . . . . . . 2A

Nutrient requirements for conidial germination
on natural soil supplemented with different
nutrients . . . . . . . . . . . 30

Effect of added plant residues on germination

of conidia of A. fumigatus on natural soil
and in aqueous soil extracts . . . . . 32

iv

 

 

LIST OF FIGURES

Page

Relation between fungal spore germination
on soil and in water . . . . . . . . 15
The leaching system in operation . . . . 19

Spore germination on A) wet Millipore filters,
B) Millipore filters placed on natural
soil and C) Millipore filters subjected to
continuous leaching with glass distilled
water or 0.01 M phosphate buffer . . . . 20

Spore germination in A) aqueous extract from
natural soil, B) synthetic extract of
natural soil, C) extract from sterilized
soil, and D) synthetic extract of sterilzed
soil . . . . . . . . . . . . . 27

Germination of macroconidia of E. solani f.
phaseoli in a series of 10—fold dilutions
of extract from natural soil and a synthetic
extract of natural soil . . . . . . . 28

Comparison of fungal spore germination on water
agar disks transferred to sterile petri
dishes after 12 hours treatment as follows:
A) without treatment, B) preincubated on
cellophane on natural soil, C) preincubated
on cellophane on sterilized charcoal, and
D) washed with glass distilled water . . 35

Agar disks supplemented with vegetable food
colors showing loss of colored material to
soil by diffusion during incubation for O—QA
hours at 24° C. . . . . . . . . . 37

Changes in amounts of carbohydrates and amino
acids in agar disks amended with glucose
and casein hydrolysate during incubation
on natural soil at l or 240 C. . . . . 39

Relation between loss of carbohydrates and
amino acids from agar disks to soil during
incubation at 24° C. and decrease in
ability of agar disks to supportgermination A0

V

 

 

 

 

INTRODUCTION

Two striking properties cf natural soil as it affects
fungal behavior are that it prevents spore germination of
most fungal species, and that it causes lysis of fungal
mycelia if germination occurs or when mycelia are added to
soil. Soil fungistasis is apparently of survival value in
preventing lysis of vegetative hyphae under starvation
condition. The purpose of this study was to investigate
the mechanism of the widespread fungistatic effect of
natural soil. The background information and techniques
of this thesis are based on previous work done in this
University (24, 26, 27, 28, 29, 30).

Antibiotics in soil were first proposed as the reason
for failure of fungal spores to germinate in soil (28),
during the pioneer work on this subject in this University°
The failure to extract inhibitory substances from soil
despite many attempts (2“) led to a consideration of other
alternatives. It was found that enhanced microbial
activity occurred very rapidly after spores were placed in
soil, and that this was due to exudation of nutrients from
the spores (2A, 27). It was further shown that germination

of conidia of Glomerella cingulata and Helminthosporium

 

 

victoriae in water was inhibited by washed cells of a

 

.variety of bacteria or streptomycetes (27). These results

1

 

 

 

 

 

suggested that soil fungistasis was a localized effect of

enhanced soil microfloral activity in the spore vicinity.
This enhanced activity might restrict spore germination in
soil either by antibiotic proauction in the spore vicinity
(2A, 27), or by depletion of spore nutrients (27).
Recently, in an extensive review of the literature on soil
fungistasis, Lockwood considered competition for nutrients
as the most promising explanation for inability of spores
to germinate in natural soil (29).

This report provides evidence supporting the hypothesis

 

that unavailability of nutrients required for spore
germination is responsible for soil fungistasis. The
necessity of exogenous nutrients for spore germination of
various fungi was studied. Model systems were designed
whereby fungistasis was reproduced without the presence
of microorganisms or soil. The nutrient status of soil

with or without added plant residues was studied.

 

   

 

LITERATURE REVIEW

Spores of most fungal species remain ungerminated in
natural soil except in the vicinity of undecomposed organic

matter or in the rhizosphere (29). The widespread

”T

occurrence of this inhibition was first established by
Dobbs and Hinson (16) and has been termed fungistasis or
mycostasis (11). The literature relating to soil

fungistasis has recently been reviewed by Lockwood (29).

lr—rr ‘ -. -

This review will only include the literature dealing with
the nature of fungistasis and important papers related to
the subject which have appeared since Lockwood‘s review.

Many mechanisms have been suggested to account for the
nature of the inhibition of fungal spore germination in
soil. None, however, has received good experimental
support.

Physical factors such as pH and redox potential seem
to have been ruled out as causes of fungistasis. Agar made
fungistatic by incubation with soil revealed no significant
difference in oxidation—reduction potential from that of
control agar which supported germination (23, 24). Moreover,
autoclaving removed soil fungistasis but only slightly
altered redox potentials (2A). Although the pH of most soils
is within the range tolerated by fungi (29), Green and his

co-workers (18, 19) have suggested that pH might play a

3

 

 

 

part in soil fungistasis With certain fungi. Their test

fungus, Trichoderma viride, germinated poorly outside the

 

pH range 3.5-5.2 in tests made on agar. In their experiments,
spore suspensions were adjusted with 0.1 N HCl to various

pH values. Spores were then placed on disks of water agar.
Tests were not done in solution or directly on soil. The
effect of pH on spore germination might have been due to the
release of soluble carbohydrates due to acid hydrolysis of

the polysaccharides present in agar.

Most workers support the view that diffusible inhibitory
substances are the cause of fungistasis. However, the
nature and source of the postulated fungistatic substances
in soil are viewed differently by different workers.
Accumulation of carbon dioxide in soil is a possibility.
But, soil fungistasis has been demonstrated under conditions
of good aeration. Besides, amendment of soil with organic
matter reduces fungistasis, yet it increases CO2 production
in soil (A).

Lingappa and Lockwood (25) once suggested that toxic
lignin decomposition products might cause soil fungistasis.
Since fungistasis occurs in soils low in organic matter, and
the effect of lignin residues was distortion and inhibition
of germ tubes rather than inhibition of germination, they
later doubted that this is the case (29).

The possibility that a volatile inhibitor may be

present has been Suggested (11, 42), but Lingappa and

 

 

 

 

 

 

 

 

Lockwood (24) failed to obtain any evidence for this.

Antibiotic substances in soil were suspected by Lockwood (28)
and Weltzien (US), but germination in soil occurs in
locations where antibiotic production is most likely to
occur. If antibiotic production in natural soil occurs
at all, the sites of production are most likely rhizospheres
or in the vicinity of organic residues. However, these are
precisely where germination occurs in soil. Furthermore,
antibiotics in soil are subjected to physical inactivation
and biological degradation. Enhanced microbial activity
in the spore vicinity was shown by Lockwood and Lingappa
(2A, 27). They suggested that this might cause the
inhibition of spore germination by virtue of antibiotic
production (2A, 27), or nutrient competition (27). Lockwood
(29, 30) recently has cautioned that it is difficult to
account for long—term inhibition with the hypothesis of
antibiotic production since the spore nutrients which would
be required to support the microbial production of
antibiotics would eventually be depleted.

An analogy between the condition of fungi in soil and
in staled fungal cultures has been proposed by Park (35, 36),
and adopted by Griffin (20). Park (36) found that
production of conidia and chlamydospores followed the cessa«
tion of mycelial growth and that the inhibition of Spore
germination and autolysis of mycelia also occured later in

the same culture. This sequence was attributed to an

‘11.. KH-h w-u n.1, -

 

 

 

. . ‘ .
- Q, . .. v
- 4-," '- m

.‘r

 

 

 

 

 

 

increasing concentration of a staling factor.produced by

the fungus in the medium.

A common staling substance produced by several
unrelated fungi has been shown to exert inhibitory and
possibly morphogenetic effects on mycelia of various
fungi tested (36, 37, 38, 39, Ml). Depletion of nutrients F_
as the cause of the staling effect, an argument raised
by Brian (4) and Lockwood (29), has been adequately ruled
out (38, 39). Vacuolation of mycelium has been used as the

index of the staling effect (39, 41). A similar

[filmi..

vacuolation was induced by soil extracts (39), but pondv
water, hypertonic solutions or toxic stains also showed the
same effect. The staling substance was only slightly
inhibitory or even had no effect on germination. Therefore,
soil fungistasis is not likely to be due to staling substances
in soil.

Dobbs and his co—workers believe that a soluble, very
unstable inhibitor of microbial origin is the cause of soil
fungistasis (12, 14, 15, 16, 17). Because of the competitive
relationship with glucose and citrate they also believe
that the inhibitor may be an antimetabolite (10, 17).

Jackson (21), on the other hand, pointed out that if the
postulated substance is of microbial origin, it must be
resistant to chemical and biological inactivation in the
soil, since the fungistatic effect persisted in fallow soil

after being subjected to repeated intense leaching for

 

 

 

 

 

 

mEarly two years. He viewed the inhibitors as complex
Organic substances, probably of microbial origin (22).
A thermostable inhibition of spore germination has
been reported to occur in certain dune sands in Britain
(13). This was termed residual mycostasis to distinguish
it from the common thermolabile microbial mycostasis. It was

suggested that CaCO and iron were responsible for the

3

residual mycostasis. However, since CaCO3 and iron are
very insoluble in water, inability<1fagar disks to support
spore germination after incubation with dune sand is
unlikely to be due to diffusion of such substances from
sand to agar disks.

Many efforts have been made to extract fungistatic
substances of whatever nature from soil, but the attempts
have usually been unsuccessful, or the results, inconclusive
(29). Many soil extracts were stimulatory instead of being
inhibitory. Some nonsterile soil extracts have been
reported to be inhibitory to mycelial growth or spore
germination. But, in most cases the inhibitory effect
disappeared partly or completely after sterilization by
heat or filtration. Sterile inhibitory extracts have been
obtained from a few soils (10, 43, 4“). However, none of
them have been shown to fulfill the characteristics of
soil fungistasis, i.e., strong inhibition, widespread
occurence and broad inhibitory spectrum. The fungistatic
substances have never been characterized, concentrated,

.nor detected by any method other than biological assay.

 

377'
I

 

 

 

 

Electrophoretic migration of an inhibitory effect in

agar made fungistatic by incubation with soil (45), as well
as induction of inhibition in agar disks by incubation with
soil at low temperature (20), have been given as indirect
evidence for the presence of fungistatic substances in soil.
However, these can also be explained as the results of
migration of soluble nutrients in agar or diffusion of
nutrients from agar to soil.

Since the discovery of the widespread soil
fungistasis, its interpretation on the basis of nutrients
required for germination has usually been ruled out by the
assumption that spores of test fungi are able to germinate
in water (16, 2M, 35). However, considerable data show
that spores of many fungi require exogenous nutrients for
germination (2, 8). That clay and some other inert
materials prevented germination when spores were placed
directly on these (2“) is also possible supporting evidence
for this interpretation. The fact that soil extracts often
support spore germination is often taken as an argument that
soil contains sufficient nutrients for germination. However,
isolated nutrient sources within a deficient soil mass may
provide enough nutrients to support germination in soil
extracts.

Cook and Schroth have suggested that competition for
available substances in soil and elaboration of fungistatic

substances by microflora are equally important to inhibition

 

 

 

0f Spore germination (9)-

Green and his co-workers (l8, l9)

believed that lack of nutrients essential for germination
and soil reaction are responsible for soil fungistasis.
However, the possibility that unavailability of nutrients
alone is sufficient to account for failure of fungal

spores to germinate in natural soil has not been ruled out.

[Emu—.muv.-. ,w .. . <

 

MATERIALS AND METHODS

Preparation of fungal spores.--Conidia of powdery

 

mildews and urediospores of rusts were kindly supplied by

Dr. A. H. Ellingboe. Erysiphe graminis DC. f. sp. tritici

 

Em. Marchal and Puccinia graminis Pers. f. sp. tritici Eriks.

 

& H. Henn. were maintained on wheat (Triticum compactum

 

Host 'Little Club'). E. graminis DC. f. sp. hordei Em.
Marchal and 3. coronata Cda. were maintained on barley

(Hordeum vulgare L. ’Manchuria') and oats (Avena sativa

 

 

L. ('Gopher'), respectively. Teliospores of Ustilago
tritici (Pers.) Rostr., g. maydis (DC.) Cada., and g. nuda
(Jens.) Rostr. were collected in Michigan during the
growing seasons of 1964 and 1965. The following spores
were obtained from cultures on potato-dextrose agar:

conidia of Thielaviopsis basicola (Berk. & Br.) Ferr.,

 

Trichoderma viride Fr., Mucor ramannianus Moller, Penicillium

 

 

 

frequentans Westling and Glomerella cingulata (Ston,)

 

 

Spauld. & Schrenk; macroconidia of Fusarium solani (Mart.)

 

Appel & Wr. f. phaseoli (Burk) Snyd. & Hans. and E. solani
f. pisi (F. R. Jones) Snyd. & Hans. One isolate of M.

ramannianus was kindly supplied by Dr. C. G. Dobbs. Conidia

 

of Helminthosporium victoriae, Meehan & Murphy, Botrytis

cinerea Pers. ex. Fr., and Aspergillus fumigatus Fresenius

 

lO

 

ifii‘a $5; ---' _‘

 

 

 

 

 

 

11

W81“? produced on V-8 Juice agar (per liter: 200 ml. V—8
Juice, 2 g, CaCOB, 20 g. agar). Helminthosporium sativum
Pam., King, & Bakke isolates #10 and #58 were kindly sent
by Dr. R. D. Tinline, and were maintained on sterilized wheat
straws in test tubes. Conidia as well as ascospores of
Neurospora tetrasperma Shear & Dodge were collected from
cultures grown on yeast maltose agar (per liter: 10 g.
maltose, A g. yeast extract, 4 g. dextrose, 20 g. agar).
Ascospores were separated from conidia by allowing them
to sediment several times through a column of distilled
water in a 100 ml. glass cylinder, and were stored under
water at 4°C. until use.

For germination tests, conidia of mildews were shaken
directly onto the medium to be tested. Urediospores of rust
and conidia of M. tetrasperma shaken from plants or agar
cultures, respectively, were suspended in water and used
without washing. Conidia of the other fungi and the smut
teliospores were washed 3 times with sterile glass distilled
water in order to remove nutrients from culture media and
plant hosts. For determination of percentage germination,
200 spores were counted for each treatment. All experiments
were done at least twice.

Direct assay for fungistasis.——The method of Lingappa
and Lockwood (26) was followed. Conover loam soil was
sifted and stored in closed glass jars. The soil was
moistened by damp paper towels placed on top of glass wool

over soil until about 25% moisture was reached. Approximately

[57 *7: -—.—--—~r——--“ ‘ '

 

 

      

r *--u
fi‘ .; ,i_...._ ,F ~—

 

 

12

E“) g. of such soil was placed on a small petri dish (50

‘x 15 mm.) and a smooth surface was made with a stainless
steel spatula. Three drops (ca. 0.1 ml.) spore suspension
were supplied onto the smoothed soil surface. After
incubation the spores were stained with phenolic rose
bengal and recovered with a solution of polystyrene. The
dried plastic film was transferred to a drop of mineral
oil on a glass slide, covered with a cover glass, and then
observed microscopically.

Indirect assay for fungistasis.——Agar disks were
prepared as follows: About 10 ml. of 2% Bacto agar (Difco)
was poured into a petri dish (90 x 15 mm.) to provide a
level surface. Another 15 ml. was pipetted onto the solidi—
fied agar. Disks 17 mm. in diam. and 2 mm. thick were cut
with a cork—borer. Each disk had a volume of 0.“? ml.

A soil block (about 70 x 6 mm.) with a smoothed surface was
carefully transferred into a sterile petri dish to avoid
contact with the edge of petri dish. The soil surface was
covered with a sterilized disk of washed, noncoated cellOphane
(110 mm. diam.). The agar disks were placed onto the
cellophane. After incubation one agar disk was transferred
and incubated with nutrient agar to test the sterility of the
disks.

Determination of carbohydrates.——Carbohydrates were

 

determined by the method of Morries (33). The anthrone
reagent was made by dissolving 0.2 g. of anthrone in 100

ml. of 7 M sulfuric acid (prepared by addition of 500 ml.

 

 

 

   

l3

C1<>ncentrated sulfuric acid to 200 ml. water). One ml. of
the solution to be assayed was mixed in a boiling water
bath for 10 minutes. The solutions were cooled and the
optical density read at 600 mu. in a colorimeter. A
standard curve was made by measuring 1 ml. of samples
containing 20, 40, 80 ug. of glucose.

Determination of amino acids and related compounds.--
Amino acids were determined according to the method of
Moore and Stein (32). Ninhydrin reagent was prepared by
dissolving 0.2 g. ninhydrin and 0.03 g. hydrindantin in
7.5 ml. methyl cellosolve, followed by addition of 2.5 ml.
4 N sodium acetate buffer (pH 5.5). To prepare 4N acetate
buffer, 54.5 g. NaOAc. 3H20 was dissolved in 40 ml. distilled
water. Ten ml. glacial acetic acid was added and the
solution was then made up to 100 ml.. One ml. of
ninhydrin reagent was mixed in test tubes containing 1 ml.
samples, and heated in a boiling water bath for 15 minutes.
The solutions were then diluted with 8 ml. of 50% ethyl
alcohol, and the optical density read at 570 mu. in a
colorimeter. A standard curve was made by measuring 4, 8

and 16 ug. of glycine in 1 ml. water.

 

 

 

 

RESULTS

Relation of nutrient requirements for fungal spore

germination to soil fungistasis.--Explanation of soil

 

fungistasis in terms of lack of nutrients required for spore
germination implies that only those fungi which require F
exogenous nutrients for germination are sensitive to
fungistasis. To test this possibility, the relation

between spore germination on soil and in glass distilled

gr.__...__."_,._.__ _._.. .

water (a nutrient free medium) was studied among various
fungi. For all the fungal species tested, spores were
germinated directly on soil. Except for conidia of mildews,
urediospores of rusts and ascospores of M. tetrasperma,
germination of fungi in water was tested at three serial
lO—fold dilutions to avoid inhibition from overcrowding.
The depth of water in the small petri dish was limited to
0.5—l.0 mm. to provide good aeration. Mildew conidia were
incubated in a moist chamber at 18°C. for 1 hour, followed
by 10 hours at 22°C. (34). All other fungi were incubated
at 24—28°C. There was a significant correlation (r = 0.94;
p < 0.5%) between germination on soil and in water when 18
of the 22 fungi tested were analyzed (Fig. 1, Table 1).
Those spores which were able to germinate in water also
germinated on soil, while those which were not able to
germinate in water also failed to germinate on soil. All

14

 

 

 

15

 

IN SOIL
m
C)

O)
(D

.5
C3

GERMINATION (96)
m
C)

 

 

 

 

1 l l l l

 

O 20 4O 60 80 IOO
GERMINATION (°/o) IN WATER

Fig. l-—Relation between fungal spore germination on
soil and in water. Closed circles indicate 18

fungi for which a significant correlation (r = 0.94;
P < 0.5%) existed between germination on soil and in
water. Open circles indicate 4 exceptions. Each
point represents a different fungus.

 

 

 

16

Table l.--Relation between spore germination on natural

soil and in glass distilled water.

 

Germination(%)a

 

 

 

 

 

 

 

 

 

 

 

 

h
o
J.)
H m
-.—1 3

o
m :3
Fungus Type a Z
L H
s p
4-3 If)
m H
z D
Neurospora tetrasperma Ascospores 94 95
Puccinia coronata Urediospores 93 76
F. graminis f. sp. tritici Urediospores 53 46
Erysiphe graminis f. sp. tritici Conidia 76 74
E. graminis f. sp. hordei Conidia 51 54
HelminthOSporium sativum #58 Conidia 25 49
H. sativum #10 Conidia l 8
Fusarium solani f.4phaseoli Macroconidia 8 7
F. solani f. pisi Macroconidia 6 O
Ustilago nuda Teliospores 4 10
g. maydis Teliospores O 0
g. tritici Teliospores 0 0
Trichoderma viride Conidia 0 0
Penicillium frequentans Conidia 0 1
Aspergillus fumigatus Conidia 0 0
Mucor ramannianus Conidia O 0
M. ramannianus Conidia 0 O
Eotrytis cinerea Conidia 0 l
Thielaviopsis basicola Conidia O 20
N. tetrasperma Conidia 0 40
H. victoriae Conidia 0 50
Glomerella cingulata Conidia 0 96

 

 

aData are from omaof two experiments with similar
results. Two hundred spores were counted in each treatment

for each experiment.

Tr“—-‘._“v‘*— -——- 2

 

 

 

 

tesyt fungi which were not able to germinate in water

geruninated on nutrient agar. H. victoriae isolate #58

 

which possessed "inherent germinability" (6, 7), a term
proposed to indicate the ability of spores to germinate

in natural soil, germinated better in water than isolate
#10 which did not possess such ability. Hence, sensitivity
of spores to soil fungistasis seems to be restricted

to those species which depend on exogenous nutrients for

germination.

 

 

Conidia of H. cingulate, H. victoriae, H. tetrasperma

 

and T. basicola were exceptional in that they germinated
completely or partially in water but not on soil. Previous
work in this laboratory showed that soil microorganisms rapidly
utilized exudates from fungal Spores including some of this
type. Moreover, incubation of spores with soil bacteria

and streptomycetes prevented germination of g. cingulata

 

and H. victoriae in water without production of any detectable

 

inhibitory substance (27). These results suggested that
microbial acitivity in the immediate vicinity of fungal
spores may result in loss of nutrients from the spores.
To test this possibility, a system which provides for more
efficient removal of nutrients from spores than occurs in
water alone was designed.

Conidia of the 4 exceptional species were placed onto
a Millipore filter (0.22 p.) in a fritted glass filter holder.

For comparison, conidia of A. fumigatus and H. frequentans

 

 

 

 

 

 

 

 

 

 

18

(vnuich are nutritionally dependent) and ascospores of H.
Efftrasperma (which are nutritionally independent and germinate

in soil) were also tested. Since conidia of g. cingulata

 

germinated on cellophane or Millipore filters over soil,
they were placed directly onto a fritted glass filter funnel
(UF), instead. The spores were leached from 6—24 hours,
according to the time required for germination, by dripping
glass distilled water or 0.01 M phosphate buffer (pH 6.9)

at the rate of 10-30 ml. per hour from a separatory funnel.
Excess liquid on the filter was continuously removed by a
small suction pressure (Fig. 2). By this means nutrients

on spores were continuously carried away by water or buffer.
The apparatus was sterilized before use and remained
uncontaminated during the experiment. Spores on Millipore
filters on soil were compared with those in the model system,
except for g. cingulata which was placed directly on soil.
For controls, spores were placed on wet Millipore filters

in petri dishes or on the wet disc of a fritted glass filter

funnel in the case of g. cingulata.

 

The nutritionally independent conidia of g. cingulata,

 

H. victoriae, H. tetrasperma and T. basicola did not

 

germinate under the leaching conditions nor on natural
soil nor on Millipore filters on soil (Fig. 3). Ascospores
of H. tetrasperma germinated in all the three test conditions,

while conidia of H. fumigatus and H. frequentans failed to

 

 

germinate.

 

Fig. 2—-The leaching system in operation.
Millipore filter in the filter holder.
ping glass distilled water or 0.01
rate of 10-30 ml per hour. Excess
removed by a small suction pressur

Spores were placed on a
Spores were leached by
M phosphate buffer (pH 6.9)
eliquid on the filter was con

drip—,

at the
tinuously

 

 

 

 

2O

 

00

as a:
O O

GERMINA TION (96)
A
O

20

 

 

 

 

Fig. 3--Spore germination on A) wet Millipore filters,

B) Millipore filters placed on natural soil and C) Milli—
pore filters subjected to continuous leaching with glass
distilled water or 0.01 M phosphate buffer. For g.
cingulata a fritted glass disc (UF) was substituted for
the Millipore filter in A) and C), and B) refers to
germination directly on natural soil. Data are from one
of two experiments with similar results. Two hundred
spores were counted in each treatment for each experiment.

 

.
p
',,
g
v
)

 

 

 

 

21

When leached conidia of H. victoriae and H. tetrasperma

 

 

'Were transferred to a nutrient medium or when a 0.1% solution
(of glucose and peptone was allowed to drip on the spores,
they germinated completely. It is, therefore, unlikely
that the inhibition was caused by some injury to the spores.
Since the germination response of each fungus when exposed
to dripping water or buffer closely resembled the germination
response of the same fungus exposed to soil itself, the model
seems a valid approximation of the soil condition inducing
fungistasis. Inability of the 4 exceptional nutritionally
independent fungi to germinate in soil, therefore, seems
related to the existence of a steep diffusion gradient from
spores to soil.

To test whether continous exposure to a steep diffusion
gradient is necessary for long—term inhibition of these

nutritionally independent fungi, conidia of H. victoriae and

 

N. tetrasperma were exposed to soil or leaching, then placed

 

in water to germinate. A small Millipore filter (0.22 u-E
25 mm. diam.) was placed on a larger one (0.22u.; 47 mm.
diam.) on soil and preincubated for 12 hours before Spores
were supplied. After 12 hours' incubation, spores were
aseptically transferred to sterilized glass distilled water
by dipping the small Millipore filter in water, and further

incubated for 12 hours. For H. victoriae, treatments were

 

also extended to 48 hours. Spores which had been exposed
to soil germinated to nearly the same extent as those which

had been leached. Three percent of the conidia of H.

 

22

te’trasperma and about 90% of those of H. victoriae

germinated in water after being exposed to either soil

or leaching. Apparently, for some fungi a short exposure

to a strong diffusion gradient is sufficient to induce

a nutritionally dependent condition which will result in

long-term inhibition in soil, while for others, a longer

or perhaps continuous exposure is necessary. This F
experiment again showed the similarity of natural soil ‘
and leaching system. i

To determine whether nutrients were lost from leached .

spores, conidia (22 mg.) of H. tetrasperma were washed 3 E
times by centrifugation, then subjected to leaching on a

Millipore filter. A total of 194 ml. leach was obtained

after 12 hours leaching. The amounts of carbohydrates and

amino acids were determined. A quantity of 6.9 mg.

carbohydrates (glucose equivalent) and 8.2 mg. amino acids

(glycine equivalent) per g. of spores were detected. There—

fore, it seems likely that removal of nutrients is responsible

for the failure of these spores to germinate under leaching

conditions.

The ability of soil to induce nutrient loss from

spores was tested further using nutritionally dependent

spores. Conidia of H. fumigatus, H. frequentans, H.

ramannianus were suspended in a nutrient solution containing

0.1% glucose and 0.1% peptone. Three drops (ca. 0.1 ml.)

of the suspension were placed on Millipore filters on soil.

After 12 hours incubation, spores were transferred to glass

 

 

 

 

 

23

diiitilled water or nutrient solution by immersing the

lflillipore filters in the liquids. Spores on moist

Millipore filters were used as control. Conidia of all the

three fungi germinated on Millipore filters, but failed

to germinate when the filters were placed on soil (Table 2).

After being transferred to water the nongerminated spores

still did not germinate, but retained their ability to

germinate in nutrient solution. The results suggest that

if spores with sufficient nutrients to germinate are placed i

in soil, these nutrients are rapidly lost by diffusion into

natural soil, and the spores fail to germinate. 3
Nutrient status of soil and its relation to fungistasis.--

In view of the above results, the nutrient status of soil

and its relation to soil fungistasis were investigated.

It is well known that sterilization of soil by heat removes

fungistasis (16). Since sterilization of soil by

irradiation is milder than autoclaving, preserving for

example enzyme activity (3, 31), fungistatic substances if

present might not be lost in soil sterilized by this means.

Soil was sterilized by gamma irradiation with a dosage of 4

megarads from a 0060 source, or by autoclaving for 40

minutes. Conidia of 5 test fungi: H. victoriae, H. sativum_

 

isolates #10 and #58, g. ciggulata and H. frequentans,

 

 

germinated completely on soil sterilized either by gamma
irradiation or autoclaving. Therefore, removal of soil

fungistasis by sterilization may be due to release of

 

 

 

 

Tatile 2.--Germination of spores carrying exogenous nutrients
CW1 Millipore filters placed on soil, and subsequent
gernnrmjion of the same spores in glass distilled water or

a nutrient solution.

 

 

Germination (%)a

 

 

 

 

 

U) (D
3 c
C: (U U)
.53 E’ 3
Treatment SIC . w m
2 ml: - m
cs 0‘ <1 l-H
E o E
m p s
a a a
l. Millipore filters on soilb 0 0 0
2. Spores from (1) placed in
distilled water 1 6 10
3. Spores from (1) placed in
0.1% glucose—peptone
solution 99 98 70
a. Millipore filter controlb 100 99 67

 

a . . . .
Data are from one of two experiments Wlth Similar
results. Two hundreds Spores were counted in each treatment
for each experiment.

bSpores were suspended in 0.1% glucose-peptone solution
before being supplied to Millipore filters.

nutrients rather than to inactivation of fungistatic
substances.

To test whether sterilization does release nutrients
or not, the nutrient content in natural and sterilized soil
were compared. Natural and autoclave—sterilized soils were
shaken for 30 minutes with an equal amount of glass distilled

water and centrifuged at 8,600 G. for 5 minutes. The

!

 

 

 

I. Ilullll Ill. Ill I

 

25

SUDEIfiiatant fluids were filtered through Millipore filters
(0.22 u.). The extract of natural soil was concentrated to
about one—third of the original volume by evaporation at
50°C. The extract of natural soil contained 4.2 pg. carbo—
hydrates (glucose equivalent) and 0.7 ug. amino acids
(glycine equivalent) per ml., whereas the extract of steri-
lized soil contained 110 ug. carbohydrates and 19 ug. amino
acids per ml. The pH range of the soil extracts was 6.7—7.0.
Thus, natural soil contained 4.1 ug. carbohydrates and 0.5
pg. amino acids, while sterilized soil contained 108 ug.
carbohydrates and 18.6 ug. amino acids per g. dry weight of
soil. Sterilization of natural soil by autoclaving increased
carbohydrates 27—fold and amino acids 37—fold. The results
did not exclude the possibility of the presence of an
inhibitor, since nutrients released by sterilization may
overcome the effect of the inhibitor if the inhibitor is an
antimetabolite. Consequently, spores were germinated in
soil extract to detect the presence of the inhibitor.
Aqueous extracts from natural soil were sterilized by

passage through a Millipore filter (0.22 u-), and tested for

germination. Conidia of H. fumigatus, H. frequentans and

H. Hgmannianus failed to germinate in this extract. Since
these fungi also did not germinate in water (Table 1),

their failure to germinate in soil extract may be due either
to the presence of an inhibitor or lack of nutrients in the

extract. The soil extract did not support germination of

the same three fungi even after the extract was autoclaved

“as.“ ~

 

 

 

 

.'—.-.'__

   

. If
F
f
. g 1’

 

j—‘_.

____~_, h _

 

26

for 140 minutes. Therefore, if there was an inhibitor, it
mustr'be a very heat stable one.
To base the inhibition on an inhibitor in the soil
extract requires the demonstration that synthetic solutions
containing the same amount of nutrients as extracts from

natural and sterilized soil would permit greater germination

 

in both cases. Synthetic soil extracts were prepared by
addition of the same amounts of carbohydrates (glucose) and
amino acids (peptone or casein hydrolysate) as the extracts

from natural or sterilized soil to a mineral salt solution

ragga-:3” ¢:——_-A_LA__a-s—.—s.—- —.. .15. _..H

i
I

containing 0.25 g. K2HPO4’ 0.08 g. NHuNO 0.25 g. MgSOu,

3’
and 0.03 g. FeCl2 in one liter glass distilled water. The
synthetic soil extracts had the pH range of 6.9—7.1, and

were autoclave-sterilized. Germination of H. solani f.

phaseoli, H. fumigatus, H. frequentans and H. ramannianus

 

 

 

in natural soil extracts was similar to germination of the
same fungi in synthetic soil extracts (Fig. 4). When

conidia of H. solani f. phaseoli were germinated in four
lO—fold dilutions of natural and synthetic natural soil
extracts, again germination in both extracts decreased
similarly with increasing dilution of the solutions (Fig. 5).
The results did not fulfill the requirements for presence

of an inhibitor. 0n the contrary, all the experiments suggest
that failure of spores to germinate in soil extract may be

due to absence of sufficient nutrients for spore germination.

 

 

 

 

 

 

 

 

 

27

 

GE RMINA TION (96)

 

I00
80 - ..
60 - -
40- < _
20 ~ ~ '
.. .7 j
0 i

.A B (I D A B (3 D A I3 C D A B (3 D

 

E A. R M.
SOLANI FUMIGATUS FREQUENTANS RAMANNIANUS

Fig. 4--Spore germination in A) aqueous extract from
natural soil, B) synthetic extract of natural soil, C)
extract of sterilized soil. Synthetic extract of
natural soil contained mineral salts, 4.2 ug. glucose
and 0.7 ug. casein hydrolysate per ml.; synthetic ex-
tract of sterilized soil contained mineral salts. 110

pg. glucose and 19 ug. casein hydrolysate per ml. Data
are from one of the two experiments with similar results.
Two hundred spores were counted in each treatment for
each experiment.

 

 

 

 

 

 

 

   
  
 

 

 

 

I00 I 1 I I
90 b "
SYNTHETIC EXTRACT

5,; 80 _ OF NATURAL SOIL .

\-

2

9 70 r ‘

L.

§

5 6° ' EXTRACT FROM ‘1

a: NATURAL son.

3 so — ‘
4o - ‘
30 I l l I l

o lo" Io'z I0’3 0
DILUTION

Fig. 5--Germination of macroconidia of H. solani f.
phaseoli in a series of lO-fold dilutions of extract from
natural soil and a synthetic extract of natural soil.
Extract from natural soil contained 4.2 ug. carbohydrates
and 0.7 ug. amino acids per ml. Synthetic extract con-
tained mineral salts, 4.2 ug. glucose and 0.7 ug. casein
hydrolysate per ml. Data are from one of two experiments
with similar results. Two hundred spores were counted in
each treatment for each experiment.

-.-z4qw. —

Ire-er ‘

 

 

 

 

 

29

Annulment of soil fungistasis by nutrient amendment

has tween demonstrated in many cases (29). Since different

fungi may require different nutrients for their germination,

annulment of fungistasis in different fungi by different

nutrients would be consistent with the view that the

nutrients are acting to satisfy a nutrient requirement.
Natural soil was dried by blowing air from an

electric fan and remoistened to about 25% moisture by

addition of a solution containing 0.1% of various nutrients.

Many kinds and combinations of nutrients were tried in
order to find the minimum requirements for complete
germination of each fungus on soil. Spores were germinated
directly on the surface of the amended soils. Conidia

of H. cingulata and H. tetrasperma required only glucose,

 

 

while thoSe of H. victoriae and H. frequentans required

 

 

glucose and ascorbic acid for complete germination on

soil. H. fumigatus required glucose, nicotinic acid and

 

NHuNO3 (Table 3). Although amendment of natural soil with

0.1% glucose completely annuled fungistasis against conidia

of g. cingulata and H. tetraSperma, addition of glucose at

 

 

concentrations from 0.1 to 5.0% failed to promote germination

of H. frequentans. The results then are consistent with the

 

view that the nutrients were satisfying a requirement for
germination rather than counteracting in some way an

inhibitor.

_i___- Mr -1,

lulu-r: ..

 

 

 

30

TaIDle 3.——Nutrient requirements for conidial germination
on Iiatural soil supplemented with different nutrients.

 

Germination (%)a

 

 

m m
Nutrients m E m g m
supplemented p m m p z
w H o: p
«H zlm - a mic - m
old a mlo 3 <|
$4 4: H
C: 4—3 O a) E
H (1) H $4 :3
o p > m m
None 0 0 3 0 0
Glucose 98 97 A7 0
Glucose + ascorbic
acid —- —- 97 100 44
Glucose + nicotinic
acid + NHuNO3 —— __ __ ___ 98

 

aData are from one of two experiments with similar
results. Two hundreds spores were counted in each treatment
for each experiment.

bSoil was supplemented with 0.1% of each kind of
nutrient.

The fact that extracts from natural soil often support
germination must be explained if soil fungistasis is to be
interpreted in terms of lack of nutrients in soil.

Possibly, natural soil contains many isolated sources of
nutrients, and fungistasis is expressed only apart from
such nutrient sources. Water extracts of natural soil may,
thereby, gather enough nutrients from the isolated nutrient
sources to support germination. Since H. fumigatus did not
germinate in extracts from natural soil, it was employed

to test this possibility. Conidia of H. fumigatus should

 

 

 

 

 

 

 

 

31

Eeruminate on soil near added plant residues but not away
ftwnn them. A water extract from such soil would be
expected to support conidial germination.

Twenty g. of Conover loam soil were placed in a small
petri dish and compressed. Twenty mg. of finely ChOpped
dry residues of mature barley or green alfalfa were placed
in a small furrow made in the center of the soil surface,
then covered with a thin layer of soil. Germination of

H. fumigatus was tested direct on soil. Conidia germinated

 

on the soil surface over the tope of the alfalfa residues,

but failed to germinate at distances greater than about 1 mm.

from then1(Table 4). Residues of mature barley did not

stimulate germination of H. fumigatus on soil. It seems

 

likely that green alfalfa residues contain more soluble
nutrients than do residues of mature barley. Soil with
0.1% barley or alfalfa residues were shaken with the same
amount of glass distilled water for 30 minutes. The soil
suspensions were centrifuged and the supernatants filtered
through Millipore filters (0.22 u.). The filtrates were
sterilized by autoclaving and tested for germination.
Extracts were also prepared from nonsupplemented natural

soil. Germination of H. fumigatus in the extract from

 

barley—amended or from nonvamended soil was very poor,
whereas germination in the extract from alfalfa—amended
soil was nearly complete. The amounts of carbohydrates

and amino acids in the extract from barley—amended soil was

 

 

 

 

32

Tablja 4.--Effect of added plant residues on germination of
'COHJJiia of H. fumigatus on natural soil and in aqueous soil

 

 

 

 

 

extracts.
r-l r-‘l r—l
H H -H
O O 0
[ID U) U)
as [U c
¢+m an: m
r—i'd (1)13 'U
as :5 as r-
HE CUE OE A
<<u m m z w _
. a ‘
Germination (%) L
L
Near residue (< 1 mm) 85 0 0 L
Away from residue )
(> 1 mm) 0 0 O L._
Soil extract 88 22 28
Nutrients (uG/ml)
Carbohydratesb 52- 6 4
Amino Acids 3.4 0.7 0.3

 

aData are from one of two experiments with similar
results. Two hundred spores were counted in each treatment
for each experiment.

bGlucose equivalent.

CGlycine equivalent.

very low and did not differ from these in plain soil extract
(Table 4). The extract from alfalfa—amended soil, however,
contained about 10 times more carbohydrates and amino acids
than the other two extracts. Evidently, extraction can gather
sufficient nutrients from organic particles in natural soil

to support spore germination in the extracts.

 

 

 

 

33

Nutrient status of agar disks and its relation to
fungistasis.—-Inability of agar disks to support spore
germination after incubation with soil has been interpreted
as the result of diffusion of fungistatic substances from
soil to agar disks (29). Like agar disks, moist filter
papers became fungistatic after incubation with soil. This
provided a more direct means to test whether a fungistatic f”
substance had diffused into such materials incubated on F
soil. Ten Whatman No. 1 filter paper disks (7 cm. diam.) }

and 7 ml. distilled water were placed in a petri dish and

”fl..- . ‘

autoclaved, then incubated with a soil block separated from

if!

the filter papers by a sheet of sterilized cellophane.
After 9 days incubation at 1°C., the soil block was removed.
The dishes were transferred to 24°C., and the cellophane
was replaced with a new sheet. H. frequentans failed to
germinate, and H. victoriae germinated poorly on the cellophane
on these filter papers. However, when liquid pressed
aspectically from such fungistatic filter papers was
transferred to fresh filter papers and tested for germination,
the same fungi germinated well. The failure of moist filter
papers or agar incubated on soil to support spore germination
is, therefore, unlikely to be due to the diffusion of
fungistatic substances from soil.

The alternative possibility that inhibition resulted
from loss of nutrients by diffusion to soil was investigated.
Attempts were made to induce a fungistatic effect in agar

disks by methods which would withdraw nutrients from the agar.

 

 

 

34

Water agar disks were incubated with charcoal or washed
with water. Thirty grams charcoal (Norit A, decolorizing
carbon) were mixed with 400 ml. ethyl alcohol and filtered
through a Whatman No. 1 filter paper to remove possible
imputities which might interfere with germination. The
charcoal was then washed and filtered 3 times with a total
of 1 liter of glass distilled water, and dried in an oven
at 80°C. for 12 hours. A charcoal block was prepared as
previously described for a soil block, then sterilized by
autoclaving. Water agar disks were placed over a sheet of
washed and sterilized cellophane on the charcoal or natural
soil. After incubation for 12 hours at 24°C., agar disks
were transferred to sterile petri dishes and tested for
spore germination. To wash water agar disks, 10 disks were
shaken or stirred with a magnetic stirring bar in 500 m1.
sterilized glass distilled water for 12 hours, then
transferred to petri dishes for germination tests. Disks
without treatment were used as controls. Sterility of the
treated agar disks was tested on nutrient agar.

Agar disks washed with distilled water or incubated
with sterilized charcoal reporduced the fungistatic effect
of agar disks incubated with soil (Fig. 6). Conidia of
g. cingulata, H. Hnguentans, H. fumigatus, and H.
ramannianus failed to germinate on agar disks incubated with
soil or sterilized charcoal. Washed agar disks also failed

to support germination of H. fumigatus and H. ramannianus

 

 

 

35

 

 

 

mo
80 - _
8
2 60 I
Q _
k .
V
3 4o - ‘
s
0: _ -
lu
Q)
20 - «
0
ABCD ABCD ABCD ABCD ABCD ABCD ABCD
*7 ‘u v v to a: 6) iv
*0, 3' ¢ " Q Q 0
v- , V“ V .I‘ V %
«5.9 see of. 03's 3. we .30:
#2340 ’~ e‘ ea} 0‘ <93 Mus \ e é‘e \
w, 90 vs So o x #o *0“
Q0 A0 Q0 0 0% Q0 v0
*4” R «Q. 0.?

Fig. 6-—Comparison of fungal spore germination on water
agar disks transferred to sterile petri dishes after 12
hours treatment as follows: A) without treatment, B)
preincubated on cellophane on natural soil, C) preincu-
bated on cellophane on sterilized charcoal, and D) washed
with glass distilled water. Data are from one of two
experiments with similar results. Two hundred spores
were counted in each treatment for each experiment.

 

 

36

arui gave reduced germination of H. frequentans and H.
cingulata. Germination of conidia of H. victoriae and H.
tetrasperma was reduced on agar disks incubated with soil
or washed with distilled water. Ascospores of H. tetrasperma
germinated on all 3 treatments. Apparently leaching of
nutrients from agar disks can render them fungistatic.
Diffusion of nutrients from agar disks to soil may be
responsible for failure of agar disks on soil to support
germination.

The rate of diffusion of substances from agar disks
to soil was tested by incubating agar disks supplemented
with colored substances on soil. Water agar disks were
immersed in red (2.5%), green (2.5%), blue (1.5%) food
colors (pH 7.0) for 30 minutes. The colored agar disks
were then placed on sterilized cellophane on natural soil,
and incubated at 1° and 24°C. Disks from each temperature
were removed after 2, 6, l2, and 24, hours incubation and
the intensity of colors compared with control disks kept in
sterile petri dishes. At 24°C., the intensity of color in
agar disks decreased with time of incubation (Fig. 7),
Within 2 hours a noticeable decrease in color intensity
had accurred, At 1°C. the rate of decrease in color
intensity was slower. The rapid diffusion of food colors
from agar disks to soil suggested that soluble nutrients

may diffuse rapidly from agar disks to soil.

 

 

 

 

 

Fig. 7——Agar disks supplemented with vegetable
food colors showing loss of colored material

to soil by diffusion during incubation for 0—24
hours at 24°C. The experiment was done twice
with similar results.

 

 

 

38

To determine the rate at which nutrients diffused
from agar disks, 2% Bacto (Difco) water agar was supplemented
with 1.7 g. glucose and 3.7 ml. casein hydrolysate (10%)
per liter in combination or separately. The agar disks were
placed on a sheet of sterilized cellophane on soil, and
were removed at different time intervals during incubation
at l or 24°C. Nutrients in the disks were extracted by
shaking 3 disks in 6 ml. distilled water in a 50 m1.
Erlenmeyer flask for 30 minutes. The extract was filtered

through a Millipore filter (0.22 u.). Rapid loss of

r1nhum n gun. a. (H.k-.‘u1$1

nutrients from agar disks was revealed. Disks supplemented
with nutrients, either singly or combined, lost 40—50%

of the carbohydrates and amino acids within 6 hours at
either 1 or 24°C. (Fig. 8, 9). When agar was supplemented
with glucose or casein hydrolysate separately, both
carbohydrates and amino acids continued to decrease after

12 hours (Fig. 9). When glucose and casein hydrolysate

were combined, however, the amount of each decreased with
time up to 12 hours incubation at either 1 or 24°C.

(Fig. 8); after 12 hours, carbohydrates in the agar continued
to decrease, but amino acids increased. The increase

in amino acids in agar disks after 12 hours may be due to
the exudation or release from living or dead microorganisms.
Nutrients naturally occurring in plain water agar disks

also were rapidly lost when disks were placed on soil.
Carbohydrates decreased for 24 hours, but amino acids de-

creased first then increased again.

GLUCOSE EQUIVALENT /DISK

Fig. 8——Changes in amounts of carbohydrates and amino
acids in agar disks amended with glucose and casein
hydrolysate during incubation on natural soil at l or

uozpe)

39

 

 

   
  

O = CARBOHYDRATES
AMINO COMPOUNDS

I°C

“~<>---—--

24°C

 
  
 

 
   

 

 

I0 20
HOURS

GLYCINE EOUVALENT/DISK

(Io'flc)

24°C. Data are from one of two experiments with
similar results.
each treatment for each experiment.

Two hundred spores were counted in

 

[riane‘fla-xfivr was . 111‘s“!
r 1

 

40

 

IO . . I00
. = CARBOHYDRATES
"‘ = AMINO COMPOUNDS “
“ o- -o = p. FREQUENTANS
3‘1 o--o =M. RAMANNIANUS ‘30
u

GLUCOSE EQUIVALENT, 102,. G/ DISK
GLYCINE EQUIVALENT, [OI/JG ”2st
GERMINATION (as)

 

 

 

 

Fig. 9-—Relation between loss of carbohydrates and
amino acids from agar disks to soil during incubation
at 24°C. and decrease in ability of agar disks to
support germination. For nutrient analysis, agar
disks were supplemented with glucose and casein hydro-
lysate. For germination tests, plain water agar disks
were removed from soil at the same time intervals.
Data are from one of two experiments with similar
results. Two hundred spores were counted in each
treatment for each experiment.

 

IF- nm;.I:-rj'~‘n-ful. fl?

 

 

 

41

Plain water agar disks without added nutrients were
incubated on soil for different time intervals to test
spore germination. Germination decreased with time and was
correlated with loss of nutrients from nutrient supplemented
disks incubated with soil (Fig. 9). After 6 hours
incubation with soil at l or 24°C., plain water agar disks
no longer supported germination of H. frequentans nor H.
ramannianus. The results further suggest that diffusion
of nutrients from agar disks to soil may account for

inability of such disks to support germination.

“ms ._ -.. z .uu. IA.‘
-'

 

DISCUSSION

Inability of fungal spores to germinate in most
natural soil could be due either to the presence of
inhibitory substances or the absence of nutrients
required for germination. Many attempts have been made rnwv
to extract fungistatic substances from natural soil. r
Although a few workers (10, 43, 44) have obtained sterile
inhibitory extracts from a few soils, most such attempts

have been unsuccessful or the results inconclusive (29).

 

The minimum requirements for the demonstration in soil
extracts of an inhibitory substance which is responsible
for soil fungistasis are the following: (i) the inhibitor
must be extractable from a wide range of soil, (ii) it
must be present in the same soil through the whole year
so long as the soil is fungistatic; (iii) it must be
inhibitory to a wide range of fungi; (iv) it must be a
very potent inhibitor; (v) spores of those fungi which
are not sensitive to soil fungistasis must not be inhibited
Thus far, none of the requirements have been met, even in
the few cases where an inhibitor has been demonstrated.
There are several other reasons for doubting that
the postualted inhibitors exist in natural soil. (i)

Sterilization by autoclaving removed fungistasis in soil,

42

 

 

43

but did not alter the fungistatic effect in the soil
extracts prepared by the method described here. (ii)
Conidia of H. victoriae germinated better in water after
being exposed to the influence of soil fungistasis.
(iii) Spores germinated in liquid expressed from

fungistatic moist filter papers. (iv) Different fungi

required different nutrients for annulment of soil E“-
fungistasis. It is very unlikely that natural soil g
contains antimetabolites against glucose, ascorbic acid, i
nicotinic acid, NHuNO3 and other compounds. (v) 3
Different propagules of the same fungus responded differently m

to soil fungistasis. Conidia of H. tetrasperma failed to
germinate on soil, while ascospores of the same fungus
germinated freely.

Several findings indicate strongly that for most
fungi inability of spores to germinate on natural soil is
due to lack of nutrients therein which are required for
germination. Most of the fungi tested required exogenous
nutrients for germination and all of these failed to
germinate on soil (Table l). Extracts from natural soil

contained insufficient nutrients to support germination of

 

spores of H. fumigatus, H. frequentans and H. ramannianus,
and presumably this is also true for other nutritionally

dependent fungi. Explanation of soil fungistasis in terms
of nutrient requirements for germination is also supported

by the germination on soil for most of those fungi which

 

 

44

were nutritionally independent, i.e., germinated in water
alone, though there were some exceptions.

The failure of four of the nutritionally independent
fungi to germinate on soil may be due to the existance of
a steep and continuous diffusion gradient from spores to
soil, resulting in greater loss of nutrients from spores
than occurs in water. This possibility was previously
indicated by the rapid utilization of nutrients from

spores by microorganisms in soil, and by the inability of

the nutritionally independent conidia of g. cingulata and
H. victoriae to germinate in water during incubation of the
washed spores with washed bacteria or streptomycetes (27).
The experiments performed in the leaching system support
the hypothesis that inhibition of these fungi occurred as
a result of a diffusion gradient. Under sterile conditions,
conidia of the 4 exceptional fungi, which germinated in
water but not on soil, failed to germinate when exposed
to dripping water. Analysis of the leachate from H. Eggggr
sperma conidia showed that nutrients were lost from the
spores by the irrigation treatment.

The existence of a strong diffusion gradient
affecting nutrinets applied to soil is further indicated
by the rapid diffusion of colored substanced and nutrients
from agar disks to soil. Moreover, when nutritionally
dependent spores carrying enough nutrients to germinate
were placed on Millipore filters on soil, nutrients were

lost and the spores failed to germinate.

 

 

45

Several lines of evidence suggest the similarity of
the leaching system to natural soil. (i) Like natural
soil, the leaching system also possesses the ability to
cause lysis of fungal mycelia. Mycelia of H. victoriae
subjected to leaching in the model system were lysed within
4 days (unpublished data). (ii) The pattern of spore
germination in the model system completely correlated
with that occurring on natural soil. The nutritionally
dependent fungi as well as the 4 exceptional nutritionally
independent ones failed to germinate both on soil and in

the leaching system. On the other hand, ascospores of

 

H. tetrasperma, which germinated on natural soil, also
germinated freely in the leaching system. (iii) Ability
of different fungi to germinate after being eprsed to
leaching was the same as that after exposure to natural
soil. For example, conidia of H. victoriae germinated
completely after exposure to soil or leaching system,
while conidia of H. tetrasperma failed to germinated
until exogenous nutrients were added.

Apparently, inability of H. tetrasperma conidia
to germinate during leaching with water was due to depletion
of nutrients, but this was not true for H. victoriae, at
least up to 48 hours. Possibly conidia of Helminthosporium
have developed the ability to respond to a strong diffusion
gradient by changing the permeability of the cell membrane
which results in preventing loss of nutrients from inside

the conidia, or in some other way postpone germination.

 

 

 

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-

 

 

‘

 

 

 

#6

This may be the means by which conidia of Helminthosporium

 

are able to survive in soil for extended perios (5).

Chinn and his co-worker (6, 7) found that some
isolates of H. sativum_were able to germinate in soil
though most were not. Genetic factors were shown to be
involved in the ability of spores of this fungus to
germinate in soil. They proposed that enzyme systems of
spores of isolates able to germinate in natural soil were
unaffected by fungistasis, or that these spores excreted
substances capable of neutralizing fungistatic substances.
However, my results suggest that isolates possessing
"inherent germinability" are resistent to fungistasis
because they contain a higher proportion of nutritionally
independent spores. More spores of isolate #58 which
possessed "inherent germinability” were able to germinate
in water than those of isolate #10 which did not have the
ability to germinate in soil.

In this report, extracts from natural soil have been
shown to contain insufficient nutrients to support spore

germination of H. frequentans, H. fumigatus, and H.

 

 

ramannianus, but contained enough nutrients for germination

 

of H. solani f. phaseoli. Extracts from fungistatic natural
soil frequently have been reported to support spore
germination, and this is often given as an argument that soil
contains sufficient nutrients for Spore germination (29).

However, Alexander (1) has pointed out that nutrients are

 

 

 

 

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“7

not uniformly distributed throughout soil, but are
compartmentalized in microsites. The kinds and amounts

of available nutrients differ from site to site. Moreover,
essential substances in natural soil may be bound in some
way by clay. Leaching the soil may release the bound
substances or gather different substances together so that
requirements for spore germination may be met in the
extracts. My results with alfalfa-amended soil show that
natural soil containing organic residues is fungistatic

apart from the residues, but that extracts from such soil

can accumulate sufficient nutrients from the residues to
support spore germination.

Cook and Schroth (9) reported that addition of
antibiotics to soil increased germination of chlamydospores

of H. solani f. phaseoli. They interpreted this increased

 

germination as due to the combined effect of reduced
competition for available nutrients in soil and reduced
elaboration of fungitoxic materials, However, the results
described here suggest that competition for nutrients alone
may be sufficient to account for inability of fungal

spores to germinate on natural soil. Recent results by
Powelson (“0) also suggest that diffusible carbon substrates
are the major factor limiting fungal spore germination in
soil. He failed to obtain germination of Verticillium
dahliae conidia, which require an exogenous source of both

carbon and nitrogen, in a silica gel medium (nutrient free)

 

 

 

#8

containing natural soil, unless the medium was supplemented
with glucose. Increased germination by addition of
antibiotics to soil may be caused by a decrease in the
diffusion gradient or by release of small amounts of
nutrients through partial inactivation of microorganisms.
Chlamydospores of Fusarium may resemble H. victoriae conidia
which failed to germinate during leaching, but retained

their ability to germinate when the diffusion gradient

 

was reduced, or they may resemble conidia of H. solani
which required only trace amounts of nutrients to trigger
germination.

Indirect evidence for the presence of fungistatic
substances in soil was given by Weltzien (45) and Griffin
(20). Weltzien showed that the inhibitory effect in
fungistatic agar migrated to the anode in gel—electrophoresis.
However, his experiment could also be interpreted as the
result of accumulation of electrolytes or migration of
essential nutrients away from the anode. Griffin obtained
inhibitory agar by placing water agar disks on cellophane
on soil for 9 days at 2°C. to allow diffusion of material
into agar from soil with minimum microbial activity. My
results indicate that nutrients from agar will diffuse
rapidly to soil under these conditions and that this may
cause inhibition of spore germination. Liquid pressed
from moistened filter papers made fungistatic by incubation

with soil at 1°C. for 9 days also was not inhibitory to

 

 

 

 

’49

Spore germination. Both colored substances and nutrients
in agar disks decreased rapidly with time of incubation on
soil at 1°C. A concomitant decrease in ability of agar
disks to support spore germination accompanied loss of
nutrients. Moreover, water agar disks washed with water
or incubated with sterile charcoal also failed to support
spore germination.

The nutrient deficiency hypothesis permits a simple
explanation for many aspects of soil fungistasis, such as
the following: (i) its widespread occurrence existing in
nearly all soil types assayed; (ii) its resistance to 3
physical inactivation and biological degradation in soil;

(iv) the restoration of fungistasis to sterilized soil by
infestation with non-antibiotic producing microorganisms;
(v) its non-lethal effect to spores; (vi) the annulment
of fungistasis by nutrients (3U); (vii) its occurrence in
inert materials such as clays.

The results described herein suggest that soil
fungistasis is a consequence of the unavailability in soil,
or loss from Spores, of nutrients required for spore
germination. Therefore, there is no necessity of postulating
the existence of fungistatic substances to account for

failure of fungal spores to germinate on natural soil.

 

IO.

.0
’.

 

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W'

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In”: f;_.’4l‘m.’..."4 -"- ‘- -_ " ,_

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