THE EFFECT OF
PENTACHLORONITROBENZENE
ON THE SOTL MlCROFLORA

Thesis for the Degree of Ph. D.
MiCHiGAN STATE UNIVERSITY
JAMES D. FARLEY
1968

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Michigan State
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This is to certify that the

thesis entitled

The Effect of Pentachloronitrobenzene
on the Soil Nicroflora

presented bg

James D..Far1ey

has been accepted towards fulfillment

of the requirements for

 

Ph°D’ degree in___:Pl.ant PathOJ-OEY
6 Major professor
4/4/68

Date

 

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ABSTRACT

THE EFFECT OF PENTACHLORONITROBENZENE
ON THE SOIL MICROFLORA

by James D. Farley

The effect of pentachloronitrobenzene (PCNB) on the
soil microflora was studied with the objective of explaining
why certain fungi insensitive to PCNB increase following
soil treatment with this compound.

Diluted soil suspensions from natural soil were
seeded onto selective agar media containing various concen—
trations of PCNB. ActinomyCete numbers were reduced 65% by
1 ppm, 9Q%'by 10 ppm and 99% at 25—200 ppm. Fungal numbers
were reduced 27%.by 10—500 ppm, but were unaffected by 1 ppm.
Bacterial numbers were unaffected at concentrations of PCNB
lower than 200 ppm PCNB; at higher concentrations bacterial
numbers were greatly reduced. PCNB at 50 ppm partially or
completely inhibited the growth in agar of 10 of 14 fungi,

7 of 10 actinomycetes and none of 10 bacteria tested.

PCNB was added to a Conover loam soil in which the
metabolic activity of heterotrophic soil microorganisms was
minimal. PCNB at 200 ppm did not affect soil respiration,
concentrations of sugars or amino compounds and numbers of

actinomycetes, bacteria and fungi in such soil. This

James D. Farley
indicated that the antimicrobial effects of PCNB observed in
agar were not expressed in soil where microbes are relatively
inactive,

Actinomycetes and fungi were inhibited by PCNB in
soil amended with nutrients, however. Five days after
addition of 0.2%.chitin and 10 ppm PCNB, there were 70%
fewer actinomycetes in PCNB-treated soil than in soil with—
out PCNB; at 10 days, there were 40%.fewer, PCNB, at 50 ppm,
suppressed respiration by 50% in chitin-amended soil during
a 33 day period. In soil treated with 0.2%,glucose, numbers
of PCNB—sensitive fungi were reduced by about 50% by 100 ppm
PCNB. Respiration in soil treated with 1% glucose increased
to a maximum of 75 ul/hr/g soil at 3 1/2 days after amendment
addition, whereas respiration in soil treated with 1% glucose
and 100 ppm PCNB reached a maximum of 58 ul/hr/g soil at the
5th day. The delay of the buildup of a respiring population
in PCNB-treated soil was probably the result of the suppres—
sion of PCNB-sensitive microorganisms.

Glucose was utilized at a slower rate in soil treated
with PCNB than in soil without PCNB, presumably because of
the inhibition of PCNB-sensitive fungi and actinomycetes,

Numbers of microbes insensitive to PCNB (e,g,,
bacteria and Fusarium spp,) increased more in glucose—amended

soil treated with PCNB than in glucose-amended soil without

James D. Farley
PCNB. Germination of chlamydospores of Fusarium solani f.
phaseoli and conidia of Helminthosporium victoriae was
greater in soil containing glucose and PCNB than in soil

with only glucose. More g, victoriae conidia germinated on

 

PCNB-treated soil amended with alfalfa residue than on
alfalfa-amended soil with no PCNB. Since germination of
these two fungi in soil was not directly affected by PCNB,
it seemed likely that PCNB treatment resulted in increased
availability of nutrients to these spores,

The following explanation is proposed for the in—
crease of PCNB-insensitive fungi following soil treatment
with PCNB: PCNB decreases competition for nutrients in soil
by suppressing actinomycetes and some fungi. The reduced
competition allows those fungi insensitive to PCNB to

flourish.

THE EFFECT OF PENTACHLORONITROBENZENE

ON THE SOIL MICROFLORA
by

James D. Farley

A THESIS

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

DOCTOR OF PHILOSOPHY
Department of Botany and Plant Pathology

1968

1D
Ax.

./J

ACKNOWLEDGMENTS

Special thanks to Dr. J. L. Lockwood for his guidance
and assistance during the course of this investigation and in
preparation of the manuscript.

Thanks are also due to Dr. M. L. Lacy, Dr. R. P.
Scheffer, Dr. N. E. Good and Dr. A. R. Wolcott for their
critical evaluation of the manuscript and their suggestions.

I would also like to extend my appreciation to Dr.

W. H. Ko for his contagious enthusiasm and ideas offered.

ii

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . ii
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . v
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . vi
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . 2
METHODS AND MATERIALS . . . . . . . . . . . . . . . . . 11
Chemical and physical properties of PCNB . . . . . . 11
Source and preparation of soil . . . . . . . . . . . ll

Microorganisms used in studies of the effect of
PCNB on growth . . . . . . . . . . . . . . . . . 12

Soil amendments . . . . . . . . . . . . . . . . . . 13
Nutrient analysis of soil . . . . . . . . . . . . . 14
Soil reSPiration . . . . . . . . . . . . . . . . . . 15
Enumeration of soil microbes . . . . . . . . . . . . l6
Germination of fungal spores in soil . . . . . . . . 17
Stability of PCNB in soil . . . . . . . . . . . . . 18
RESULTS . . . . . . . . . . . . . . . . . . . . . . . . 20
Stability of PCNB in soil . . . . . . . . . . . . . 20
The effect of PCNB on soil microbes in agar . . . . 20
Selective medium for bacteria . . . . . . . . . . . 27
Effect of PCNB on microbes in natural soil . . . . . 31

The effect of PCNB on actinomycetes in chitin-

amended soil . . . . . . . . . . . . . . . . . . 33
The effect of PCNB on fungi in glucose-amended soil 4O
Stimulatory effect of PCNB on F. oxysporum f.

melonis and F. solani f. phaseoli. . . . . . . 43
The effect of PCNB on bacterial numbers in glucose—

amended soil . . . . . . . . . . . . . . . . . . 46

 

iii

Page

The effect of PCNB on the rate of glucose utili-

zation in soil . . . . . . . . . . . . . . . . . 48
The effect of PCNB on soil respiration in glucose—
amended soil . . . . . . . . . . . . . . . . . . 51

Germination of conidia of Helminthosporium
victoriae and chlamydospores of Fusarium solani
f. phaseoli on PCNB-treated soil . . . . . . . . 53

 

 

 

DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 6O

LITERATURE CITED . . . . . . . . . . . . . . . . . . . 68

iv

LIST OF TABLES
Table Page
1. Stability of PCNB in Conover loam soil . . . . . 21

2, Inhibitory effect of PCNB on microorganisms in
nutrient agar . . . . . . . . . . . . . . . . 24

3. Effect of 200 ppm PCNB on numbers of microbes
in natural soil . . . . . . . . . . . . . . . 34

4. Effect of 200 ppm PCNB on germination of
Fusarium solani f. phaseoli chlamydospores in
glucose-amended soil . . . . . . . . . . . . . 58

Figure

1.

LIST OF FIGURES

Numbers of actinomycetes, fungal, and bacterial
colonies from diluted soil suspensions plated
on media amended with various concentrations
of PCNB . . . . . . . . . . . . . . . . . . .

The effect of 50 ppm PCNB on numbers of
actinomycetes and bacterial colonies on 3
different media seeded with a diluted soil
suspension . . . . . . . . . . . . . . . .

Colonies of bacteria and actinomycetes on soil
extract agar with or without 50 ppm PCNB . .

The effect of 50 ppm PCNB on numbers of
actinomycete and bacterial colonies on soil
extract agar seeded with diluted soil sus—
pensions prepared from muck, forest, and loam
soils . . . . . . . . . . . . . . . . . . . .

Effect of PCNB on number of actinomycetes in
natural soil and chitin-amended soil . . .

Effect of PCNB on the colonization of colloidal
chitin by actinomycetes in soil . . . . . .

Effect of PCNB on oxygen uptake in soil with or
without chitin . . . . . . . . . . . . . .

Effect of PCNB on numbers of fungi in natural
soil and glucose-amended soil . . . . . . . .

Effect of 100 ppm PCNB on numbers of Fusarium
oxysporum f. melonis, I-5 and other fungi in
natural soil or in soil amended with 0.2%
glucose . . . . . . . . . . . . . . . . . . .

 

vi

Page

26

28

29

32

36

38

39

41

44

Figure Page

10. Effect of PCNB on numbers of bacteria in natural
soil with or without glucose . . . . . . . . . 47

11. Effect of PCNB on the rate of glucose (1%)
utilization in soil . . . . . . . . . . . . . 49

12. Effect of PCNB on the rate of glucose (0.05%)
utilization in soil . . . . . . . . . . . . . 50

13. Effect of PCNB on oxygen uptake in soil with or
without glucose . . . . . . . . . . . . . . . 52

14. Effect of PCNB on germination of conidia of
Helminthosporium victoriae on glucose-amended
soil . . . . . . . . . . . . . . . . . . . . 55

15. Effect of PCNB on germination of conidia of
Helminthosporium victoriae applied to soil
0—6 cm from an alfalfa residue . . . . . . . . 56

16. Summary of the effects of PCNB on the soil
microflora . . . . . . . . . . . . . . . . . . 6?

vii

INTRODUCTION

When pentachloronitrobenzene (PCNB) - a narrow
spectrum fungicide principally used for the control of

damping-off or root rot caused by Rhizoctonia solani - is

 

added to soil, a significant increase in seedling damage by
other fungal pathogens insensitive to PCNB often occurs.
The two fungi most commonly associated with this increase
have been Fusarium Spp. (6, 12), and Pythium spp. (13, 16,
22, 46). It has been suggested, although supporting data
are generally lacking, that the increased activity of these
pathogens following soil treatment by PCNB may be due to the
suppression by PCNB of Specific microbial antagonists of
these pathogens.

The effect of PCNB on the soil microflora was studied
with the purpose of explaining why fungi insensitive to PCNB

increase following soil treatment with this compound.

LITERATURE REVIEW

When a fungicide is added to soil to control patho—
genic fungi, significant alterations in the biophase often
occur. Soil fungicides usually make little distinction
between the target pathogen and its neighbors (22). Actino-
mycetes, bacteria and saprophytic fungi, and microfauna as
well, may be inhibited or killed outright, and because of
reduced biological competition for nutrients unaffected
organisms tend to multiply. The well equilibrated and stable
biological system of soil is changed, for better or worse.
The subject of this literature review concerns the deleterious
effects of soil treatment on the soil biophase. The emphasis
is on how a soil fungicide (PCNB) promotes disease accentu—
ation and disease exchange.

Disease accentuation: Disease accentuation, also

 

called the 'boomerang effect' (22), involves the disappearance
of the dominant pathogen after treatment, which is soon
followed by its reappearance in often greater amounts.
Kreutzer (22), for example, has observed reinvasion of soils

treated with chloropicrin and chlorobromopropene by Rhizoctonia

 

2

3
solani and Pythium spp. Gibson (15) reported that R, E21222
and Pythium ultimum rapidly recolonized ethyl-mercury
phosphate-treated soil, and diseases caused by these pathogens
were more severe in treated soil than in untreated soils. 3,
solani and P, ultimum were noted to be more tolerant to the
fungicide than other common soil saprophytes. KenKnight (19)
noted that mercury compounds used for soil treatment to con-
trol Streptomyces scabies generally caused, instead, a marked
increase in scabbing. Certain isolates of S, scabies were
very tolerant to mercury compounds and he concluded that these
mercury-tolerant isolates were able to increase in treated
soil because of reduced competition from mercury—sensitive
organisms. For a more complete review of disease accentuation,
see Kreutzer (22, 24).

Disease exchange: Disease exchange is a situation in

 

which the dominant pathogen is controlled but a previously
unimportant pathogen is elevated to major importance thus
becoming the new dominant pathogen. For example, Gibson (14)
found that treatment of peanut seeds with organo—mercurial
compounds reduced preemergence losses, but damage due to

crown rot caused by Aspergillus niger increased. Certain

 

isolates of A, niger were mercury-tolerant and multiplied

in the area of mercury—treated seeds. It was speculated that

4
they increased because of the reduction of competition from
mercury-sensitive soil microorganisms.
A soil fungicide often cited as causing disease
exchange is PCNB (6, 12, l3, 16, 22, 46). The most common
use of PCNB is for the control of damping—off or root rot

caused by Rhizoctonia solani. Many workers have treated the

 

soil with PCNB to control 3, solani only to find an increase
in damping-off or root rot caused by other pathogens. The
most common pathogens elevated to new importance by PCNB
treatment are Pythium spp. (l3, 16, 22, 46) and Fusarium Spp.
(6, 12).

The first published report of disease exchange caused
by soil treatment using PCNB was by Fulton in 1956 (12).
Fulton studied the activity of various fungi pathogenic to
cotton seedlings in soil treated with various fungicides. In
plots treated with PCNB a marked reduction in the pathogenic

activity of R, solani and Sclerotium bataticola was evident,

 

whereas the incidence of disease in cotton seedlings caused

by Fusarium moniliforme and Colletotrichum gossypii increased.

 

 

Fulton speculated that PCNB decreased microbial competition
in the soil, thus allowing the two PCNB-insensitive pathogens
to increase their activity.

Bird et 31, (6) also tested various fungicides as

5"
control measures for the cbtton seedling disease complex, the

most important pathogens of which were 3, solani, Fusarium spp,

 

and Pythium spp. Bird observed no change in the incidence of
damping-off or root rot following PCNB treatment of soil. It
was noted, however, that PCNB treatment caused marked decreases
in the number of plants infected with R, solani and Pythium

spp., whereas the number of plants infected with Fusarium spp.

 

increased with PCNB treatment. Bird thought that PCNB
'conditioned' the soil microflora so as to permit a more rapid

development of Fusarium spp. No data were presented to support

 

this hypothesis.

Others have reported an increase in the pathogenic
activity of Pythium spp. following soil treatment with PCNB.
Garren (13), for example, observed increased pod rot on peanuts
caused by Pythium spp. following soil treatment with PCNB for
the control of R, solani. Vaartaja et_§l, (46) also observed
disease increases with the use of PCNB to control damping-off
of conifers. For example, Dexon (sodium p-dimethylamino-
benzenediazo sulfonate) gave significant damping-off control
when used alone, but failed when mixed with PCNB. No
phytotoxicity was observed and chemical interaction seemed
unlikely as these two materials are commonly mixed. Pythium

Spp. were the organisms most often associated with diseased

6

seedlings after PCNB and Dexon treatment. They attributed the
increased activity of Pythium spp. to the inhibition by PCNB
of organisms antagonistic to Pythium, but no conclusive data
were presented to verify this conclusion. Kreutzer (22) has
also observed disease exchange with sugar beets. PCNB was
applied to soil to control R,solani and the activity of
Pythium spp. increased.

The only paper which has dealt with PCNB-induced
disease exchange in detail is that by Gibson g£_§l, (16).
They reported that PCNB was effective as a soil treatment
against E, solani damping-off on pine seedlings, but was of
little use against Pythium spp. Gibson showed that PCNB not
only failed to control disease caused by Pythium spp. but also
that appreciable increases inPythium disease followed use of
the fungicide. To investigate the possibility that the in-
crease of disease caused by Pythium might be due to the action
of the fungicide on a competitor of Pythium in the soil, soil
suspensions were plated onto Czapek—Dox amended with PCNB.
When the colonies were three days old, inocula of Pythium were
introduced on the side of each plate. Pythium made faster
growth through soil plates treated with PCNB than through
those untreated. 0n agar media without PCNB, Pythium was noted

to»be inhibited in the vicinity of colonies of Penicillium

7
paxilli, whereas on agar containing PCNB, P, paxilli was inhib—
ited. It was postulated that Pythium increased in PCNB-treated
soil because PCNB inhibited an antagonist of Pythium, namely
.P. paxilli. The addition ofg, paxilli to soil to control
Pythium disease was unsuccessful, however.
Specificityfiof PCNB: PCNB has been used to control

diseases caused by B, solani, Streptomyges scabies, Botrytis

 

 

cinerea, Sclerotinia spp., Sclerotium spp., Plasmodiophora

 

 

brassicae and Tilletia caries (17, 23). It is relatively

 

 

ineffective against diseases caused by Pythium spp., Fusarium
spp., Verticillium albo-atrum, Colletotrichum gossypii,

Phytophthora infestans, Aphanomyces euteiches and Thielaviopsis

 

basicola.

 

There have been several in yiE£2_studies of the effect
of PCNB on the germination, sporulation and/or growth of
various fungi (2, 10, ll, 32, 34, 36, 37, 45). PCNB has been
found to inhibit spore germination and sporulation of some
fungi (32, 34). Roy (34), for example, reported that PCNB at
a concentration of about 5,000 ppm strongly repressed spore
germination of Botrytis cinerea, Fusarium caeruleum, Ascochyta

rabiei, Trichoderma viride, Rhizopus nigricans and Alternaria

 

 

spp. Sclerotial formation of Rhizoctonia solani as well as

 

Sporulation of the above mentioned fungi was also almost

8
completely inhibited by PCNB. Reavill (32) using PCNB in the
gaseous phase also found that sporulation of B, cinerea, F,

caeruleum and T, viride was repressed. No one, however, has

 

comprehensively studied the effect of PCNB on germination and
sporulation on a broader spectrum of pathogenic and saprophytic
soil fungi.

PCNB has been reported to inhibit the growth in agar
of R, solani (2, 10, ll, 32, 34, 36, 37), Botrytis spp. (2, 31.

32, 34), Rhizopus spp. (34, 45), Trichoderma viride (32, 34, 45),

 

Mucor ramannianus (45), Alternaria sp. (34), Ascochyta rabiei

 

 

 

(34), Pencillium spp. (2, l6),_Sclerotinia sclerotiorum (2),

 

 

and Sclerotium rolfsii (2). PCNB was relatively ineffective

 

against Fusarium spp. (2, 32, 34, 45), Pythium spp. (2, 10,

38, 45), Phytophthora spp. (2) and Thielaviopsis basicola (30,

 

 

43).

Little work has been done on the effect of PCNB on
soil bacteria and actinomycetes. Takahashi e£_al, (41) found
that when 500 ppm PCNB was incorporated into Martin's rose
bengal agar, numbers of actinomycetes from soil dilutions were
drastically reduced. At 1000 ppm, PCNB also reduced numbers
of soil bacteria. Davis (8) also found a very great reduction
of numbers of actinomycetes colonies on soil dilution plates

with water agar amended with 50 ppm PCNB. Davis also observed

9.
a reduction of bacterial coloniés on soil dilution plates
treated with 50 ppm PCNB. For the enumeration of soil bacteria,
he used soil extract agar, a medium which will also support
actinomycetes. Because many actinomycetes were macroscopically
indistinguisable from bacterial colonies, both actinomycetes
and bacteria were reported as bacteria. Therefore, the reduc—
tion of colonies observed may have been due to suppression of
actinomycetes rather than bacteria.

PCNB is often cited in the literature as an example of
a narrow spectrum soil biocide, affecting only soil fungi (22).
Strangely enough there have been no reports dealing with the
effects of PCNB soil treatment on actinomycetes and bacteria.
However, because of the recent Work of Davis (8) and Takahashi
gt_al, (41) showing that PCNB drastically inhibits growth of
actinomycetes in_vitro, the selective activity of PCNB in soil
might be questioned.

Stability of PCNB: PCNB has been reported to have a

 

long lasting effective residue in soil. Scheffer and Haney
(35) found that PCNB allowed good seed germination and growth
of sweet pea throughout 24 weeks in steamed soil artificially

infested with Rhizoctonia solani. Kennedy and Brinkerhoff

 

(20) reported that PCNB controlled R, solani over a 4—6 week

period in nonsteamed sandy loam soil at ph 6.0. Other

10
investigators have reported disease control of R, solani 2-12
months after soil treatment with PCNB (3, 17, 33).
Recent evidence from gas chromatographic assay methods
suggested that PCNB may be changed in soil. Chacko (7) has
reported that PCNB can be altered in_yi£r2_by several soil

Streptomyces. Most or all of the PCNB was converted to

 

pentachloroaniline (PCA). Very recently W. H. Ko (unpublished
data) found that PCNB was rapidly converted to PCA in soil
submerged with water. Apparently, anerobic conditions are
essential for conversion. Ashworth gt_al, (4) suggested that
PCNB may be changed in soil into one or more fungitoxic prod-
ucts which may account for the long term disease control by
PCNB. Of 100 ppm of PCNB added, only 12—32 ppm could be
detected by gas chromatographic analysis after 160 days in a
non-sterile field soil. During this time PCNB effectively
controlled 3. solani root rot on bean seedlings. Unidentified
substances were recovered from PCNB-treated soil, but not from

untreated soil.

MATERIAL AND METHODS

Chemical and physical properties of PCNB: PCNB is
produced by Olin Chemical Corporation under the trade name of
Terraclor. Commercial formulations containing 10-75%.PCNB
are available. A 20% wettable powder was used in most experi-
ments discussed in this thesis. A crystalline preparation
(99% purity) was used when PCNB was incorporated into agar
media. PCNB is readily soluble in most nonpolar solvents,
such as acetone, benzene, toluene, xylene, carbon tetrachloride,
carbon disulfide and chloroform, and is less soluble in more
polar solvents such as methanol, ethanol and isopropanol. PCNB
was dissolved in acetone prior to adding to water in some
experiments. It is relatively insoluble in water. The melting
point is 142-145 C. The vapor pressure at 20 C is 4 mm of Hg.
(32) .

Source and preparation of soil: Conover loam from

 

Michigan State University Botany farm was used in all tests
unless specified otherwise. Water holding capacity (WHC) of
this soil was 42.7% and organic matter content was 3.8%; pH
was 6.7. The soil contained 7.5% clay, 42.8% silt and 49.7%

11

12
sand. The soil was collected from an area which has been
fallow for several years and to‘which pesticides had not
recently been applied. Soil was sieved and stored in closed
plastic containers at 15%.moisture (35%.WHC) at 23-27 C. Such
soil will be referred to as natural soil. Unless otherwise
stated, soil moisture was maintained at 35%.WHC throughout
experiments.

Microorganisms used in studies of the effect of PCNB

 

on growth: Fungi used were: Aphanomyces euteiches Drechs.,

 

 

Aspergillus terreus Thom., Fusarium solani (Mart.) Appel & Wr.

 

 

f. phaseoli (Burk) Snyd. & Hans., E, oxysporum Schlecht. f.

 

melonis (Leach & Currence) Snyd. & Hans., Glomerella cingulata

 

(Ston.) Spauld. & Schrenk, Helminthosporium victoriae Meehan

 

& Murphy, Mucor ramannianus Moller, Pythium ultimum Trow, g,

 

 

debaryanum Hesse, Rhizoctonia solani Kuhn, Thielaviopsis

 

 

 

basicola (Berk. & Br.) Ferr., Trichoderma viride Fr., Verti—

 

cillium albo-atrum Reinke & Berth. All fungi were maintained

 

on potato-dextrose agar, except H, victoriae, which was grown

 

on V-8 juice agar (per liter: 200 ml V-8 juice, 2 g CaC03,
20 g agar).

Actinomycetes used were: Actinoplanes philippinensis

 

Couch, Micromonospora sp. Orskov, Nocardia erythropolis (Gray

 

 

& Thornton) Waksman & Henrici, Streptomyces aureofaciens

 

13

Duggar, S, ggiseus (Krainsky) Waksman & Henrici, S, lavendulae

 

(Waksman & Curtis) Waksman & Henrici, S, scabies (Thaxt.)

Waksman & Henrici, S, venezuelae Ehrlich §E_§1,, S, virido-

 

chromogenes (Krainsky) Waksman & Henrici, Streptosporangium

 

 

roseum Couch. All actinomycestes were maintained on a
nutrient agar (per liter: 10 g maltose, 4 g yeast extract,
4 g dextrose, 20 g agar).

Bacteria used were: Agrobacterium tumefaciens (Smith

 

& Townsend) Conn., Bacillus licheniformis (Weigmann) Chester,

 

.S. subtilis Cohn emend. Prazmowski, Corynebacterium fascians

 

(Tilford) Dowson, Escherichia coli (Migula) Castellani &

 

Chalmers, Pseudomonas angulata (Fromme & Murray) Holland, 2,

 

fluorescens Migula, Rhizobium trifolii Dangeard, Serratia

 

marcescens Bizio, Xanthomonas phaseoli var sojensis (Hendges)

 

 

Starr & Burkholder. All bacterial were maintained on potato-
dextrose agar (PDA).

Soil amendments: Glucose or chitin and PCNB (20%

 

»wettab1e powder) were usually added in particulate form to
moist soil (35%.WHC). Chitin amendment was colloidal chitin
dried at 100 C and ground by mortar and pestle into a fine
powder. Colloidal chitin was prepared by the method of
Lingappa and Lockwood (26) with modifications of Lloyd (27).

In some experiments glucose and PCNB were added to soil dried

14
to l-2%.moisture by an electric fan. In these experiments
glucose was added in aqueous solution, PCNB as 20%.wettable
powder.

Nutrient analysis of soil: Carbohydrates and amino
compounds were extracted from soil by shaking equal parts of
soil and distilled water in a 250 ml erlenmeyer flask on a
wrist action shaker for 30 min. The soil suspension was
centrifuged at 10,000 X G. for 5 min. and the supernatant
was passed through a 0.22 n Millipore filter and stored at
4 C until analyzed.

Carbohydrates were determined by the anthrone method
(29). The anthrone reagent was prepared by dissolving 0.2 g
anthrone in 100 ml 7 M sulfuric acid. One ml of the sample
was added to 9 ml of the reagent and placed in a boiling
water bath for 10 min. Optical density was read at 600 mu
in a colorimeter. A standard curve was made by using glucose
at concentrations of 20, 40 and 80 ug/ml.

Amino acids and related compounds were determined by
the ninhydrin method (28). Two-tenths g ninhydrin and 0.03
g hydrindantin were dissolved in 7.5 ml methyl cellosolve,
followed by the addition of 2.5 ml of 4 N sodium acetate
buffer (pH 5.5). One ml of the sample was mixed with 1 m1
of the reagent and placed in a boiling water bath for 15 min.

The solution was then diluted with 8 ml of 50% ethyl alcohol

15
and the optical density read at 570 mu in a colorimeter. A
standard curve was prepared by using glycine at concentrations
of 4, 8 and 16 ug/ml.

The amount of glucose in extracts of glucose-amended
soil was determined by the Glucostat reagent (Worthington
Biochemical Corporation). “Glucose was extracted from the
soil by adding 20 m1 of water to 20 9 soil and shaking by
hand for 1-2 min. The soil suspension was centrifuged at
10,000 X G. for 10 min. and the‘supernatant was passed through
a 0.22 n Millipore filter. One ml of the sample, diluted such
that 1 ml contained 0.05-0.3 mg glucose, was added to 9 ml of
the Glucostat reagent, prepared accordingly to the manufac—
turer's instructions. After 10 min. one drop of 4 M HCL was
added to stop the reaction and to stablize the color. The
optical density was read at 400'mu in a colorimeter. A
standard curve was prepared by using glucose at concentra—
tions of 20, 40 and 80 ug/ml.

Soil respiration: Oxygen uptake was measured by

 

standard Warburg manometric techniques (44). Six 9 moist
soil was added to each flask and 3 or 4 flasks were used per
treatment. Experiments were run for 10-30 days at a constant
temperature of 270 C, with KOH (20%) being changed in the
center well every 2-6 days. Manometers were read twice daily.

the period of oxygen uptake measurements varying from 1/2—3

16
hrs, depending on respiratory activity of the soil. The rate
of oxygen uptake was expressed in ul per g of oven dry soil
per hour.

Enumeration of soil microbes: The numbers of propa—

 

gules of soil bacteria, fungi and actinomycetes were estimated
by the soil dilution plate technique. Soil suspensions were
prepared by adding the equivalent of l g oven-dry soil to 100
m1 sterile 0.85% saline solution for bacteria, or to sterile
distilled water for actinomycetes and fungi, followed by
blending in a Servall Omnimixer‘for l min. at approximately
4,000 rpm. Further dilutions were made with sterile 0.85%
saline solution or water. For bacteria and actinomycetes,

1 ml diluted soil suspensions was mixed with 15 ml of molten
agar at 43 C in petri dishes. For fungi, 0.5 m1 diluted soil
suspension was applied to the surface of 10-15 ml cooled agar.
Chitin agar (26) was used to support the growth of actino-
mycetes. A modified soil extract agar containing 25-50 ppm
PCNB to suppress actinomycetes was used for the enumeration
of soil bacteria (1). This method will be discussed in the
results. A modified acidified PDA medium, similar to that

of Steiner and Watson (40), was used for enumerating fungi.

A detergent, NPX (nonyl phenyl polyethylene glycol ether,
Union Carbide Corporation), at a concentration of 1000 ppm,

was added to FDA prior to autoclaving to retard fungal growth.

17

Bacteria were suppressed by acidifying the agar, after auto-
claving, by adding 1 m1 of“50% lactic acid to 200 m1 agar.

Germination of fungal spores on soil: PCNB (200 ppm)
in the form of a 20% wettable powder was mixed with air—dried
natural soil. Twenty g of the treated soil or soil without
PCNB were added to 250 m1 flasks and 3 m1 of 0.34% glucose
solution were pipetted on the soil surface, to give a final
concentration of 500 ppm. ”Soil moistened with distilled
water served as the control. The moistened soil was mixed
throughly with a spatula and passed through a sieve with 2
mm diam. meshes. At 3-hr intervals after glucose addition
the soil was added to a small petri dish (50 X 15 mm).
One-1.5 ml of distilled water was added to the soil and a
smooth surface was made with a stainless steel Spatula.

Spore suspensions of Helminthosporium victoriae, washed 3

 

times by centrifugation in glass distilled water, were then
added to the smoothed surface. ”After incubation at 23-27 C
for 6—8 hrs, the spores were stained with phenolic rose
bengal, recovered with plastic film and observed microscop-
ically (25).

In other experiments 75 g of moist soil (48%.WHC)
treated with various concentrations of PCNB (20%.wettable
powder) were placed in a 150 mm diam. petri dish, and the

surface was smoothed. Seventy five mg of moistened, finely

18
chOpped alfalfa residue were placed in a small trough
(4 X 0.5 X 0.5 cm) made at the edge of the soil surface, then
covered with a thin layer of soil. Spore suspensions of S,
victoriae, washed 3 times in glass distilled water, were
applied to the soil surface on top and at varying distances
from the residue. Spores were incubated at 23-27 C for 6-8
hrs and recovered and observed as previously described.

Stability of PCNB in soil: The stability of 100 ppm

 

PCNB (20% wettable powder) was studied in Conover loam soil
(35%.WHC) amended with 1%,g1ucose or 0.2% chitin. Treated
soil was added to 250 m1 flasks and PCNB was extracted after
0, 2, or 4 weeks incubation at 23-27 C.

PCNB was extracted from the soil in the following way:
40 ml of a mixture of hexane and isopropyl alcohol (3:1) were
added to 20 9 soil in a 250 ml flask. The mixture was shaken
on a wrist action shaker for 30 min. After sedimentation, the
solvent mixture was decanted. The extraction procedure was
repeated once. The combined extracts were diluted with the
solvent mixture such that comparisons with a 1 ppm PCNB stan—
dard could be made. An Aerograph model 600-D gas chromatograph
(Varian Aerograph Co.) with an electron-capture detector was
used to detect PCNB in the extract. A column (5 ft. X 1/8
in.) containing 3%,SE-30 on 60-80 Chromosorb W was used. In—

jection, column, and detection temperatures were 260 C, 155 C,

19
and 170 C, respectively, and the flow rate of nitrogen was

maintained at 60 ml/min.

RESUDTS

Stability of PCNB in soil: It was necessary to deter-
mine whether the microbial effects observed in experiments
with PCNB were due to PCNB or some byproduct(s) of PCNB.
Experiments were therefore designed to test the stability of
PCNB in soil under the conditions of tests done in this
research on the effects of PCNB on soil microflora.

The stability of PCNB was tested in Conover loam soil
amended with 0.2%.chitin or 1% glucose and 100 ppm PCNB.
Analyses of soil amended with glucose and PCNB were made 1
and 2 weeks following amendments. Analyses of soil amended
with chitin and PCNB, or with PCNB only were made 1, 2, 3, and
4 weeks after treatment. No detectable alteration of PCNB
occurred during the time periods tested (Table l).

The time intervals and amendments are similar to those
used in experiments reported in this thesis and thus it appears
likely that PCNB and not some byproduct was responsible for
the microbial effects to be reported.

The effect of PCNB on soil microbes in agar: The
effects of PCNB on growth, germination and sporulation of

20

2.1

Table 1.-—Stability of PCNB in Conover loam soil.a

W

PCNB recovered from soil treated as
Incubation period, indicated, %b
weeks

 

PCNB GlUCose + PCNB Chitin + PCNB

 

0 95 95 96
1 95 97 101
2 92 97 98
4 93 98

 

aPCNB (100 ppm) as a 20% wettable powder was added to
soil and recovered 0, l, 2, and 4 weeks later by extracting
the soil with hexane and isopropyl alcohol (3:1). The
extracts were analyzed for PCNB by gas chromatrography.

bGlucose = 1%q chitin = 0.2%.

22

several soil-borne fungi in agar have been studied (32).
Little work, however, has been done onrthe effects of PCNB
on soil bacteria and actinomycetes. In order to get a
general idea of the antimicrobial Spectrum of PCNB in agar,
2 kinds of experiments were done: 1) The inhibitory effect
of various concentrations of PCNB was tested on identified
cultures of 10 soil bacteria, 10 actinomycetes and 14 fungi,
and 2) diluted soil suspensions from natural soil were seeded
onto selective media containing various concentrations of
PCNB and the number of colonies of bacteria, actinomycetes
and fungi counted.

For experiments with identified soil microbes, diluted
spore or cell suspensions were plated onto PDA containing 1,
10, and 50 ppm PCNB. Mycelial disks from agar, rather than

spores, were used as inocula for Aphanomyces euteiches,

 

Pythium ultimum, S, debaryanum and Rhizoctonia solani. Agar

 

 

 

containing 25 ppm Triton X—100 but without PCNB served as a
control. Growth was estimated visually after 3—10 days in-
cubation at 23—25 C and compared with growth on the control.
PCNB was added to agar by the following method:
Fifty-100 mg of PCNB (technical grade or an equivalent
amount of 20% wettable powder) were dissolved in one ml of
acetone in a stoppered test tube. The acetone sterilized

the PCNB within two hr. The acetone solution of PCNB was

23

then added to 100 ml of sterileidistilled water to which
0.05 ml of a wetting agent, Triton X-100 (Rohm and Haas Co.,
Philadelphia), was previously added; PCNB was precipitated
as a milky suspension. Suitable amounts of the PCNB suspen—
sion were then added to molten agar at 43-50 C. .Tribon X-100
at the concentrations used, 15-25 ppm in the agar, had no
suppressive effect on fungal, actinomycete or bacterial
numbers.

PCNB partially or completely inhibited growth of 10
of 14 fungi tested (Table 2). At a concentration of 10 ppm,

PCNB completely inhibited growth of S, victoriae and S,

 

solani (isolate 2). S, euteiches, S, cingulata, S, ultimum,

 

 

R, solani (isolate l) and T, viride were partially inhibited

by 10 ppm PCNB. S, oxysporum f. melonis, g, ramannianus and

 

 

:2. albo-atrum.were partially inhibited at 50 ppm. Fungi un—

 

affected by PCNB at 50 ppm were 5, terreus, S, solani, S,

debaryanum and T, basicola.

 

 

The actinomycetes A, philippinensis, Micromonospora

 

 

sp., S, gTiseus, S, scabies and S, venezuelae were completely

 

inhibited by PCNB at 50 ppm. Micromonospora Sp. was also

 

completely inhibited by 10 ppm and partially inhibited at 1

ppm. S, erythropolis and S, roseum were partially inhibited

 

at 50 ppm. Three other actinomycetes were unaffected. Thus,

PCNB at 50 ppm completely or partially inhibited growth of 7

24

Table 2.—-Inhibitory effect of PCNB on microorganisms in
nutrient agar.

 

 

 

PCNB (ppm)
Microorganism
0 l 10 50
Actinomycetes
Actinoplanes philippinensis +a + _ _
Micromonospora Sp. + :_ _ _
Nocardia erythropolis + + + +
Streptomyces aureofaciens + + + ‘1
S. griseus + + + _
S. lavendulae + + + +
S. scabies + + + _
S. venezuelae + + + _
S. viridochromogenes + + + +
Streptosporangium roseum + + + +
Fungi

Aphanomyces euteiches + + i. +
Aspergillus terreus + + + ‘:
Fusarium solani f. phaseoli + + + +
F. oxysporum f. melonis + + + +
Glomerella cingulata + + i. :E
Helminthosporium victoriae + + _ _
Mucor ramannianus + + + +
Pythium ultimum + + + +
P. debaryanum + + + ':
Rhizoctonia solani (Isolate 1) + :_ i. ‘1
R. solani (Isolate 2) + :_ - _
Thielaviopsis basicola + + + +
Trichoderma viride + + i. ‘:
Verticillium albo-atrum + + + ‘:

 

a . . . . . . . . .
+, no inhibition of growth; :, partial inhibition; -,
complete inhibition.

25

of the 10 actinomycetes. Only Micromonospora sp. was inhib-

 

ited at concentrations lower than 50 ppm.
Of the 10 bacteria tested none was affected by 50

ppm PCNB. Bacteria tested were: Agrobacterium tumefaciens,

 

Bacillus licheniformis, S, subtilis, Corynebacterium fascians,

 

 

Escherichia coli, Pseudomonas angulata, S, fluorescens,

 

 

 

Rhizobium trifolii, Serratia marcescens, Xanthomonas phaseoli

 

 

var sojensis.

 

In experiments using diluted suspensions of natural
soil, actinomycete numbers were reduced by 65% at 1 ppm PCNB,
90% by 10 ppm and 99% at 25-100 ppm (Fig..l). Fungal numbers
were reduced 27-40% by 10-500 ppm, but unaffected by ppm.
Bacterial numbers were unaffected at concentrations of PCNB
lower than 200 ppm PCNB; at higher concentrations bacterial
numbers were greatly reduced.

That PCNB inhibits the growth of some soil fungi con-
firms the reports of others (2, 10, 11, 32, 34, 36, 37, 45).
However, the evidence that PCNB dramatically inhibits actino—
mycetes in agar is contrary to the widely held belief that
PCNB is a narrow spectrum fungicide affecting relatively few
soil microbes (22). That PCNB inhibits actinomycetes at
lower concentrations than it inhibits bacteria appears to
conflict with the opinion that bacteria are generally more

sensitive to a toxicant than actinomycetes (23).

NUMBER OF COLONIES

26

 

 

 

 

 

 

I I I 1 fl '
‘00 O O u
‘ o
BACTERIA
O
M... . .
so ' -
ACTINOMYCETES
IO . .
\
k I "Q l l
0' 1 10 ‘ so 100 200 500
PCNB (PPM)

Fig. 1.-—Numbers of actinomycete, bacterial and fungal

colonies from diluted soil suspensions plated

on media amended with various concentrations of
PCNB. Chitin agar was used for actinomycetes,
soil extract agar for bacteria and acidified PDA
+ NPX for fungi. For comparison, numbers of
colonies were adjusted on the basis of 100
colonies per plate without PCNB.

27

Selective medium for baCteria: Media used for esti-
mating numbers of soil bacteria also permit the development
of actinomycete colonies (26). Since PCNB inhibits actino-
mycetes, but not bacteria in agar, the use of PCNB in media
commonly used for the enumeration of soil bacteria was studied
in an attempt to find a medium selective for soil bacteria.

Soil suspensions were prepared as previously described.
Six plates were used for each treatment. Plates were
incubated at 23—27 C and colony counts were made after 2
weeks incubation. Microscopic examination of all non—sporing
colonies was made to ascertain if they were actinomycetes or
bacteria. All experiments were repeated at least twice.
Indicated differences were Significant at the 1% level.

Thornton's standardized agar (42), soil extract agar
and sodium albuminate agar (48) were amended with 25-50 ppm
PCNB. All media which contained PCNB had more bacterial and
fewer actinomycete colonies than media without PCNB (Fig. 2).
For example, in a typical test, the numbers of bacteria and
actinomycetes on soil extract agar without PCNB were 200 and
70 respectively, whereas there were 270 bacteria and 5 actino-
mycetes on PCNB-amended soil extract agar (Fig. 3). The
increase in numbers of bacteria'on PCNB—amended agar was
probably the result of reduced competition due to the

inhibition of actinomycetes. This was indicated by the

COLONIES I PLATE

28

 

- PCNB

250 [2:] NO PCNB

200

150

TOO

50

 

 

 

BACT AC T
THORNTON 'S
AGAR

 

 

 

 

 

‘4

BAC T ACT
SOIL EXTRACT
AGAR

 

 

 

 

BACT ACT
SODIUM ALBUMINATE
AGAR

Fig. 2.—-The effect of 50 ppm PCNB on numbers of
actinomycete (ACT) and bacterial (BACT)
colonies on 3 different media seeded with

a diluted soil suspension (10’5).

29

Fig. 3.--Colonies of bacteria and actinomycetes on soil
extract agar without or with 50 ppm PCNB. Most
of the large colonies on soil extract agar with-
out PCNB (bottom) are actinomycetes. Nearly all
the colonies on soil extract agar treated with
PCNB (top) are bacteria.

 

31

absence of any increase in bacterial numbers on PCNB—amended
agar when the distance between colonies was increased by
using soil dilutions greater than 10-5.

.To establish the usefulness of the PCNB medium with
different soil types, soil suspensions of muck, forest, or
loam soil were mixed with soil extract agar containing 25-50
ppm PCNB. For each soil type, agar with PCNB had more
bacteria and fewer actinomycetes than agar without PCNB

(Fig. 4).

Effect of PCNB on microbes in natural soil: Kreutzer

 

(24) has divided soil in which higher plants are growing into
three substrate zones; an outer zone, representing the soil
external to living roots and beyond the direct influence of
their exudates, the rhizosphere zone and the rhizoplane. In
the outer zone all utilizable substrates are occupied. This
is an area where available energy is low and organisms are
approaching or are in a state of quiescence. Soil of this
type, which I will call natural soil, was treated with 200
ppm PCNB. If PCNB treatment killed microbes in this 'quies—
cent zone,‘ leakage of nutrients from dead cells Should
prompt microbial growth and multiplication. Soil respiration,
population, and nutrient analyses were used as parameters to
estimate the effect of PCNB on soil microflora.

Oxygen uptake was measured in natural soil treated

 

 

 

 

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

     

 

 

3.; czgpcna
250 _
F' ‘ E2] NO PCNB

. w

200.. ‘ t ' ,. ..
“J I 1
2 4 i
.1 i T Q
a. T _, . T

x 150., ‘ ’ .-
W . - tr 1
u, .1
'7: I ~‘

0

U i i . I .
4
T j i" 1

so _ ‘ F _
t 1
1 1

.4 - ‘ T. J .1.
BACT ACT BACT ACT BACT ACT

MUCK ‘ FOREST lOAM

Fig. 4.--The effect of 50 ppm PCNB on numbers of
actinomycete and bacterial colonies on soil
extract agar seeded with diluted soil sus—
pensions prepared from muck, forest, and
loam soils. ’

33
with PCNB and in soil without PCNB. Respiration rates were
unaffected by PCNB during a 30 day incubation period at 27 C
(Fig. 9). Natural soil with or without PCNB took up oxygen
at a rate of 0.7 - 2.0 ul/hr/S 9 soil during this 30 day
period.

The concentration of amino compounds and sugars was
determined in natural soil with or without PCNB. Extracts
were made at daily intervals for 2 weeks after PCNB treatment.
An average of 12 ug of anthrone positive substances (sugars)
per g soil and 2.5 ug of ninhydrin positive materials (amino
acids and related compounds) per g soil were recovered from
natural soil untreated with PCNB. Concentration of nutrients
recovered from PCNB-treated soil did not differ from those
from untreated soil.

The numbers of actinomycetes, bacteria and fungi in
PCNB—treated soil and in soil without PCNB were estimated one
and two weeks after PCNB addition. PCNB had no effect on
microbial numbers (Table 3).

The results of the respiration, nutrient, and popula—
tion studies suggest that 200 ppm PCNB does not effect microbes
in natural soil.

The effect of PCNB on actinomycetes in chitin-amended
.Sgii: The effect of PCNB on the development of actinomycetes

in chitin—amended soil was studied to determine if PCNB

34

Table 3.--Effect of 200 ppm PCNB on numbers of microbes in
natural soil.

 

Colonies/g soil (X 105)a

 

 

 

 

7 days 14 days
Microorganisms
Natural Nat. soil Natural Nat. soil
soil + PCNB soil + PCNB
Actinomycetes 83 90 73 71
Bacteria 279 260 200 196
Fungi 0.42 0.48 0.44 0.44

 

aAt each time period numbers of microorganisms in
natural soil with or without PCNB did not differ signifi—
cantly (1% level).

35
inhibits actinomycetes in soil in the presence of a suitable
substrate for their growth. Numbers of actinomycetes, soil
respiration and the colonization of chitin dr0ps applied to
soil surfaces were studied. All experiments were repeated
at least twice.

The numbers of actinomycetes were estimated in soil
treated with 0.2% chitin and 10 or 100 ppm PCNB and in chitin-
amended soil without PCNB 0, 5, and 10 days after soil
treatment. Controls were unsupplemented natural soil and
natural soil treated with 200 ppm PCNB.

There were about 7 X‘106 actinomycetes per g unsupple-
mented soil. This number remained constant throughout the
experiment in natural soil and in natural soil treated with
200 ppm PCNB (Fig. 5). In chitin-amended soil, there were
about 3.4 X 107 actinomycetes 5 days after amendment and
approximately 8 X 107 10 days after chitin addition. ,Thus
the increase was approximately 5-fold over unsupplemented
soil at the 5th day and 11-fold at the 10th day. In soil
containing both chitin and PCNB there was approximately

1 X 107 at the 5th day, a 1.7-fold increase over the numbers

in natural soil, and 4 X 107 at the 10th day, a 6-fold in-
crease over natural soil. Thus in PCNB-treated soil

actinomycete numbers were suppressed by about 70% 5 days

after amendment and by about 40% at 10 days.

36

 

 
   
 

 

 

 

 

 

so
.3"
o CHITIN (0.2%) u
E. .0 -
=2
9.
a CHITIN(0.2%) +
5
).
g 20" «CHInN(o.2%)+ -1
5 PCNB(IOOppm)
<
441 4;
CONTROL or PCNB(200 ppm)
. a s: .19
DAYS

Fig. 5.——Effect of PCNB on numbers of actinomycetes in
natural soil and chitin—amended soil.

37

After ten days the surface of chitin-amended soil
was covered with white colonies of actinomycetes, whereas
few colonies appeared on the surface of soil treated with
chitin and PCNB. To further substantiate this observation,
6 drops of a thick colloidal chitin suspension were placed
on the surface of natural soil treated with 0, 10, 50 and
100 ppm PCNB. One week later it was noted that actinomycetes
had heavily colonized the chitin drops on natural soil
without PCNB, but colonization of drops on PCNB-treated
soil was suppressed (Fig. 6). Suppression was progressively
greater as the concentration of PCNB increased. The observa—
tion that PCNB suppressed colonization of chitin by
actinomycetes further indicates that actinomycetes are
inhibited by PCNB in soil.

Another approach to estimate the effect of PCNB on
actinomycetes was to measure the rate of oxygen uptake in
chitin-amended soil treated with PCNB. Chitin was added to
soil with-50 ppm PCNB or without PCNB and respiration was
measured at daily intervals for 33 days. Natural soil with
200 ppm PCNB and without PCNB served as controls.

PCNB had no effect on respiration in soil treated
with chitin during the first 9 days. From the 9th to the
25th day after chitin amendment, soil without PCNB respired

more than soil with PCNB (Fig. 7). During this interval

38

N0 PCNB 10 pm

 

50 PPM 100 PPM

Fig. 6.—-Effect of PCNB on the colonization of colloidal
chitin by actinomycetes in soil. Six drops of
a thick colloidal chitin suspension were applied
to the surface of soil treated with PCNB. Pictures
were taken after 7 days incubation. White zones
on the soil surface are actinomycetes colonizing
the chitin.

39

 

 
 
   
  

 

 

 

l 1 l T I l I l I— I 1 I I I F I
14. u
a
8 ‘2 '- I:
2:
a.
an ab .
>4
:5
S 6 - CHITIN (0.2%) + PCNB (SOppm) "
5 4
1- C1
9.
><
C) 2F. ‘\\pe"‘~o——of’AEfftifi:::f:ffffj:f—o”.“‘-o .
l l l l l l 1‘ l l l J l 1 l l I
0 3 9 15 21 27 33
DAYS

Fig. 7.-—Effect of PCNB on oxygen uptake in soil with or
without chitin.

40
chitin-amended soil without PCNB took up oxygen at the
average rate of 10 ul/hr/S 9 soil, whereas chitin-amended
soil with PCNB took up 5 ul/hr/S 9 soil. Thus, on the
average, PCNB caused about a 50% reduction of respiration
in chitin-treated soil. Natural soil treated with 200 ppm
PCNB respired at the same rate as natural soil without PCNB
during the 33 day period of the experiment.

The effect of PCNB on fungi in glucose-amended soil:
To study the effect of PCNB on fungal numbers in soil, soil
treated with 100 ppm PCNB was amended with 0.2%.or 1% glu—
cose. Controls were unamended natural soil and natural soil
plus 100 ppm PCNB. The numbers of fungi were estimated by
the soil dilution plate technique 0, 2, 4, 6, 8 and 14 days
after amendments.

The numbers of fungi increased more in soil contain-
ing 0.2%.glucose and PCNB than in soil with glucose only
(Fig. 8). Especially striking was the rapid early increase
in numbers of fungi in soil treated with glucose and PCNB.
The numbers of fungi in this soil more than doubled during
the first 2 days whereas the fungal numbers in soil treated
with glucose alone did not increase during this period.
After 2 days, the rate of increase in the numbers of fungi
were nearly the same in both treatments, although numbers

in PCNB-treated soil continued to be higher than those in

41

 

 

 

 

 

 

 

 

 

I I I I I
20 I. all
——c
u, . \oLucoss (0.2%) + PCNBOOO ppm) .
'2
x 12 4 ..
5 Kowcosetoms)
m
S a - ~
0
z
E
4 .. (CONTROI. or PCNB (100m) ..
4—0
l l l 1 1
o 2 4 a a 14
DAYS

Fig. 8.—-Effect of PCNB on numbers of fungi in natural
soil and glucose-amended soil. Fungi from
soil treated with glucose and PCNB were of
2—3 types. A Fusarium sp. was predominant.
Dilution plates from natural soil and soil
treated with glucose supported many different
fungal genera.

42‘
soil amended with glucose only. Similar results were obtained
in 5 other experiments using 0.2%.glucose and in 3 additional
experiments using 1%.glucose. 'Numbers of fungi were un-
affected when 100 ppm PCNB were added to natural soil.

The observation that numbers of fungi increased more
in soil treated with glucose and PCNB than in glucose only
seems to conflict with the results in agar that fungi were
inhibited by PCNB. However, nearly all of the fungi from
soil treated with glucose and PCNB were of 2-3 types, as
indicated by colony growth and pigmentation on agar. One
of the types, representing about 50% of the total number of
colonies, was a Fusarium Sp. whiCh proved to be unaffected
by 100 ppm PCNB in agar. The other types could not be in-
duced to Sporulate and therefore identifications were not
made. Dilution plates from glucose-amended soil without
PCNB supported many different types of fungi, not differing
largely from those from nonsupplemented natural soil.

Other evidence that Fusaria increase in PCNB-treated
soil was obtained with the use of Fusarium oxysporum f.
melonis, isolate I-5. One g of sandy soil infested with
approximately 1 X 106 chlamydospores of S: oxysporum f.
melonis was added to 9 g of dry sandy loam soil. The soil
'was adjusted to 15%.moisture. The infested soil was treated

‘with 0.2%.glucose and 100 ppm PCNB or with 0.2% glucose only.

43
The numbers of fungi were estimated 2 and 7 days after amend-
ment by the soil dilution plate technique using an acidified
PDA + TMN (trimethyl nonyl ether of ethylene oxide) (5). The
medium was prepared as previously described for the selective
medium for fungi, except that TMN was used instead of NPX.

On this medium S, oxysporum f. melonis isolate I-5 deve10ps

 

a purplish color, which aids in its distinction from other
fungi.

No significant increases in fungal numbers occurred
up to 2 days. Seven days after the addition of PCNB and glu-

cose the numbers of S, oxysporum f. melonis propagules were

 

greater and other fungi were fewer in soil treated with
glucose and PCNB than in soil treated with glucose only
(Fig. 9). For example, in one experiment, 7 days after soil

treatment, there were 3 X 105 propagules of S, oxysporum f.

 

melonis per g of PCNB-treated soil and 1.8 X 105 propagules
of this fungus in soil treated with glucose only. On the

other hand there were 4 X 105 fungi other than S, oxysporum

 

per g of soil treated with glucose and 2 X 105 per g in soil
treated with glucose and PCNB. Similar results were obtained
in 2 other experiments.

Stimulatory effect of PCNB on F. oxysporum f. melonis

and F. solani f. phaseoli: It seemed likely that the in-

 

crease in the number of propagules of Fusarium was related

44

 

4° " - F. OXYSPORUM f. MELONIS FT
S OTHER FUNGI

0
O
T

 

 

FUNGI lg (X104)
N
O
I

3
r

 

 

 

 

 

 

 

NATURAL GLUCOSE + GLUCOSE
SOIL PC NB

 

Fig. 9.——Effect of 100 ppm PCNB on numbers of Fusarium
oxysporum f. melonis, I-5 and other fungi in
natural soil or in soil amended with 0.2%
glucose. There were more S, oxysporum f.
melonis and fewer other fungi in soil treated
with glucose and PCNB than in soil treated
with glucose only (1% level).

 

45
to the suppression of other fungi PCNB. However there was
the possibility that Fusarium spores or mycelia were directly
stimulated by PCNB and the increased Fusarial activity was
not related to reduced competition. This possibility was
tested in 3 ways: 1) In three experiments chlamydospores

or conidia of S, oxysporum f. melonis and S, solani f. phaseoli

 

failed to germinate on the surface of natural soil contain-
ing 100 ppm PCNB. 2) No increase in germination of
chlamydospores or conidia, or in growth of mycelium of

either fungus was observed on agar with 100 ppm PCNB as
compared with agar without PCNB in 3 tests. 3) In 2 experi-
ments chlamydospores of either fungus were added to autoclaved
soil with or without 100 ppm PCNB. The numbers of propagules
of Fusarium were estimated by the soil dilution plate tech-
nique 3 and 7 days after infestation. There was no evidence
of Fusarium stimulation by PCNB; in fact the converse was
true, numbers of propagules were usually less in the PCNB-
treated soil. .For example, in the case of S, solani f.
phaseoli, 7 days after infestation there was a 48—fold
increase in numbers in PCNB-treated soil over the number
originally added and an 84-fold increase in autoclaved soil

without PCNB. Results with S, oxysporum f. melonis were

 

similar.

46

The effect of PCNB on bacterial numbers in glucose—

 

amended soil: Numbers of bacteria were estimated in soil
amended with 1%.glucose and 100 ppm PCNB or with glucose only
at intervals up to 10 days after amendment. Natural soil
with or without PCNB served as controls.

In unsupplemented natural soil there were 5 X 106
bacteria per g of soil. During the time intervals tested
the number of bacteria were unaffected when 100 ppm PCNB
only was added to this soil (Fig. 10). From 1-10 days after
amendments numbers of bacteria were greater in soil treated
with glucose and PCNB than in soil treated with glucose only.
The increase in bacterial numbers with PCNB was especially
striking at the 5th day when there were 2.5 X 108 bacteria
in soil treated with glucose and PCNB, compared with 7 X 107
in soil treated with glucose only. At the 7th and 10th days
there were still more bacteria per g of PCNB-treated soil
than in soil without PCNB.

The results with bacteria were very similar to those
with fungi, in showing that those soil microbes insensitive
to PCNB, i.e., bacteria and certain fungi, such as Fusarium
Spp., increased more in PCNB-treated soil than in soil with-
out PCNB. PCNB treatment evidently created a partial 'bio-

logical vacuum' in glucose—amended soil by the inhibition of

actinomycetes and fungi. Those microbes insensitive to PCNB

 

 

 

 
     

 

 

 

I T I I I
25
(GLUCOSE (1%)
+ PCNB" 00)
20-
C
2
X
g 15~
0*
as
Q oLucosstm)
< A!
E 10-
E; 4;,
.<
I.
5 :-
[CONTROL or PCNB (IOOppm)
t s—e_r ‘ 4e—4 43
L 1 | —L i l
O I 3 5 7 10
DAYS
Fig. 10.—-Effect of PCNB on numbers of bacteria in soil

with or without glucose.

 

48

quickly increased to fill such a vacuum.

The effect of PCNB on the rate of glucose utilization
in soil: Inasmuch as PCNB partially suppressed the metabolic
activity of PCNB-sensitive actinomycetes and fungi in
nutrient-amended soil, the rate of utilization of an energy
source by soil microbes may be less in PCNB-treated soil
than in soil without PCNB. Thus, as another measure of the
effect of PCNB on soil microbes, 0.05% or 1%.glucose was added
to soil with or without 100 ppm PCNB. At varying intervals
after glucose addition the amount of glucose remaining was
determined.

During the first 2 days, the rate of 1% glucose
utilization was the same in the PCNB-treated soil and in soil
without PCNB. From the 3rd to the 5th day glucose disappeared
faster in glucose-treated soil than in soil treated with glu-
cose and PCNB (Fig. 11). At the 6th day, glucose had almost
disappeared from both soils. The lowered rate of glucose
utilization in PCNB-treated soil may be related to the suppres-
sion of PCNB-sensitive actinomycetes and fungi in that soil.,

The effect of PCNB on the rate of glucose utilization
was also tested in soil treated with 0.05% glucose and 200
ppm PCNB. Glucose was utilized at a slightly lower rate in
PCNB-treated soil than in soil without PCNB (Fig. 12).

Similar results were obtained in 4 experiments. The largest

mg GLUCOSE lg SOIL

49

 

 

 

Fig.

 

ll.——Effect of PCNB on the rate of glucose
utilization in soil.

 

50

 

 
 
   

 

 

 

m I l I I F l _
400
«GLUCOSE(0.05%) + PCNB(200ppm)
:‘
8 300 J
o
\
“J
3 cwcose (0.05%) J
S
a 2G3 —
g
100- _
0t fl
L 1 1 l t
o s 45 - 12 15 T1

HOURS

Fig. 12.——Effect of PCNB on the rate of glucose
utilization in soil.

51

difference in the amount of glucose was detected at the 15th
hour. For example, in one experiment 100 ug of glucose re-
mained per g in soil treated with glucose only and 150 ug/g
in soil treated with glucose and PCNB.

The effect of PCNB on soil respiration in glucose—

 

amended soil: To study the effect of PCNB on the activity

 

of microbes in glucose—amended soil, respiration was measured
at daily intervals for an 8 day period in soil amended with
1%,glucose and 100 ppm PCNB and in soil treated with glucose
only. Natural soil with 100 ppm PCNB and without PCNB
served as controls. .
For the first 2 days soil treated with glucose and
PCNB respired at the same rate as soil treated with glucose
only (Fig. 13). Respiration in soil treated with glucose
increased to a maximum of 75 ul/hr/g soil at 3 1/2 days.
After 3 1/2 days the rate of respiration rapidly declined,
until at 7 days, it remained at about 10 ul/hr/g. In PCNB-
treated soil oxygen uptake reached a maximum of 58 ul/hr/g
at the 5th day, then declined rapidly. Respiration rates
in both soils were about the same at the 7th and 8th day.
Evidently, in soil with glucose but no PCNB the popu—
lation of microorganisms able to utilize glucose increased
rapidly and a peak of maximum respiration occurred very

quickly. In soil treated with glucose and PCNB, the buildup

80

b 0
O O

OXYGEN UPTAKE (pl/hrlg sou)
N
O

52

 

 

GLUCOSE (1%) + _
PCNB (100 ppm)
‘CONTROL or PCNB (100 ppm) ‘
O

GLUCOSE(T%)

i

 

 

-

 

DAYS

Fig. l3.——Effect of PCNB on oxygen uptake in

soil with or without glucose.

53'
of a large respiring population was probably delayed by the
suppression of PCNB-sensitive microorganisms. The rapid
decline in both soils was probably due to exhaustion of glu-
cose.

Germination of conidia of Helminthosporium victoriae

 

and chlamydospores of Fusarium solani f. phaseoli on PCNB-

 

treated soil: Results from the previous two sections have

 

indicated that competition for glucose may be suppressed in

soil containing PCNB. S, victoriae and S, solani f. phaseoli

 

 

are fungi which are relatively insensitive to soil fungi—
stasis, i.e., their Spores require very low nutrient levels

to germinate in natural soil (G. W. Steiner, unpublished
data). Because very small differences in the amount of
available nutrients are reflected in the amount of germination,
spore germination of these fungi was used as a bioassay to
detect the effect of PCNB on nutrient competition. Two types
of tests were conducted: 1) Germination of conidia of S,

victoriae and S, solani was tested in glucose-amended soil

 

with or without PCNB, and 2) Germination of S, victoriae was

 

tested in alfalfa residue-amended soil with or without PCNB.

Tests with S, victoriae will be discussed first.

 

Glucose «105%) and ascorbic acid (0.05%) were added
to soil with or without 200 ppm PCNB. Ascorbic acid was

added to insure complete germination on soil (21). Natural

54"
soil with or without 200 ppm PCNB served as controls. After

amendment, washed Spores owa, victoriae were placed on the

 

surface of the soil at 3-hr intervals for 15 hrs. Germination
was determined after 6-8 hrs incubation.

No Spores germinated on natural soil with or without

PCNB. The germination of S, victoriae spores was greater,

 

at all time intervals, on soil treated with glucose and PCNB
than on soil with glucose only (Fig. 14). For example, 72%
germination occurred at the 0, 3, and 6 hr intervals in soil
treated with PCNB and glucose, whereas germination in soil
treated with glucose only declined from 57%.at 0 hr to 29%
at 6 hr. The difference in germination at time 0 was probably
due to the fact that spores took 6-7 hrs for complete germi-
nation; during this time period glucose may have been
utilized faster in glucose—amended soil than in soil with
PCNB and glucose. From the 6th—15th hrs, germination was
still higher on PCNB-treated soil than on soil without PCNB.
In another test using conidia of S, victoriae, 75 mg
of moistened alfalfa residue were buried in a trough in soil
treated with 50, 100, or 200 ppm PCNB, or in soil without

PCNB. Washed Spores of S. victoriae were applied to the

 

soil surface at varying distances from the residue.
More Spores germinated near the residue in soil amended

with PCNB than in untreated soil (Fig. 15). For example,

55

 

(GLUCOSE (0.05%) + PCNB (200m)

 

    

aw- .
2
Q L 4
'é
- 4o- 4
5
:5 .. (mucosa (0.05%) _.
20-

 

-
-
n
h
b

 

 

HOURS

Fig. l4.--Effect of PCNB on germination of conidia of
Helminthosporium victoriae on glucose—amended
soil. Washed conidia were placed on the soil
surface at 3—hr intervals for 15 hrs after
soil amendment. No spores germinated on
natural soil with or without PCNB.

56

 

80-

o
O
I

    
 

PCNB (200ppm)

GERMINA‘I’ION ( %)
b
O
I I

20-

 

_
b
p
_
h
b

 

 

DISTANCE FROM assuous (cm)

Fig. 15.——Effect of PCNB on germination of conidia
of Helminthosporium victoriae on soil.
Seventy—five mg of moistened alfalfa resi—
due were buried in a trough in soil treated
with PCNB and conidia of S, victoriae were
added to the soil surface at varying
distances from the residue. Soil without
PCNB served as control.

 

57

germination at 1 cm was 60, 50, 40, and 31% in soil treated
“with 200, 100, 50 ppm PCNB and untreated soil, respectively.
Differences of this magnitude continued until the 5th cm.
No differences in germination were detected directly on top
of the residue. These data indicated that nutrients diffusing
from the alfalfa residue were utilized more slowly by micro-
organisms in soil treated with PCNB than in soil without
PCNB, thus providing more nutrients for spore germination.

Tests using 1%,glucose indicated that the rate of
glucose utilization in PCNB-treated soil was slower than that
in soil without PCNB (Fig. 11). Chlamydospores of S, solani

f. phaseoli were used as a bioassay to confirm these data.

 

Chlamydospores were applied at various time intervals to soil
treated with 1% glucose and 200 ppm PCNB or with glucose only.
Natural soil with or without 200 ppm PCNB were controls.

No spores germinated on natural soil either with or
without PCNB. During the 0-10 day interval 1.5—2 times as
many spores germinated on PCNB-treated soil amended with glu-
cose than on soil with glucose only (Table 4). The low
germination in the 4-5 day interval may be the result of the
production of antibiotic or'staling products from soil
microbes, or the unavailability of essential mineral elements,
since sufficient glucose was present to support germination

(Fig. 11). Large numbers of chlamydospores germinated at the

58

Table 4.——Effect of 200 ppm PCNB on germination of Fusarium
solani f. phaseoli chlamydospores in glucose-
amended soil.

 

. . a
Germination (%)

 

 

Days after
amendment of soil Glucose + PCNB Glucose

0 96 94

2 56 51

4 5 0

5 0 0

6 58 24

7 ' 54 32

8 68 0

9 29 2

10 0 0
Total i 366 203

 

aData are from one of two experiments with similar
results. Two hundred spores were counted in each treatment
for each experiment. No spores germinated in natural soil
with or without PCNB.

59
6-9 day interval in PCNB-treated soil even though glucose

could not be detected in these soils. Their germination might
have been supported by metabolic or autolytic byproducts of

microbes.

DISCUSSION

The present study was initiated to clarify some con—
flicting and controversial aspects of past research on PCNB.
Three areas needing clarificatiofi were questions of the
stability of PCNB in soil, the antimicrobial spectrum of
PCNB, and PCNB-induced disease exchange.

The stability of PCNB in soil has been tested by 2
different methods: 1) Determination of the viability of
Rhizoctonia solani in soil by seedling tests, and 2) chemical
analyses of the extracted fungicide by gas chromatrography.
In almost all earlier studies the stability of PCNB has been
studied by method 1. Results from these tests indicated that
PCNB was relatively persistent in soil (3, 17, 20, 33, 35).
However, this method would provide no information as to
changes in the chemical structure of PCNB if such changes did
not affect the toxicity of the fungicide against S, solani.
In other words, the persistence of a degradation byproduct of
PCNB may have been estimated, rather than the stability of
PCNB itself. A recent unconfirmed report in which gas chro—
matrography was used for analysis, indicated that PCNB was

60

61 ‘
changed in soil into one or more fungitoxic products which
may account for the long term disease control by PCNB (4).

Results from my research*generally confirmed the re-
sults from bioassay methods; PCNB was not changed in soil,
at least not in 4 weeks. The purpose of stability tests in
the present research was to determine if effects on micro-
organisms were due to PCNB or some byproduct. It is obvious
that more work needs to be done in the greenhouse and field
over longer periods before the question of PCNB stability is
solved. It will be difficult to interpret data from soil
treated with PCNB until we are sure that effects observed
are due to PCNB and not to some byproduct of PCNB.

PCNB has gained the reputation of being inhibitory
to only a few organisms (9, 22, 23). The evidence supporting
this premise is that PCNB strongly inhibits some soil fungi
but is relatively ineffective against others (23); perhaps
more importantly, it is generally regarded as innocuous to
soil bacteria and actinomycetes 19).

Two reports that PCNB may affect microbes other than
fungi were those of Davis (8) and Takahashi gE_ST, (41), who
found that actinomycetes were drastically inhibited in agar
by PCNB. Results from my research confirmed these observa-
tions, and therefore, were contrary to the generally accepted

idea that PCNB acts solely as a selective soil fungicide.

62
For example, 7 out of 10 species of actinomycetes were par—
tially or completely inhibited in agar by 50 ppm PCNB. More
significantly, the number of actinomycete colonies arising
from diluted soil suspensions was suppressed by about 90%
on agar containing 10 ppm PCNB. Results of respiration and
population studies in chitin-amended soil also Showed that
actinomycetes were inhibited by PCNB. Thus, it was obvious
that actinomycetes were greatly affected by PCNB in agar and
in soil. It is strange that this fact has generally been
overlooked, for PCNB is a very pOpular fungicide with a
hundred or so papers published on its antimicrobial activi-
ties (23). The fact that Davis' work is unpublished and
Takahashi's paper was published in a Japanese journal probably
account for the lack of notice of their works.

In some kinds of experiments actinomycetes were more
strongly inhibited by PCNB than in others. For example,
actinomycete numbers from a natural soil population in soil
dilution plates were reduced 90% by 10 ppm PCNB, whereas
numbers of actinomycetes in chitin—amended soil were only
reduced 40 to 70% at this concentration. More significantly,
only 1 of the 10 identified actinomycetes was inhibited at
this concentration. The differences in sensitivity in these
tests could be explained in several ways: 1) The heavy

inoculum load (mycelia and Spores) in chitin—amended soil and

63

in tests with identified cultures may have been sufficient to
overcome the biostatic effect of PCNB. The source of colonies
of actinomycetes from natural soil suspensions, on the other
hand, was probably a single spore (39). Mycelial growth may
have been less affected by PCNB than was spore germination.
3) The 10 identified actinomycetes chosen may not represent
a random sample in terms of sensitivity to PCNB. 4) There
may be a buildup of a PCNB-tolerant population of actinomycetes
in chitin-amended soil treated with PCNB. 59 PCNB may be less
effective in inhibiting actinomycetes in soil than in agar.

PCNB inhibited the growth of several fungi in agar
and soil. These results generally agree with data from other
workers. PCNB is generally regarded as innocuous to soil
bacteria. The evidence supporting this premise is that PCNB
had no effect on respiration in natural soil (9). This
observation was interpreted to mean that PCNB had no effect
on actinomycetes or bacteria in soil and that the fungi in-
hibited by PCNB contributed little to soil respiration.
Results obtained in the present work confirmed Vredeveld's
(47) observation that the antimicrobial effects of PCNB are
not expressed unless microbes are metabolically active.
Thus, the testing of the effects of PCNB on soil microbes by
measuring soil respiration in unsupplemented natural soil

where the activity of heterotrophic microorganisms is minimal

64
is of little significance. The premise that soil bacteria
are unaffected by PCNB was substantiated by the observations
in this work that bacteria were unaffected by PCNB in agar
or glucose—amended soil; at least at concentrations lower
than 200 ppm.

When PCNB is added to soil, an increase in seedling
damage by pathogenic fungi not sensitive to PCNB occasionally
occurs (6, 12, l3, 16, 22, 46). It has been suggested that
the increased activity of these pathogens following soil
treatment by PCNB may be due to the suppression of specific
fungal antagonists of these pathogens (6, 12, 16, 46). This
hypothesis is not unreasonable considering the widely-held
view that the inhibitory activity of PCNB was restricted to
soil fungi. However, the evidence that PCNB suppresses growth
of actinomycetes as well as growth of fungi in nutrient-
amended soil, suggested that certain fungi insensitive to
PCNB may benefit by soil treatment with PCNB because of re—
duced competition for nutrients rather than by suppression
of specific antagonists.

Two lines of evidence indicated that microbes inseni-
tive to PCNB benefited by soil treatment with PCNB: l)
Bacteria and Fusarium Spp., both of which are relatively
insensitive to PCNB, increased to a greater extent in PCNB—

treated soil amended with glucose than in soil with glucose

65

only. 2) Conidia of Helminthospgrium victoriae and chlamydo—

 

spores of S, solani f. phaseoli germinated more in PCNB-treated
soil with glucose than in soil with glucose only. Since
germination of these two fungi was not directly affected by
PCNB, it seemed likely that PCNB treatment resulted in
increased availability of glucose to these spores.

Two additional lines of evidence indicated that PCNB
reduces nutrient competition in soil: 1) Glucose was
utilized slower in soil treated with PCNB than in soil with-
out PCNB and 2) the buildup of a respiring population was
delayed by PCNB in soil treated with glucose.

In natural soil, the rhi20sphere or the vicinity of
dead organic matter are zones where the effects of PCNB on
the microbial population would be expressed, for they provide
energy sources capable of supporting metabolic activity and
growth. It would seem likely that one of the factors involved
in PCNB-induced disease exchange'would be the reduction of
competition for nutrients by the inhibition of actinomycetes
and fungi in the rhizosphere, thus permitting fungal pathogens
insensitive to PCNB to flourish in the infection court.
Possibly, by the same means, PCNB—insensitive pathogens
could also increase their inoculum density in dead organic
matter.

Other factors than microbial competition might also

66
play a role in disease exchange. For example, the possibility
of phytotoxicity due to PCNB and'its effects on disease must
be considered. In at least 2 instances, disease increases
occurred when plants were injured by PCNB (18, Y. Katan,
personal communication). In a preliminary experiment in this
research, muskmelons were injured by 100 ppm PCNB in soil and
the stunted melons were much more susceptible to Fusarium

oxysporum f. melonis than melons in soil without PCNB.

 

In brief summary the results of this research are as
follows: 1) PCNB had no effect on soil microbes in nonsupple—
mented natural soil. 2) PCNB suppressed the activity of
sensitive actinomycetes and fungi in soil supplemented with
nutrients. 3) The suppression of PCNB—sensitive microorgan-
isms reduced competition for organic carbon sources. 4)
Because of reduced competition, PCNB-insensitive fungi and
bacteria increased more in nutrient—amended soil with PCNB
than in soil without PCNB. These conclusions are illustrated

in Fig. 16.

67

EFFECTS OF PCNB ON THE SOIL MICROFLORA

O \0/
$ 1
9"“
\ :3“ 3,}
o o NUTRIENTS 9 o o.-—
..‘0/ If.
4'0
r
*/6%
‘ac
flc
4M9“ 0
o o\° o/
O
°oP
O o O
. — FUNGAL SPORE °/ ‘0:
(INSENSITIVE To PCNB)
—— - ACTINOMCYCETE

o — BACTERIUM
—- - FUNGUS

Fig. 16.——A summary of the effect on numbers of
actinomycetes, bacteria, fungi and
germination and sporulation of a PCNB-
insensitive fungus by A) PCNB in
natural soil, B) nutrients in natural
soil, and C) PCNB in nutrient—amended
soil.

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Arndt, C. H. 1953. Evaluation of fungicides as protec—
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Ashworth, L. J. Jr., J. D. Price and B. C. Langley.
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Banihashemi, Z. 1968. The biology and ecology of
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Bird, L. S., C. D. Ranney and G. M. Watkins. 1957.
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68

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71

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32. Reavill, M. J. 1954.
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some fungi.

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