EFFECTS OF FUNGICIDE FREAFMENT: OF 7 ‘

SOIL ON CERTAEN COMPONENTS
OF THE SOIL MICROFLORA

, Thesis fair-{ho nghé of pit. D 7
MICHEGAN STATE UNIVERSITY
Robert A; Davis »
1965 I

THEBlS

LIBRARY

Michigan ~'

 

This is to certify that the

thesis entitled

EFFECTS OF FUNGICIDE TREATMENT OF SOIL
ON CERTAIN COMPONENTS
OF THE SOIL MICROFLORA

presented bg

Robert A. Davis

has been accepted towards fulfillment
of the requirements for

Ph. D. _ Plant Pathology
____ degree 1n_____

flue/,0 .44

Majdrjrofessog/

Date November 18, 1965

0-169

  

‘W W" (WY

 

ABSTRACT
EFFECTS OF FUNGICIDE TREATMENT OF SOIL
ON CERTAIN COMPONENTS
OF TEE SOIL MICROFLORA

by Robert A. Davis

Various narrow and broad spectrum non-volatile fungicides were
investigated to determine their effects on soil microflora. iMineral
and organic muck soils were treated with fungicides, sampled at approx-
imately weekly intervals for four to six weeks,and assayed for changes
in numbers of actinomycetes, bacteria and fungi. Additional observat-
ions were made on the effects of the fungicides on disease producing
soil fungi, using baiting-recovery techniques and evaluation of damping-
off disease in a test crop°

Of the narrow spectrum fungicides, Dexon (p-dimethylamindbenzene
diazo sodium sulfonate) at 20 lbs./Acre and Chemagro 3-29957 (an experi-
mental fungicide selected for activity against Fusarium.sp.) at
35 lbs./Acre had no persistent effects on numbers of actinomycetes,
bacteria and fungi in mineral soil over a four week sampling period.
PCNB (pentachloronitrdbenzene) treatment of mineral soil (3O lbs./Acre)
increased counts of actinmmycetes, bacteria and fungi in natural soil

artificially infested with.Rhizoctonia. Observations on cucumber

 

damping-off disease indicated that PCNB afforded some protection against

pre-emergence damping-off by Rhizoctonia. There was no further evidence

 

of toxicity to soil microflora. On the other hand, Difolatan, a broad
spectrum fungicide (N-(l,1,2—tetrachloroethyl sulfenyl)-cis-z:-h-cyclo
hexene-l,2-dicarboximide), at 30 lbs. /Acre reduced counts of all three

groups of soil microflora for a least four weeks after soil treatment

Robert A. Davis

and there was no indication of a decrease in toxicity during that period.
A combination of two narrow spectrum fungicides, Dexon + DAC-h69

(octachloro tetrahydro thiophene) at 20 + 30 lbs./Acre respectively,

had no apparent effect on colony counts of fungi in a highly organic

muck soil artificially infested with.Rhizoctonia. In contrast, colony

 

counts of bacteria were increased by soil treatment and counts of actino-
mycetes were decreased by this fungicide combination during the three
week period. Damping-off disease of red beet seedlings in this soil

was partially controlled by the same fungicide mixture during the three
week test period.

Results indicate that the soil dilution colony count method can be
adapted to measure gross effects of fungicide treatment on soil micro-
flora. By this method a contrast was shown between the relatively
slight effects of narrow spectrum.fungicides on the soil microfloral
composition and the more severe effects of a broad-spectrum fungicide,
such as Difolatan.

In the selection of Rhizoctonia isolates for the microfloral

 

studies, a number of isolates were tested for pathogenicity to red beets
and for sensitivity to certain fungicides. A wide range of responses
was obtained. Some isolates responded differently to fungicides in

agar tests than in the soil environment. Isolate R-Bh was of particular
interest because it was inhibited by 1 ppm PCNB in fungicide agar but
was much less sensitive to PCNB in soil. Observations on control of
damping-off caused by this isolate showed a lack of disease control by
PCNB at 30 lbs./Acre. The response of another isolate was also differ~

ent from that expected as a result of the laboratory tests. This

Robert A. Davis

isolate, which was less inhibited than others by PCNB in vitro, appeared
to be controlled by PCNB in soil. It seems possible that PCNB may‘be
changed to other fungitoxic compounds in soil and this may account for
the differential response of the above isolates. The variability in

pathogenicity and sensitivity to fungicides among Rhizoctonia isolates

 

may help to explain the variability of results of fungicide tests and
will be an important factor in selecting fungicides for control of

diseases caused by'Rhizoctonia.

 

EFFECTS OF FUNGICIDE TREATMENT OF SOIL
ON CERTAIN COMPONENTS

OF THE SOIL MICROFLORA

By

“30

Robert A. Davis

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

1965

Approved /dg;¥Ld :SJ’dfl lglz“*//
'K/ CV

ACKNOWLEDGMENTS

The author extends his sincere appreciation to Dr.
Donald J. de Zeeuw, committee chairman and advisor, for the
advice and suggestions given in the course of this study,
and for assistance and criticism in the preparation of the
manuscript.

Sincere appreciation is also extended to Dr. John L.
Lockwood for suggestions relative to this work and for edit-
ing of the manuscript.

Acknowledgment is also made to the following companies
who have sponsored an assistantship for seed and soil treat»
ment research, employing the author. The funds, materials,
and technical information supplied by these companies have
been valuable aids in this research.

American Cyanamid Company

California Spray-Chemical Corporation

Chemagro Corporation

Chipman Chemical Company, Inc.

Corona Chemical Div., Pittsburgh Plate Glass Company
Diamond Alkali Company

B. I. duPont de Nemours & Company, Inc.

Eli Lilly Company

Ferry—Morse Seed Company

Gallowhur Chemical Corporation

Gerber Products Company

Haviland Chemical Company

Monsanto Chemical Company

Naugatuck Chemical Company

Niagara Chemical Div., FMC Corporation

Nitrogen Div., Allied Chemical and Dye Corporation
Norwich Pharmacal Company

Olin-Mathieson Chemical Corporation

ii

Panogen Div., Morton Salt Company
Shell Development Company
Tennessee Corporation

Union Carbide Chemicals Company
Upjohn Company

Velsicol Chemical Corporation

iii

CONTENTS

Page
ACKNOWLEDGMENTS o o o o o l o o o 0v 0 o o o o o o o o 0 ii
LIST OF TABLES o o o o o o o o o o o o o o o o o o o 0 Vi

LIST OF ILLUSTRATIONS 9 O O O 0 0 O O O 0 0 0 O 0 0 O Viii

 

 

 

 

 

 

 

INTRODUCTION 0 O 0 0 0 O O O 0 0 0 O 9 O O 0 0 O O O 0 1
REVIEW OF LITERATURE O O O O O 0 O O 0 0 0 0 O O O O 0 3
MATERIALS AND METHODS . . . . . . . . . . . . . . . . 26
EXPER IMENTAL RESULTS 0 O O O O O O O O O 9 0 O O 0 0 0 3 9
In-Vitro Studies of Fungicides. . . . . . . . . . 39
Treatment of Mineral Soil . . . . . . . . . . . . 45
PCNB
Assays for Fungi, Bacteria and Actinomycetes. 45
Rhizoctonia Recovery from PCNB Treated Soil . 55
Disease Control . . . . . . . . . . . . . . . 57
Dexon
Assays for Fungi, Bacteria and Actinomycetes. S9
Pythium Recovery from Dexon Treated Soil. . . 63
Bayer 29957
Assays for Fungi, Bacteria and Actinomycetes. 64
Rythium, Fusarium and Nematode Recovery . . . 67
Rhizoctonia, Pythium, Fusarium and Nematode
Recovery . . . . . . . . . . . . . . . . . . 68
Difolatan
Assays for Fungi, Bacteria and Actinomycetes. 69
Pythium, Fusarium and Nematode Recovery . . . 73
Rhizoctonia, Eythium, Fusarium and Nematode
Recovery . . . . . . . . . . . . . . . . . . 73
Treatment of Organic Muck Soil. . . . . . . . . . 74

Dexon+DAC-469
Assays for Fungi, Bacteria and Actinomycetes. 79

iv

Rhizoctonia and Pythium Recovery.

Disease Control

Rhizoctonia Isolates

 

Pathogenicity in Muck Soil.
Sensitivity to PCNB In-Vitro.
Sensitivity to Difolatan In-Vitro

DISCUSSION. . . .

LITERATURE CITED.

0

O

O

O

O

O

86
87

89
102
102
108

118

LIST OF TABLES

 

 

 

 

Page
Procedure for washing soil samples. Mineral
and muck soils. . . . . . . . . . . . . . . . . 33
Growth response of Rhizoctonia solani (R-54),
Pythium_i£regulare (P—lO), and Fusarium oxy—
sporum_f. cucumerigum (F—83) inocula to various
concentrations of Dexon, PCNB and OM-l921 fungi-
cides in the nutrient medium. . . . . . . . . . 41

Colonies of fungi, bacteria and actinomycetes
assayed per gram of mineral soil (air-dry
equivalent). Natural, Rhizoctonia-amended and
Rhizoctonia + PCNB soils sampled at 0, ll, 33

and 44 days after treatment . . . . . . . . . . 47

 

 

Recovery of Rhizoctonia from Dexon treated and
non-treated buckwheat stems buried in Natural,
Rhizoctonia—amended and Rhizoctonia + PCNB

soils . . . . . . . . . . . . . . . . . . . . . 56

 

 

 

Colonies of fungi, bacteria and actinomycetes
assayed per gram of mineral soil (air-dry
equivalent). Control and Dexon—treated soils
sampled at O, l, 2, 3 and 4 weeks after soil
treatment . . . . . . . . . . . . . . . . . . . 6O

Colonies of fungi, bacteria and actinomycetes
assayed per gram of mineral soil (air—dry
equivalent). Control and B-29957 treated soils
sampled l, 2, 3 and 4 weeks after soil treatment 64

Colonies of fungi, bacteria and actinomycetes
assayed per gram of mineral soil (air-dry
equivalent). Control and Difolatan-treated

soils sampled l, 2, 3 and 4 weeks after soil
treatment . . . . . . . . . . . . . . . . . . . 72

Colonies of fungi, bacteria and actinomycetes
assayed per gram of muck soil (air-dry equiv-
alent). Natural, Rhizoctonia-amended and 32;:
zoctonia + (Dexon + DAC-469) soils sampled at

 

O, l and 3 weeks (time of treatment, emergence,
and harvest). . . . . . . . . . . . . . . . . . 75

vi

Table Page

9. Overall means of fungus colony counts for
various soil treatments and sub—treatments at
the time of harvest (3 weeks after treatment
with DAC~469 + Dexon). Analysis of variance. . 76

10. Overall means of bacterial colony counts for
various soil treatments and sub-treatments at
the time of harvest (3 weeks after treatment).
Analysis of variance. . . . . . . . . . . . . . 77

11. Overall means of actinomycete colony counts for
various soil treatments at the time of harvest
(3 weeks after treatment). Analysis of variance 78

12. Stands, survival and yields of red beet seed—
lings in natural muck soil amended with Rhizoc—
tonia isolates and treated with Dexon . . . . . 9O

13. Stands, survival and yields or red beet seed—
lings in natural muck soil amended with various
Rhizoctonia isolates. Part A. No chemical soil
treatment. Part B. Dexon + Difolatan soil
treatment . . . . . . . . . . . . . . . . . . . 92

14. Stands, survival and yields of red beet seed—
lings in natural muck soil amended with 3
Rhizoctonia isolates and treated with fungicide-
chemicals. Comparison of isolate—chemical com-
binations . . . . . . . . . . . . . . . . . . . 99

 

15. Stands, survival and yields of red beet seed-
lings in natural muck soil amended with 3
Rhizoctonia isolates and treated with fungicide-
chemicals. Part A. Comparison of isolates.
Part B. Comparison of chemical treatments . . . 101

16. Inhibition of growth of various Rhizoctonia
isolates exposed to 100 ppm PCNB or 50 ppm
Difolatan in agar medium. . . . . . . . . . . . 103

 

vii

Figure

1.

LIST OF ILLUSTRATIONS

Fungus colonies assayed per gram (air—dry
equivalent) of washed and non—washed soil
samples. Natural, Rhizoctonia and Rhizoctonia
+ PCNB mineral soils at 0, ll, 33 and 44 days
on standard medium without PCNB . . . . . . . .

 

 

Fungus colonies assayed per gram (air-dry
equivalent) of washed and non-washed soil
samples. Natural, Rhizoctonia—amended and
Rhizoctonia + PCNB mineral soils at 0, ll, 33

 

 

and 44 days on standard medium containing 50
ppm PCNB. O O O O O O O O O O O 0 O O O O 0 o 0

Bacterial and actinomycete colonies assayed per
gram (air-dry equivalent) of Natural, Rhizoc—
tonia-amended and Rhizoctonia + PCNB mineral

 

soils at 0, ll, 33 and 44 days on standard

medium without PCNB . . . . . . . . . . . . . .

Bacterial and actinomycete colonies assayed per
gram (air—dry equivalent) of Natural, Rhizoc—

tonia-amended and Rhizoctonia + PCNB mineral

 

soils at 0, ll, 33 and 44 days on standard
medium containing 50 ppm PCNB . . . . . . . . .

Fungus colonies from washed and non-washed
samples of Natural, Rhizoctonia-amended and
Rhizoctonia + PCNB mineral soils at 33 and 44
days after fungicide treatment. Comparison of
the number of colonies resulting from plating
on standard medium vs. medium containing 50 ppm
PCNB. . . . . . . . . . . . . . . . . . . . . .

 

Bacterial and actinomycete colonies from samples

of Natural, Rhizoctonia—amended and Rhizoctonia

 

 

+ PCNB mineral soils at 33 and 44 days after

treatment. Comparison of the number of colonies

resulting from plating on soil extract or water
agar vs. the same medium with 50 ppm PCNB . . .

Survival of cucumber seedlings in unsteamed
mineral soil amended with Rhizoctonia (R—54)
and treated with PCNB . . . . . . . . . . . . .

 

viii

Page

46

49

51

52

53

54

58

Figure

8.

10.

ll.

12.

13.

14.

15.

l6.

l7.

Fungus colonies assayed per gram (air-dry
equivalent) of washed and non-washed soil
samples. Control and Dexon treated mineral
soils sampled at O, l, 2, 3 and 4 weeks after
fungicide treatment . . . . . . . . . . . . . .

Bacterial and actinomycete colonies assayed per
gram of Control and Dexon treated mineral soils
sampled at O, l, 2, 3 and 4 weeks after fungi-
cide treatment. . . . . . . . . . . . . . . . .

Fungus colonies assayed per gram of washed and
non—washed soil samples. Control and B-29957

treated mineral soils sampled at l, 2, 3 and 4
weeks after fungicide treatment . . . . . . . .

Bacterial and actinomycete colonies assayed per
gram of Control and B-29957 treated mineral
soils sampled at l, 2, 3 and 4 weeks after
fungicide treatment . . . . . . . . . . . . . .

Fungus colonies assayed per gram of washed and
non-washed soil samples. Control and Difolatan
treated mineral soils sampled l, 2, 3 and 4

weeks after fungicide treatment . . . . . . . .

Bacterial and actinomycete colonies assayed per
gram of Control and Difolatan treated mineral
soils sampled at l, 2, 3 and 4 weeks after
fungicide treatment . . . . . . . . . . . . . .

Fungus colonies assayed per gram of non-washed
soil samples. Natural, Rhizoctonia-amended,

and Rhizoctonia + (Dexon + DAC~4697 muck soils
at O, l and 3 weeks after fungicide treatment .

 

 

Fungus colonies assayed per gram of washed soil
samples. Natural, Rhizoctonia—amended and Rhi-
zoctonia + (Dexon + DAG-469) muck soils at O, 1

 

 

and 3 weeks after fungicide treatment . . . . .

Bacterial colonies assayed per gram of Natural,
Rhizoctonia—amended, and Rhizoctonia + (Dexon +
DAG-469) muck soils at O, l and 3 weeks after

fungicide treatment . . . . . . . . . . . . . .

 

Actinomycete colonies assayed per gram of Nat-
ural, Rhizoctonia-amended, and Rhizoctonia +
(Dexon + DAG—469) muck soils at O, l and 3
weeks after fungicide treatment . . . . . . . .

 

ix

Page

61

62

65

66

70

71

81

82

83

85

Figure

18.

19.

20.

21.

22.

23.

24.

25.

26.

Page

Survival of red beet seedlings in Natural and
Rhizoctonia—amended muck soils treated with

 

Dexon + DAC-469 fungicides. . . . . . . . . . . 88

Progress of post-emergence damping-off of red

beet seedlings in Dexon-treated muck soil nat—
urally infested with Pythium or additionally
infested with various Rhizoctonia isolates. . . 91

Survival of red beet seedlings in natural muck
soil (control) treated with Dexon, Difolatan
and PCNB. . O O O O C O O O C O O O O O O O O O 94

Survival of red beet seedlings in natural muck
soil amended with Rhizoctoni§,R—45 and treated
with Dexon, Difolatan and PCNB. . . . . . . . . 95

 

Survival of red beet seedlings in natural muck
soil amended with Rhizoctonia R—51 and treated
with Dexon, Difolatan and PCNB. . . . . . . . . 96

 

Survival of red beet seedlings in natural muck
soil amended with Rhizoctonia R-54 and treated
with Dexon, Difolatan and PCNB. . . . . . . . . 97

 

Cultural characteristics of Rhizoctonia isolates
R-41 to R-47 and R-49, R-Sl, R—53, R-S4 and
R-56, on potato dextrose agar medium. . . . . . 104

 

Growth response of Rhizoctonia isolates R-41,
R-43, R-47 and R-56 to 50 ppm Difolatan fungi-
cide incorporated in the culture medium . . . . 105

Growth response of Rhizoctonia isolates R—Sl
and R-52 to 50 ppm Difolatan fungicide incor-
porated in the culture medium . . . . . . . . . 107

 

INTRODUCTION

Application of fungicides to soil for the control
of damping-off and root rotting fungi is becoming an accepted
agricultural practice. But little is known about the effect
of this practice on soil microbes and soil borne disease
problems over a period of time.

Since most fungicides are not highly specific in
their action, there is a possibility that frequent or even
occasional application of fungicides to soils may result
in deleterious effects on organisms other than the target
organism. If beneficial organisms, such as nitrifying bac-
teria, saprOphytic fungi and saprophytic bacteria, and ac-
tinomycetes that decompose plant and animal debris are ad-
versely affected, a loss of soil fertility may occur. In
addition, removing large groups of organisms along with
the “target" organism may produce ecological shifts that
allow undesirable competitors to gain supremacy. Such com-
petitors may be as harmful or perhaps more harmful than the
target organism (57, 22).

Information on the effects of soil fungicides on
soil microorganisms is a prerequisite for safe, intelligent
and economical use of fungicides for agricultural disease

problems. In this study several fungicides, with broad or

restricted ranges of activity against fungi, were studied

for their general effect on populations of fungi, bacteria

and actinomycetes in the soil. Studies on control of damping-

off disease were made in conjunction with certain experiments.
Studies were made on the in—vitro effects of certain

fungicides on damping—off fungi and on the response of

Rhizoctonia isolates to fungicides used in this study.

REVIEW OF LITERATURE

General Effects of Pesticides on Soils

 

Effects on Physical Structure

Desirable physical structure of soil is directly
related to the aggregation of soil particles brought about
by microbial decomposition of organic residues in the soil

and some species such as Aspergillus niger, Azotobacter

 

indicum and Cunninghamella blakesleeana were found to be

 

more effective than others (66). Fungi, as a group, were
found to be most effective followed by actinomycetes, cer—
tain bacteria, yeasts, then the majority of bacterial spe—
cies tested (72). McCalla (73) later found that inocula—
tion of the soil with an effective fungus, such as Stachy—
botrys atgg, after fumigation improved soil binding. Fumi—
gants such as DD (dichloropropane—dichloropropene), chloro—
picrin, and EDB (ethylene dibromide) in laboratory tests
were reported to have no effect on aggregation (65). But
in field experiments with DD and EDB, Hansen and McCalla
(38) discovered a decrease in soil aggregation with EDB

soil treatment.

Effects on Chemical Properties
There is also considerable evidence that chemical

properties of soil may be altered by soil treatment. Such

changes may be beneficial or detrimental depending on the
properties of the soil. Use of fumigants has sometimes

led to an increased growth response greater than can be
explained on the basis of disease control alone (80).
Waksman and Starkey (106) indicated that the effect was

due largely to increased release of plant nutrient elements,
especially nitrogen, from inorganic soil constituents. Soil
microbes also contain relatively large amounts of plant
nutrient elements (107). Decomposition of the nutrient—
rich non—living fraction of the soil organic matter may

also be stimulated by soil fumigation (106, 69). Nutrient
elements may also be released from decomposing pesticide
residues such as SO4 from oxidation of carbon disulfide,

Cl from dichloropropene-dichloropropane mixture or chloro-
picrin (2, 40), P04 from oxidation of Pestox (105), and

NH3 from cyanamide (74). The acidifying effects of these
ions will also tend to solubilize plant nutrients from the
organic soil fraction (69, 70) and occasionally soil pH

may be changed (77). Several workers have measured increases
in soluble inorganic constituents, Mn, Cu, Zn, Ca, and K,
following soil fumigation as reviewed by Martin (69). In—
creases in soluble trace elements following partial soil
sterilization with fumigants or fungicides are usually not
sufficient to retard plant growth but occasionally temporary
toxicity to some plants may occur. Tobacco plants are in—

jured by 15 ppm Mn and concentrations of 1—5 ppm of copper

may be injurious to some plants. QIn soils low in micro—
elements, fumigation may stimulate plant growth by increas-
ing availability (68). Accumulation of chloride, bromine
or copper ions from pesticides may result in phytotoxicity,
especially to plants which are particularly sensitive to

them (68, 69). *

Effects on Biological Processes

Effects on Ammonification and Nitrification——Ammoni-
fication and nitrification are biological processes of pri-
mary importance in soil. The soil bacteria which oxidize
ammonia to nitrites and nitrites to nitrates are relatively
sensitive to soil fumigants and fungicides (54, 96, 98,

106, 110).

Although organisms which release ammonia from or-
ganic nitrogenous complexes quickly return to soil follow-
ing treatment, the activity of the nitrifiers may be checked
for several weeks to several months depending on the chem—
icals used, dosages, and soil properties. During this period,
ammonia accumulates from the decomposing organic fraction
of the soil and from the remains of the organisms killed
by the chemical. The inhibition of nitrification may or
may not affect plant growth. Many plants, for example pine—
apple, utilize ammonia nitrogen and some utilize it more
readily than nitrate nitrogen (69).

Pesticides other than fumigants have been found

inhibitory to nitrification and to nitrogen fixation by

leguminous plants. Metallic dithiocarbamates such as ferbam,
zineb, ziram, maneb and nabam suppressed nitrification (8,

9, 48). Dichlone, chloranil and Ceresan M were toxic to
Rhizobia and reduced nodulation of legumes (53, 46, 87).

Thiram was also quite toxic to Rhizobium (87). Captan was

 

reported to be slightly inhibitory to nodulation when used
as a soil drench (39). Species of Rhizobium, legume nodule
formers, appear to be sensitive to thiram, dichlone and
chloranil and slightly sensitive to captan when these mate-
rials are used as seed protectants on non-inoculated seed

(57), but seed treatment of Rhizobium-inoculated seed with

 

either captan or thiram did not affect nodulation (47).
BHC (benzene hexachloride) and chlordane insecticides have

also been found inhibitory to nitrification (4, 37).

Effects on Decomposition of Organic Matter and Fer-
tility—-Some chemicals kill or reduce growth of numerous
soil microbes and thereby drastically influence microbial
numbers and activity for varying periods of time. The ex-
tent of reduction of microbial activity depends on soil type,
moisture content, temperature, soil physical condition,
amount and kind of chemical applied and method of applica-
tion. After the initial reduction in numbers, certain groups,
usually bacterial species, return very quickly and reach
numbers far in excess of those in untreated soils. In gen—
eral, the greater the initial kill, the greater the subse-

quent peak in numbers. Fungi and other microbes become

 

reestablished at various periods and may not exceed numbers
in the original soils (67, 108, 69). With time, which may
be a year or longer, total numbers approach those in the
untreated soil. The fungi, as a group, are usually more
readily killed by soil fumigants and fungicides than are
some bacterial types (20, 78, 106). Following treatment,
they may increase in numbers shortly after the bacteria

and attain higher numbers than in the original soil (51)

or they may remain at low numbers for relatively long periods
of time (67, 75). Warcup (108) observed that 18 months
after treatment of a field soil with formalin, the numbers
of fungi were still lower than in untreated soils.

Very little specific information is available con—
cerning the effects of fumigants and fungicides on numbers
of protozoa, algae, yeasts and other saprophytic soil forms
(69). Protozoa appear to be affected in a manner similar

to fungi (88).

1) Effects on Microbial Respiration——The measure—
ment of CO2 evolution from pesticide treated soils has been
used to assess the effect of pesticides on decomposition of
organic materials in soil. With soil fumigants, the soil
respiration was inhibited for a short time after applica—
tion, then surviving microorganisms recover or recolonize
and the respiration rate rises to high values and exceeds
even that of an untreated soil. The same holds true gener—

ally for non—specific soil fungicides. Among the compounds

tested (23, 24) captan, mercuric chloride and methylarsine
sulfide caused a depression lasting about 24 hours after
which the soil respiration increased to about the level of
the control, but the total oxygen consumption remained less
than the control throughout the experiment. The return to
normal respiration probably occurs as a result of fungicide
inactivation and buildup of the fungicide resistant microbial
population. Fungicides with specific action, such as Dexon
and PCNB, showed no influence on soil respiration.

Studies of C0 evolved from nabam (200 ppm in soil)

2
and Mylone (3,4 dimethyl dihydro—l,3,5, 2H—thiadiazine —2
thione) at 150 ppm in soil, showed respiration inhibition
for 28 days; the effect lessened at 42 days and at 56 days
the treated soils evolved more carbon dioxide than the con-
trol. Nitrification was inhibited similarly over the same
time period. Parallel studies of population numbers showed
greatly reduced total numbers of fungi in each of the four
soil types tested for at least 30 days following treatment.
Numbers of bacteria and Streptomyces were decreased to a
lesser extent. At 60 days the mold and bacterial popula-
tion had increased significantly (8).

Thiazine and carbamate herbicides caused a reduc—
tion in C02 evolution over a 28 day period (7). Similar
results had previously been noted by Kratochvil (56) but
the depression occurred only during the early stages of

incubation. No effect of 2,4—D or 2,4,5-T was evident.

Effects of applications of 10 chlorinated hydrocarbon
insecticides (heptachlor, chlordane, methoxychlor, lindane,
aldrin, dieldrin, toxaphene, TDE (l,l-dichloro-2,2-bis
(p-chlorophenyl) ethane), DDT, and BHC (benzene hexachloride))
to fine sandy soil at rates of 12.5, 50, and 100 ppm each
were studied for a 16 month period by soil—dilution colony
counts and soil carbon dioxide evolution (29). In soil
examined one month after application, bacterial numbers
were normal. None of the insecticides had affected numbers
of fungi except dieldrin, which increased numbers of fungi.
CO2 evolution was increased by toxaphene, dieldrin, TDE,
DDT and BHC only. Nitrate production was decreased by
heptachlor, lindane and BHC but was increased by toxaphene,
TDE and DDT and was unchanged by the remaining chemicals.
Sixteen months after application, no significant changes
were observed in numbers of bacteria, fungi or carbon diox—
ide evolved. Nitrate production was reduced by DDT and
BHC. Growth of stringless beans, as indicated by reduced
root and top weights, was more affected by the insecticides
than were the soil organisms. The authors concluded that
toxicity to higher plants will be the earliest warning of

toxic accumulation of chlorinated hydrocarbon insecticides.

2) Effect on Composition of Soil Microflora—-In the
discussion of the effect of pesticides on the composition
of soil microflora, certain closely related phenomena such

as "disease trading," biological control, and accumulated

10

phytotoxic residues have been included.

a) general Effects on Fungi, Bacteria, and Actinomyf
cetes——Kreutzer (57) has reviewed the effects of fungicides
on soil microorganisms according to various classes of chem-
icals: l) Volatile general eradicants, 2) Volatile selective
eradicants, and 3) Non-volatile protectants. Volatile gen—
eral eradicants such as chloropicrin, methyl bromide, allyl
alcohol, nabam and Mylone, were moderately or strongly toxic
to fungi but generally less toxic to bacteria and actinomy—
cetes at the same dosage. Nabam and Mylone at fungicidal
levels were either non—toxic to bacteria and actinomycetes
(8) or increased numbers of actinomycetes (20). Chloropicrin
at fungicidal levels increased numbers of bacteria in soil
(55). Actinomycetes showed even greater tolerance than
bacteria to fumigation with Chloropicrin (110).

Volatile selective eradicants such as DD (dichloro—
propene—dichloropropane), ethylene dibromide and dibromo-
chloropropane also caused an increase in bacterial numbers,
but produced moderate inhibition of fungi (55). DD was
relatively non—toxic to actinomycetes (71).

Captan, thiram and nabam as non-volatile protectants
show similar patterns. Both captan and thiram applied as
soil drenches to control Pythium spp. increased bacterial
populations (11). Nabam was considerably less toxic to
actinomycetes and bacteria than to fungi (8).

b) Effects of Toxicants on Fuggi in Soil—~Among

the volatile eradicants, Chloropicrin is non—selective in

 

 

 

11

its toxicity to soil fungi but there is evidence that methyl
bromide is somewhat selective varying in toxicity to various
fungi. Those most susceptible to methyl bromide were As:

pergillus, Penicillium, Sclerotinia sclerotiorum, Sclerotium

 

gglphinii, Rhizoctonia solani, Pythium spp., Phytophthora

spp., Fusarium oxysporum f. callistephi, and Verticillium

 

albo atrum. Those most resistant were Thielavia, Chaetomium

spp., Penicillium vermiculatum and Trichoderma viride.

 

Allyl alcohol and formaldehyde were toxic to many fungi

but were relatively non—toxic to Trichoderma viride (57)°

 

As reviewed by Kreutzer (57), Pythium and Phytoph-
thora species appear to be the most susceptible to volatile

eradicants; Rhizoctonia, Fusarium, Sclerotium spp., Myrothe-

 

cium, Cladosporium, Mortierella, Phoma, and Paecilomyces
are somewhat resistant; and Verticillium albo atrum is even

more resistant. Aspergillus, Penicillium and Trichoderma

 

viride appear to be the most resistant. The volatile se—
lective eradicants, DD, ethylene dibromide and carbon di—
sulfide appear to have a selectivity range similar to that

of the volatile general eradicants. Pythium and Phytophthora
were more sensitive than other soil fungi while Fusarium

and Verticillium were moderately resistant (57). The non—
volatile protectants as represented by captan and the di—
thiocarbamates also appeared to follow a range-of—toxicity
pattern similar to that of the volatile eradicants. Thiram
was quite effective against P thium, especially 3. ultimum,

but a little less toxic to Phytophthora spp. However, water-

12

soluble nabam was highly effective on species of Phytoph—

thora. Thiram was more toxic to Rhizoctonia than to Pythium

 

but the reverse was true for captan. Verticillium was mod—

erately resistant to nabam while Typhula, Trichoderma and

 

Penicillium appear to be quite resistant to thiram (57).

c) Effects of Toxicants on Bacteria--Of four physio—
logical groups of bacteria studied, nitrifiers and cellulose
decomposers were most severely reduced in numbers by methyl
bromide fumigation (110). The effects of various toxicants
on the other groups, ammonifying and nitrifying bacteria
and on legume nodulation, have already been discussed.

Kreutzer (57) has stated that most toxicants showed
little or no effect on the general bacterial population
or may actually increase numbers of bacteria in soil. Methyl
bromide, chloropicrin, DD, thiram and captan are among those
chemicals reported to increase numbers of bacteria in soil.
Thiram, ferbam and nabam at 200 ppm also increased bacterial
numbers considerably (86).

d) Effects of Toxicants on Actinomycetes——Observa-
tions on over 200 chemicals indicate: that actinomycetes
are affected in a manner similar to bacteria. Occasionally
they appear to be more resistant to toxicants than bacteria.
They appeared to be more tolerant of low dosages of toluene
than were most bacteria, but when bacterial numbers increased
after treatment, relative numbers of actinomycetes decreased
(106). Actinomycetes showed a greater tolerance to methyl

bromide than did bacteria or fungi (110).

13

DD did not reduce numbers of actinomycetes (81, 71).
Chandra (8) reported that nabam and mylone had little effect
on soil actinomyces but Domsch (20) found that these com—
pounds increased numbers of actinomycetes when used at fungi-
cidal levels. Numbers of actinomycetes were also increased
by Vapam treatment (13). Thiram had no effect on soil ac—
tinomycetes (86).

e) Effects on Microbial Balance-~Effects of fumi—
gants on microbial "balance" has been discussed by Wilhelm
(112). Experiments with Verticillium recolonization in soils
fumigated with chemicals of known biocidal activity showed
that the less broadly biocidal the chemical was, the slower
the colonization rate of Verticillium. In this way, a form
of biological control resulted from minimal disturbance of
the microbiological balance. Wensley (110) also pointed
out that fumigation with methyl bromide, DD, and ethylene
dibromide altered the microbiological balance in favor of
fungi that were normally suppressed by a predominance of
Aspergillus and Penicillium spp.

In addition to biological control, changes in microbial
balance has led to "disease trading“ in some instances (22).
Bird (4) found that furrow treatments with various combina~
tions of fungicides (captan+dithane, captan+zineb, captan+
PCNB and others) controlled Rhizoctonia and Pythium seedling
infection on cotton but resulted in increased infection by
Fusarium spp. Vaartaja (104) has discussed the influences

of chemical treatments on biological control and offers the

14

hypothesis that certain fungicides interfere with biolog-
ical control. For example, treatment with Dexon gave sig-
nificant damping-off control for pine when used alone, but
control failed when Dexon was combined with PCNB. Since
phytotoxicity or chemical reaction were not logically re—
sponsible, Vaartaja suggested that PCNB may have interfered
with the biological aspects of disease control obtained

by Dexon treatment alone. He also pointed out other ex—
amples of selective and "anomalous" effects of soil treat-
ments in controlling seedling diseases. Others have noted
anomalous effects of soil fumigants on disease development

(85, 99).

Effects of Particular Fungicides on Soil Microflora

Effects of Captan and Related Compounds

Physical Structure and Chemical Properties-—No in-
formation on the influence of captan on soil aggregation
or on chemical properties was found. Its stability in soil
has been studied by Domsch (16) and by Burchfield (6). Ac—
cording to Domsch, captan 1000 ppm has a fungicidal (not
chemical) half-life greater than 50 days as measured by
disease control in soil inoculated with Pythium and cropped
with peas. At concentrations ensuring disease control,
captan and zineb had a half—life of 70-75 days; Spergon,
nabam and an organic mercury,15—20 days;and ziram and thiram
35—45 days. Burchfield indicated that captan was about

seven times more stable than dyrene in a silt loam soil.

15

Kennedy and Brinkerhoff (52) found that captan retained

its effectiveness for 4—6 weeks in a non-steamed moist sandy
loam of pH 6.0. Another aspect which may influence the
stability of captan in soil is its reaction to ultraviolet
light (90). Polyethylene disks coated with captan, Phaltan
and Difolatan subjected to sunlight and plated on agar seeded
with Penicillium expansum showed a degradation of fungicidal
activity. Activity of Phaltan was reduced to 67% of initial

activity, captan to 42% and Difolatan 21.6%.

Biological Properties-~The study of effects of cap—
tan on microbes in the soil has largely been confined to

effects on soil microflora.

1) Effects on Nitrification--At present there is no
evidence that captan has an adverse effect on nitrification
in the soil. But as a 300 ppm soil drench, there is some

evidence that legume nodulation by Rhizobium spp. was re-

 

duced (39). Yet nodulation of alfalfa seed preinoculated
with root nodule bacteria and treated with captan or thiram

was not affected (47).

2) Effects on Decomposition of Organic Matter and
Fertilitye—Soil treatment with captan appears to affect
both soil respiration and numbers and kinds of soil micro-
flora.

a) Effects on Microbial Respiration--Soil treatment

 

with captan, mercuric chloride or methyl arsine sulfide

 

16

caused a depression of C02 evolution for about 24 hours,
after which soil respiration increased to about the level

of the control, but the total oxygen consumption remained
less than that of the control throughout the experiment
(23). Domsch (24) has recently completed a study of the
influence of captan on decomposition processes in soil using
respiration techniques. Breakdown of cellulose and chitin
are most inhibited by soil treatment with captan while de-
composition of glucose, starch, tannin, esculin, pectin

and chitin is retarded for only a short period-of time.

b) Effects on Composition of Soil Microflora-—Re—
sponses of soil fungi to captan have been variable. Lyons
and Leach (63), using captan as a furrow treatment for sugar
beets, found that Rhizoctonia solani and Pythium ultimum
were controlled but Aphanomyces cochlioides was not. In
other studies with cotton and certain ornamentals, captan

was more effective against Pythium ultimum and P. debaryanum

 

than against Rhizoctonia solani (S2, 89). Yet in-vitro

 

studies indicated effectiveness of captan against Rhizoc-

tonia solani (103). The apparent discrepancies may be due

 

to differences between in—vitro and in—vivo tests but, ac—
cording to Thomas (97), it may also be due to inherent vari-
ation between isolates in their susceptibility to captan.
Captan does not appear to be particularly effective against

species of Sclerotium (58) or against species of Phytophthora

 

 

(76, 49, 84). Based on an analysis of the soil fungus flora,

Domsch (21) found the following fungi to be particularly

 

17

sensitive to captan, thiram, an organic arsenical compound,

nabam, vapam and allyl alcohol: Rhizoctonia solani, Dorato-

 

myces microsporus, Monilia pruinosa, Mucor heimalis, Rhizopus
nigricans, Mortierella alpina, Fusarium solani and Asper—

gillus fumigatus. Those with marked tolerance to these

 

fungicides were: Penicillium nigricans, Pullularia pullu-

lans, Chaetomium sp. and Aspergillus versicolor.

 

Numbers of actinomycetes and bacteria, as well as
fungi, were reduced significantly by normal application
rates of protectant-type soil fungicides such as captan
and thiram (22). On the other hand, when captan and thiram
were used as weekly drenches for control of damping—off
of pine and spruce seedlings, there were no deleterious
effects on bacteria (11). In this case bacterial growth
was apparently encouraged. The low application rates may
not have affected the bacterial population and may even
provide increased amounts of nutrients by selective kill—
ing of soil fungi. It was found that Penicillium and
Fusarium were less affected by this treatment than the
pathogens Rhizoctonia solani and Pythium sp.

With respect to anomalous effects, Solel (95) de-
scribed an increase in cotton seedling damping-off with
furrow or hopper box treatment with captan, similar treat-
ments with Quintozene (PCNB) and zineb adequately controlled

post emergence damping-off.

 

18

Effects of Dexon on Soil Microflora

 

Studies of the effect of Dexon on soil microflora
have been limited to its effect on certain soil fungi.
Dexon has been characterized by a number of rather unique
properties. It has been highly effective when used to con—
trol seedling diseases caused by certain Phycomycetes (59).
It has also been effective as a chemotherapeutant against

Phytophthora root rot of avocado when used as a soil drench

 

(114). It has been shown that Dexon is absorbed by roots
of sugar beet seedlings and translocated through xylem and
phloem to the primary leaves (41). Such movement may con-
tribute to control of seedling disease but there is some
evidence that Dexon is changed to other compounds in the
plant. Hills indicated that Dexon is probably most effec~
tive in controlling damping—off disease at the site of ap—
plication. Further investigation by Hills and Leach (42)
revealed that aqueous Dexon was unstable in soil and in~
Vitro when exposed to air and light. Studies suggested a
two-stage decomposition; a photochemical reduction of the
diazonium group evolving N2 and subsequent oxidation and
polymerization. Decomposition products were non—toxic to
Pythium in—vitro or in soil (42, 44). Dexon is highly

fungistatic to Pythium ultimum but is much less effective

 

against 3, solani (42). Dexon is an effective inhibitor
of the mitochondrial DPNH-cytochrome C-reductase system

of Pythium, but Rhizoctonia and sugar beets have a mitochon—

 

drial system that decomposes Dexon in the presence of DPNH

19

(100, 101).

Soil respiration studies with Dexon treated soil
showed no inhibition of respiration rate (23). In contrast,
Torgeson (102) showed that Dexon in-vitro reduced oxygen
consumption of Phytophthora parasitica, Sclerotium rolfsii,
and Fusarium oxysporum. This occurred at Dexon concentra-
tions greater than that required to inhibit fungal growth.

Certain anomalous effects have been observed with
Dexon soil treatment,as mentioned previously. Dexon gave
good control of damping-off in pine when used alone, but
control failed when Dexon was combined with PCNB (104)o
Vaartaja indicated that PCNB probably interfered in some
way with a combination of biological and chemical control

given by Dexon alone.

Effects of PCNB and Related Compounds on Soil Microflora

From the standpoint of stability in soil, it was
found that PCNB, captan and zineb retained their fungicidal
effectiveness for 4—6 weeks in a non—steamed moist sandy
loam at pH 6.0 (52). PCNB has a low vapor pressure in sus—
pension or solution and does not produce fungitoxic vapors
in soil (79).

Domsch (23) and Torgeson (102) obtained the same
results with PCNB as with Dexon (above), in regard to soil

respiration and in—vitro inhibition of certain fungi.

Effects on Soil Fungi—~Kreutzer (57) has pointed

 

out that PCNB and TCNB (tetrachloro nitrobenzene) have rather

 

 

 

20

unusual properties exemplified by their specificity toward
a number of comparatively unrelated organisms, among which

are Rhizoctonia solani, Streptomyces scabies, Sclerotinia

 

spp., Sclerotium spp., Plasmodiophora brassicae, Rhizopus

 

nigricans, Penicillium paxilli, Trichoderma viride and

 

 

Olpidium brassicae. PCNB is relatively ineffective against

 

Pythium spp., Fusarium sp., Verticillium albo atrum, Col—

letotrichum ggssypii and Typhula itoana.

 

 

However, even the homologous PCNB and TCNB differ
in their degree of specificity. TCNB was effective against

Fusarium and Phytophthora while PCNB was not, but PCNB con-

 

trolled Sclerotinia and TCNB did not. PCNB appeared to be

 

more toxic to Trichoderma viride than TCNB and the latter

 

was more toxic to Rhizoctonia (57). Eckert (25) reported

 

that 2,3,4,5 and 2,3,5,6 isomers of TCNB were less toxic

to Rhizoctonia than PCNB.

 

As a soil treatment for potato Rhizoctonia control,

 

PCNB at 25, 50 and 100 #/A. reduced the number of tuber
sclerotia (61). The PCNB was thought to have acted selec~
tively on soil populations and preferentially reduced the

number of sclerotium-forming strains of Rhizoctonia.

 

Effect of PCNB on Microbial Balance—~Failure to

 

control certain diseases with PCNB in combination with other
fungicides was discussed earlier (page 19). Gibson (35, 36)
also reported increased damping—off of pine seedlings after

repeated applications of PCNB to soil. He suggested that

 

21

the effect was due to selective action against the compet-
itors of Pythium species in the soil, one of which was iden—

tified as Penicillium paxilli.

 

Further evidence that PCNB may alter the soil micro~
flora in an undesirable way was provided by Garren (30).
Several fungicides including Dexon, reduced peanut pod rot
caused by Pythium sp. but pod rot was increased with PCNB

alone or in combination with each of three other chemicals.

Sensitivity of Rhizoctonia Isolates to Chemicals
Sinclair (93) was among the first to note the differ-

ential reaction of Rhizoctonia isolates to certain chemicals.

 

Highly significant differences were found between four

Rhizoctonia isolates in their response to soil treatment

 

with captan + PCNB and nabam + PCNB. In later work (94)

five different Rhizoctonia isolates differed in their sensi-

 

tivity to PCNB, captan, and dichlone alone and in combinam
tion, when applied in furrow in greenhouse flats. In-vitro

tests with 12 Rhizoctonia isolates and 12 chemical treat~

 

ments at three rates yielded wide variation in sensitivity.
The greatest mean inhibition for all isolates at all rates
was in response to PCNB, PCNB + Dexon, Dexon, and thiram.
Of the 12 isolates, nine were inhibited by PCNB at 240 ppm,
and seven at 60 and 120 ppm. PCNB-Dexon inhibited eight
isolates at 120 and 240 ppm and six at 60 ppm. Dexon in—
hibited seven isolates at 240 ppm, six at 120 ppm and five

at 60 ppm. No chemical inhibited all isolates and no isolate

22

was inhibited by all chemicals at the concentrations tested.
There was no correlation between pathogenicity to cotton
and sensitivity to chemicals.

Mycelial growth of four biotypes of Rhizoctonia from

 

Pinto bean differed significantly when placed on potato dex-
trose agar sprayed with .25% captan. Two races were com—
pletely susceptible and one was highly resistant (97). In
soil treated with captan 100 ppm and PCNB 100 ppm, the inn
cidence of disease caused by the same isolates varied with
each fungicide. Three races were partially resistant to
the effect of PCNB on cotton and beans and those races which
were resistant to captan in—vitro were innocuous to Pinto
bean and cotton.

Shatla and Sinclair (91) examined 13 isolates of

Rhizoctonia solani from diseased cotton seedlings, for tol-

 

erance to PCNB in plate culture studies. Five isolates
representing the growth patterns of the original 13 were
used to determine maximum and minimum tolerances. These
isolates gave three types of reactions to PCNB: l) Sus~
ceptible...growth inhibited at 0-3600 ppm, 2) Moderately
tolerant...growth inhibited at 3600-4200 ppm, 3) Highly
tolerant...growth inhibited at 11,000 ppm or more. Cul-
tural differences were noted and some isolates were more
virulent in greenhouse tests. Further tests with 36 iso-
lates on agar containing PCNB produced significant varia~
tion in mycelial growth during test periods of four, seven

and 10 days (92). The isolates varied in growth on PCNB

 

23

agar from highly tolerant (growth at 10,000 ppm) to sensi-
tive (growth at 600 ppm). A frequency distribution plot

of sensitivities produced a normal curve. Under greenhouse
conditions nine isolates varied in their pathogenicity from
slightly to highly pathogenic and a correlation was found
between pathogenicity and tolerance to PCNB. When the data
for tolerance to PCNB for the nine isolates tested in the
greenhouse were compared with laboratory results for the
same isolates, all but two of the isolates showed a posi~
tive correlation. One isolate, moderately tolerant in the
greenhouse acted as a sensitive isolate in the laboratory.
Another isolate showed considerable tolerance in the labora—
tory but only moderate tolerance in the greenhouse. Iso~
lates from PCNB treated plots showed significantly higher
tolerance to PCNB than did isolates from nonwtreated plots.
Although cultural characteristics varied among isolates,

no correlation was found between culture characteristics
and pathogenicity or tolerance to PCNB.

The apparent discrepancies relative to correlation
of sensitivity to chemicals with pathogenicity to seedlings
may be explained by results obtained by Daniels (12). She
found a considerable variation in pathogenicity and morphol-

ogy among 33 isolates of Rhizoctonia (Corticium) solani.

 

From her observations on the branching and anastomoses of
the multinucleate hyphae, she suggested that the field pop—
ulation of E, solani may be continually varying in patho-

genicity and other characters. Even when 35 single

24

basidiospores from a single isolate from Poa annua were

 

tested on cabbage, lettuce and tomato, every symptom from
black leg, damping—off, root rot and growth reduction to
no infection was obtained.

Elsaid and Sinclair (26, 27) suggested that strains,
adapted to higher concentrations of fungicides, might arise
under field conditions through continuous use of these fungi-
cides. This hypothesis was supported by later work (92)

in which Rhizoctonia isolates from cotton growing in PCNB

 

treated field plots tended to be more tolerant to PCNB than
isolates from untreated plots. A Rhizoctonia isolate from
cotton also became more tolerant to captan, dichlone, maneb,
thiram and PCNB through serial transfers on medium contain~
ing the fungicides (28). The isolate maintained its toler-
ance to PCNB and maneb, but not to captan and dichlone when
the isolate was cultured for a time on fungicide-free medium.
Only the dichlone tolerant strain maintained its tolerance
after three serial transfers on agar without fungicides.
Georgopoulos (31) described similar occurrences with Sclerw

otium rolfsii, when it was subjected to repeated fungicide

 

applications. Two strains from a parent isolate became re-
sistant to PCNB and copper oxychloride respectively. Later

studies with Fusarium (Hypomyces) solani f. cucurbitae

 

(32, 33, 34) revealed 12 mutants tolerant to PCNB and 2,3,5,6
Tetrachloronitrobenzene and to seven more chlorinated nitro-
benzenes, as well as 2,6—dichloro~4~nitroanaline. In this

case the tolerance was genetically determined by three known

25

genes which were inherited independently. A linkage rela-
tionship was recognized between genes for chlorinated nitro-

benzene—tolerance and virulence to Cucurbita pepo.

 

 

MATERIALS AND METHODS

Experimental Fungicides

Several fungicides were chosen for their selective

range of activity against certain groups of fungi and one

was chosen for its broad activity range.

were as follows:

Fungicide Source
PCNB (pentachloro— Olin-Mathieson
nitrobenzene) Corp.
DAC—469 (octachloro Diamond Alkali
tetrahydro thiophene) Co.

Dexon (p-dimethylamino Chemagro Corp.
benzene diazo sodium
sulfonate)

Bayer-29957 (a straight Chemagro Corp.
chain organic compound)

Difolatan California Spray
(N-(l,l,2—tetrachloro- Chemical Co.
ethyl sulfenyl )-cis-A
~4-cyclohexene—l,2~
dicarboximide)

Olin-Mathieson 1921 Olin Mathieson

(p-isopropyl benzalde- Corp.
hyde, 3-semicarbazone)

In-Vitro Effects of Fuggicides

 

These fungicides

Activity

Rhizoctonia sp.

 

Sclerotinia sp.
Sclerotium sp.

Rhizoctonia sp.

 

Phycomycetes

Fusarium sp.

Broad Spectrum

Fusarium sp.

In-vitro tests of the effects of Dexon, PCNB and

Olin—Mathieson 1921 on mycelium of Pythium irregulare Buisman,

mycelium and sclerotia of Rhizoctonia solani Kfihn, and the

 

26

27

mycelium and conidia of Fusarium oxysporum (Schlect.) Snyder

 

& Hanson f cucumerinum (J. H. Owens) were made at four fungi—

 

cide concentrations: 1, 10, 100 and 1000 ppm. Since some
heating was required to kill bacterial contaminants in both
the wettable powder and chemically purified preparations,

a study was made of the effect of heat treatment on the
activity of the fungicides. The heat treatments for the
fungicides in water agar were: 1) Autoclaving 15 min. at
15 PSI (122° C.); 2) Steaming the melted medium containing
the fungicide for 20 minutes at 100° C; and 3) Adding the
fungicide to the medium immediately after removal from the

steamer. Growth tests with Rhizoctonia, Pythium and Fusarium

 

indicated that steaming the fungicide—agar 20 minutes was
sufficient to kill contaminating bacteria without impairing
fungicidal activity of the compounds. Blocks 12 mm square
were cut from the fungicide agar plates and placed on flamed
glass microscope slides enclosed in petri plate humidity
chambers. One block of each of the four fungicide concen~
trations was included in each plate of a fourmreplicate set.
Each of the blocks was implanted with mycelium, conidia or
sclerotia of the test fungus and blocks were examined periodm
ically for mycelial growth or condial germination.

Further in—vitro tests of the effect of Difolatan
(50 ppm) and PCNB (100 ppm) on growth of various Rhizoctonia
isolates were done in conjunction with a study of the effects

of these fungicides on the same isolates in soil.

 

 

 

28

Experimental Soils

Two types of soil were chosen, a Conover loam min—
eral soil and a highly organic muck soil. The Conover loam
mineral soil was selected from the Department of Botany
and Plant Pathology field plots which had been used for a
number of years for seed treatment damping-off studies.
This soil is characterized as a grey brown Podzolic soil,
mostly loam but in part sandy loam, light in color, low
in humus with medium fertility, and imperfectly drained.
Measured moisture holding capacity was .347 grams of water
per gram of oven dried soil and the soil pH ranged between
6.8 and 7.2.

Highly organic muck soil was taken from cultivated
plots of the Michigan State University muck farm at Corey
Marsh near Bath, Clinton County. This soil is classified
as Carlisle muck with a rather low acidity (pH 6.4). MoiSw
ture holding capacity was measured at approximately 3.9

grams of water per gram of oven dried soil.

Rhizoctonia-Amendment

 

Since both soil types were found to be relatively

free of Rhizoctonia, it was necessary to artificially in-

 

fest them with Rhizoctonia for certain experiments. The

 

soil was infested by mixing l%-liters of a Rhizoctonia R—54
vermiculite culture with three flats of soil. After 3-5
days incubation the soil was treated with the fungicide.

In a later study with Dexon-Difolatan, muck soil

29

was infested with a number of Rhizoctonia isolates. Since

 

previous experiments had shown that R—54 was not particu~
1arly sensitive to certain fungicides, a comparison of iso-
lates was made to ascertain their pathogenicity to red beets
and their sensitivity to Difolatan and PCNB. Three to five

days after infestation with Rhizoctonia, the soil was treated

 

with Dexon (30 lbs./acre, 4" depth) or Dexon + Difolatan
(30 + 30 1bs./acre, 4" depth). Dexon was used to control
Pythium-induced damping-off which would otherwise obscure

that caused by Rhizoctonia. Detroit Dark Red Table beet

 

seeds were planted as a test crop and a daily record of
damping-off was made for both natural and artificially inn
fested soils. At the end of the test period, seedlings
were removed, observed for root rot, and weighed for com-

parison of pathogenicity of isolates.

Fungicide Treatment of Soils

 

Soils were treated individually in halfmflat lots
(0.28 cu. ft.) by tumbling the soil with the fungicide in
a hand-turned mixer. Fungicides were applied at the followm
ing rates:

Rate: (lbs. active ingredm

Fungicide ient/acre, 3" depth)
PCNB 20% W.P. 30 lbs.
Dexon 5% vermiculite granules 20
Bayer 29957 Technical liquid 35
Dexon + DAC 469 (5% gran—5% dust) 20 + 30

Difolatan 10% vermiculite granules 30

30

After treatment, the soil was placed in flats and
each flat enclosed in a polyethylene bag to control loss
of soil moisture and at the same time provide for gaseous
exchange from soil to air. The flat units were covered to
exclude light and to insulate them against temperature vari-
ations induced by radiant heat from the sun. Soils were
kept in the 70 degree F. temperature-controlled greenhouse.
Soil moisture was maintained at nearly constant weight by
periodic weighing of the flats and spraying the soil surm

face with a hand watering bulb.

Microbiological Assays of Soils

To evaluate the general effect of fungicides on
soil microorganisms, agar plate colony counts of the three
major groups of microorganisms, actinomycetes, bacteria and
fungi were made before and after treatment of the soil with
fungicides. This was accomplished by the soil—dilution
plate method (50) using media appropriate for the three
groups of organisms.

A further evaluation of the effect of the fungicides

on Pythium and Rhizoctonia was attempted using bait isolam

 

tion techniques.

Sampling and Preparation—~Soils were sampled at
the time of treating and at various intervals up to four
weeks after treatment. Samples were made by taking repre~
sentative three—inch cores from the soil in the flats with

a % inch diameter cork borer. Borings were bulked in

31

polyethylene bags to form a minimum sample of 1200 grams
of soil. The soil was screened through wire mesh screen
(18 holes/inch) to remove stones and debris. After mixing
and dividing, one to 10 gram samples were weighed for the
soil-dilution plate experiments and the remaining soil was

assayed for Pythium or Rhizoctonia by baiting methods.

 

Colony counts were expressed as equivalents of colonies
per gram of air—dried soil.

A soil washing technique described by Watson (109)
was used to remove a large portion of fungus spores from
the soil samples, thus making the plate count estimates
of viable mycelial propagules in soil more nearly repre-
sentative.

An amount of soil equivalent to one gram of air—dry
soil was added to 200 ml of water in a 500 ml flask. The
contents were swirled at least 10 times and the flask canted
at 45 degrees for several minutes to allow the soil particles
to settle. A settling time of one minute was prescribed
for this method (92) but it was found that many of the soil
particles were poured off in this way. Also, the amount to
be decanted off each time was not specified. If the entire
amount was decanted off, many of the finer particles were
lost. An attempt was made to standardize the procedure
by allowing the particles to settle and pouring off the
suspension until the visible particles began to flow out.
Approximately one-third of the liquid remained in the flask

and fresh tap water was added to restore the liquid to the

 

32

original volume. The contents were swirled and allowed
to settle for the next washing.

The method was later modified to handle larger soil
samples. Ten gram samples of soil were added to two liters
of water in a three liter Fernbach flask. After settling
of the soil particles, a portion of the solution—suspension
was poured off and fresh tap water was added to restore
the liquid to the original volume. The washing procedure
is tabulated in Table 1. When the washing was completed,
the water was then decanted very slowly and almost completely

and the dilutions were made with the remaining soil.

Dilution and Platinge—Preliminary experiments indim

 

cated that dilutions of 1:5000 would produce a favorable
range for counting fungal colonies, and 1:500,000 would
give a suitable range for counting both bacteria and actin—
omycete colonies. A dilution of 1:1000 was used for countm
ing fungi from washed soil samples.

For one gram (air—dried equivalent) samples, washed
or non—washed soil samples were added directly to the first
99 ml dilution blank (sterile 0.1% water agar (111)) to
give the initial soil dilution of 1:100 and successive dilu~
tions of 1:1000, 1:5000 and 1:500,000 were made. The 1000
and 5000 dilution blanks were placed on a mechanical shaker
for 5—10 minutes to thoroughly mix the soil suspension before
further dilution. For 10 gram soil samples, the washed or

non—washed soil samples were placed in 500 m1 of sterile

33

Table 1. Procedure for washing soil samples. Mineral and
Muck soils.

 

Mineral Soil-~Washing Procedure
(10 g. soil sample in 3 liter Fernbach Flask)

 

 

(min.) ml

Wash No. Settling Time Poured Off Notes

1,2 15,12 1350,1550 Root and plant debris,
insects, other floating
debris removed. Sus—
pension very cloudy.

3,4 12,12 1650,1650 Some floating debris,
suspension cloudy.

5,6,7 12,10,10 1700 ea. Suspension slightly
cloudy.

8,9 10,10 1700,1800 Suspension appears
clear.

10-15 7 1800 Suspension appears
clear.

Muck Soil-—Washing_Procedure
(10 g. soil sample in 3 liter Fernbach Flask)

 

1,2,3 25,20,20 800 ea. Much plant debris, in~
sects and other floating
debris removed. Sus~
pension very dense.

4,5,6 15,15,15 800,900, Some debris still
900 floating out. Sus—
pension dense.
7,8,9 15,15,15 900,900, Some debris still
1000 floating out. Sus-
pension becoming
clearer.
10—15 15,10,10, 1100,1200, Little debris. Sus-
10,10,10 1300,1400, pension appears clear

1500,1500 or slightly cloudy.

 

34

0.1% water agar in an alcohol washed 600 m1 beaker. The
soil suspension was thoroughly mixed with a magnetic stir-
rer for 3-5 minutes. For transfer of the initial suspension
(1:50), a 5 ml stainless steel dipper was introduced into
the agitated soil suspension and removed quickly to sample
a representative range of soil particles. The dipper con-
tents were transferred to the first dilution flask (1:1000)
containing 95 ml of 0.1% water agar and the dipper was rinsed
repeatedly in the dilution blank to remove the heavier soil
particles that remained. Further dilutions were made as
previously described.

Ohio (OAES) medium (113) was selected for estima-
tion of numbers of fungi in soil. This medium inhibits
bacterial growth and retards the growth of fast growing
spreading type colonies.

Soil extract medium for isolation of soil bacteria
was prepared by extracting Conover loam mineral soil as
described by Allen (1). The medium was phosphate buffered
to pH 7.0 at .01 M final concentration.

The actinomycete assays were made on plain water
agar (2%) instead of chitin medium (60) which is slightly
more selective but more difficult to prepare. A purified
Difco agar was used because it supported less bacterial
growth than crude agar.

Soil dilutions were plated by adding 1 ml of the
appropriate dilution to each plate of a six—replicate set.

Melted medium, cooled to 40° C., was poured into the plates

 

35

and mixed thoroughly with the soil suspension. To reduce
spreading surface growth, a second layer of medium was added
after the first had solidified. A surface layer of water
agar containing only sodium propionate and sodium taurocho~
late, at the same concentration as in the base layer, was
used for some fungus assays. The rapid spread of actinomy—
cetes, bacteria and fungi on the surface of the agar was

reduced, but for fungi the advantage gained was slight.

Colony Counts and Analysis—~Fungus colony counts
were begun at about 3-4 days, and bacterial and actinomy—
cete counts at about seven days after plating and continued
daily until plates were covered or no new colonies appeared.
Counting was facilitated by marking the plate bottom with
India ink at the site of each newly developed colony. Trans-
mitted light and a magnification of 3X was sufficient to
locate all growing colonies. Because some actinomycete
colonies grew on the soil extract medium and were indis~
tinguishable from bacterial colonies in their early developm
ment, counts of these colonies were included with the bac~
terial colonies. At the end of the counting period, the
number of distinguishable actinomycete colonies was sub—
tracted from the total bacterial count for each plate.

An analysis of variance of colony counts was comm
pleted for each of the groups, fungi, bacteria and actin-
omycetes, to determine whether differences between colony

counts for various soil treatments were significant. Most

36

of the experimental results were analyzed with the CDC 3600

computer.

Phycomycete Assaye-A method similar to that described

 

by Hine and Luna (45) was used for isolation of Pythium from
soil. Three mm square potato cubes were soaked for one hour
in a combined solution of 100 ppm Streptomycin and 100 ppm
Mycostatin. Then 120 cubes were spaced uniformly through
the 1 Kg soil sample, and the soil was kept at room temperam
ture. After 12—15 hours, cubes were retrieved by passing
the soil through an 18 holes/inch wire screen. The cubes
were washed in running tap water for 25-30 minutes, placed
either in sterile water or the Streptomycin—Mycostatin solu~
tion, and then transferred, one cube per block, to 8 x 10 mm
agar blocks, containing 100 ppm Streptomycin, 100 ppm Mym
costatin and 2% agar. The blocks were incubated on flamed
glass slides supported by a U—shaped glass rod in the bottom
of a glass petri plate.

The potato cubes were removed from the agar blocks
when mycelium appeared on the surface of the blocks (20~24
hrs.). Later the blocks were examined directly with the
compound microscope for fruiting structures of Pythium or
other Phycomycetes and the number of blocks containing Pythium

was recorded.

Rhizoctonia Assaye-Papavizas and Davey's (82) method

 

for measuring the inoculum potential of Rhizoctonia solani

Kfihn in soil using buried buckwheat stem segments was modified

37

to compare Rhizoctonia recovery from fungicide—treated and

 

non-treated Rhizoctonia-amended soils. Preliminary experim

 

ments using 5 mm dried mature stem segments,as they described,
gave very low recovery rates and other fungi, particularly

Alternaria, were frequent colonizers. In a search for more

 

selective material, stems aged10%3 l2, 14%-and 16 weeks were
tested. Recovery percentages were variable but green stem
segments, agele-l4% weeks, were more efficient than mature
dried segments. However, Pythium was often recovered on

the green stem segments. In view of the possibility that

this might interfere with Rhizoctonia recovery, an attempt

 

was made to reduce Pythium colonization by vacuum infiltratm
ing the segments with 50 ppm Dexon solution. From previous

work it was found that Rhizoctonia isolate R-54 would tolerm

 

ate 100 ppm Dexon in the culture medium without apparent
inhibition. Dexon 50 ppm was also incorporated in the iso~
lation medium, but it may have reacted with Streptomycin

in the medium. The characteristic yellow-brown Dexon became
colorless after about 8 hours. Recovery of Rhizoctonia was
not improved materially by this method, even though Pythium
was controlled.

The buckwheat stems were segmented just prior to
burial in the soil. One kilogram soil samples were selected
from treated and non-treated soils and 120 segments were
uniformly spaced in each sample. The segments were incu-
bated four days at room temperature, removed from the soil

by screening, washed in running tap water 25—30 minutes,

38

and drained on sterile paper towel. The isolation medium
was prepared by adding 50 ppm each of Streptomycin sulfate,
Aureomycin hydrochloride, and Neomycin sulphate to 2% water
agar just before the plates were poured. The 8 x 10 mm agar
blocks were cut and lifted from the ruled plate and placed
on flamed glass microscope slides in Petri dish chambers
previously described.

Each stem segment was transferred to a single agar
block and incubated for 24-48 hours at room temperature.
The stem segments were then removed from the agar blocks
and the blocks examined periodically with the compound micro~

scope. The number of blocks with Rhizoctonia mycelium was

 

recorded and the percentage recovery was used as an index

of the level of Rhizoctonia inoculum in Rhizoctonia-amended

 

soil vs. Rhizoctonia soil treated with fungicides.

 

EXPERIMENTAL RESULTS

In—Vitro Studies of Fungicides
Fungitoxic effects of Dexon, PCNB and Olin—Mathieson
1921 on mycelial growth and spore germination were studied
by an in-vitro agar plate test. Since the fungicides were
contaminated with bacteria, a series of heat treatments to

eliminate the contaminants was investigated initially.

Stability of Fungicides to Heat Treatment—~Toxicity

 

of Dexon (100, 1000 ppm) to Pythium irrggulare was not ap~
preciably altered by the heat treatments and the character=
istic yellow color was unchanged.

Autoclaving of PCNB almost completely destroyed
the inhibitory prOperties at the 1000 ppm concentration.
Steaming for 20 minutes reduced effectiveness somewhat but
Rhizoctonia growth inhibition was clearly retained at 100
ppm, and growth of contaminating bacteria was checked by
this treatment. Adding the fungicide after steaming did
not reduce fungitoxicity, but bacterial contaminants grew
profusely in the medium.

Olin-Mathieson 1921 (100 ppm) was not affected by
the heat treatments. It completely inhibited growth of

all three test fungi: Rhizoctonia, Pythium and Fusarium.

 

The 20 minute steaming treatment was used for the

in—vitro agar plate tests.

39

 

40

Dexon—Inhibition of Fungus Growth——Mycelium of

 

Pythium irregulare grew moderately well at 1 ppm but growth

 

did not equal that of the control (Table 2). At 10 ppm
Dexon, mycelial growth was very limited compared to that
of the control. At 100 ppm, there was no mycelial growth
but when the inoculum plug was transferred from the Dexon
block to fungicide-free agar, the mycelium grew slightly.
Dexon may be fungistatic to Pythium at this concentration.
At 1000 ppm, there was no growth in the treated block or
the mycelial plugs when they were transferred to fungicidem
free agar. Dexon appears to be fungicidal to Pythium at
this concentration.

Rhizoctonia grew at each concentration, including
1000 ppm Dexon, but the mycelial growth was slightly less
at the higher concentrations. It appears that Dexon acted
as a growth retardant, since even at 1 ppm Dexon, growth

of Rhizoctonia did not equal that of the control. Rhizoc~

 

tonia sclerotia responded much the same as the mycelial
inoculum discs (Table 2).

Fusarium oxysporum f. cucumerinum grew about equally

 

 

well at all four concentrations of Dexon, but growth was
not as extensive as that of the control. Fusarium spores
grew equally well at all four concentrations, but growth
was considerably less than that on the control medium (Table

2).

 

41

 

 

 

 

 

 

 

 

 

 

Table 2. Growth response of Rhizoctonia solani (R-54),
Pythium irregulare IP-lO), and Fusarium oxygporum
f. cucumerinum (F-83) inocula to various concen-
trations of Dexon, PCNB and OM—1921 in the nutri-
ent medium
Growth Index at 4
Fun i— concentrations of fungicide
. g . (rated l—5...5 represents control)
Cide in Inoculum
Blocks Used 0gppm 1 ppm 10 ppm 100 ppm 1000 ppp_
Dexon R-54 Mycelium 5 4 4 4 3 '12
" P-lO " 5 3 2 0 + _p. 0 ~
" F-83 " 5 4.5 4.5 4.5 4.5
" ‘ R—54 sclerotia 5 3-4 3—4 3-4 2-3
“ F-83 conidia 5 4 4 4 3.5
PCNB R-54 mycelium 5 2 + 0 + 0 + 0 +
" P-10 " 5 4 + 3 + 3 + 3 +
" F-83 " 5 l + 1 + l + l +
” R—54 sclerotia 5 2 + 0 + 0 + 0 +
" F—83 conidia 5 0 0 0 0
OM—l921 R-54 mycelium 5 3 + 0 + 0 + 0 +
" P—10 " 5 2 + 1 — 0 - 0 ~
" F—83 " 5 2 0 + 0 + 0 ~
" R-54 sclerotia 5 3 + 0 + 0 + 0 +
" F-83 conidia 5 2 ‘— 0 *— 0 *— 0 '11;
£§_Mycelial growth of R—54 on Dexon medium was largely

surface growth without formation of a thick mat.

/b +

indicates growth resumed when inoculum disk
was transferred to water agar without the
fungicide.

indicates no growth, when transferred as above.
indicates growth resumed when the fungicide
treated block was overlaid with a water agar
block.

indicates no growth of mycelium in fresh water
agar block.

 

42

PCNB—Inhibition of Fungus Growth—-Rhizoctonia R~54

 

mycelial growth on medium containing 1 ppm PCNB was less than
that of the control (Table 2). PCNB at 10, 100 and 1000 ppm
completely inhibited growth of Rhizoctonia R~54 in agar.

When mycelial inoculum discs from the PCNB blocks were trans»
ferred to funcicide-free water agar, there was some growth

on all blocks, indicating that inhibition on PCNB medium

was at least partly fungistatic. Sclerotia placed on PCNB
reacted in a similar manner.

Pythium irregulare was not affected by 1 ppm PCNB

 

in the medium and mycelial growth at 10, 100 and 1000 ppm
was inhibited only slightly. Inoculum discs from these conm
centrations produced rapid mycelial growth when transferred
to fresh water agar blocks.

Fusarium oxysporum f. cucumerinum mycelial growth

 

 

was inhibited at all four concentrations of PCNB, but some
growth did occur. Growth from the inoculum discs resumed
when discs were removed from the PCNB medium and transferred
to fresh water agar. Germination of the Fusarium conidia
was completely inhibited even at 1 ppm. Since the spores
could not be transferred readily to fresh water agar blocks,
the water agar blocks were placed on the tops of the treated
blocks in an attempt to reduce the fungicidal concentration
at the locus of the conidia. No germination or growth oc»

curred (Table 2).

 

 

43

Olin-Mathieson 1921--Inhibition of Fungus Growth—n

 

Fusarium oxysporum f. cucumerinum mycelium grew moderately
on water agar containing 1 ppm of this fungicide but did not
grow at higher concentrations (Table 2). Inoculum discs
from the 10 and 100 ppm concentrations grew slightly when
transferred to water agar, but discs from 1000 ppm concen—
tration did not. Fusarium conidia germinated and produced
moderate growth on 1 ppm 0M-l921, but no germination occurred
at 10, 100 or 1000 ppm. Fresh agar blocks placed on the
treated blocks failed to reduce the toxic effect of the
fungicide and there was no growth after two days. Some
growth was noted on several of the blocks after four days.

Rhizoctonia R—54 mycelium and sclerotia produced
moderate growth on agar containing 1 ppm OM~1921, but there
was no mycelial growth at higher concentrations (Table 2).
Yet inoculum discs from all four concentrations grew freely
when transferred to fresh water agar blocks. The action
appears to have been fungistatic.

Pythium irregulare grew moderately well at 1 ppm

 

but only slightly at 10 ppm OM-1921. No growth occurred
at 100, and 1000 ppm and little or no growth resulted when
inoculum discs from any of the four fungicide concentrations

were transferred to water agar (Table 2).

Summary—~In-Vitro Fungicide Study-mResults for these

 

three fungicides show their range of activity against various

morphologic forms of the test fungi. Pythium was most

44

sensitive to Dexon with partial inhibition at 1 ppm. 337
sarium mycelial growth was only slightly inhibited by Dexon

and Rhizoctonia was inhibited somewhat more. Germination

 

of Fusarium F-83 conidia and Rhizoctonia R—54 sclerotia

 

was inhibited more than their corresponding mycelial forms.
PCNB (1 ppm) inhibited mycelial growth of Rhizoc~

tonia R—54 and Fusarium F-83, and inhibited germination

of R-54 sclerotia and F—83 conidia. Growth of Fusarium

was inhibited more than Rhizoctonia at 1 and 10 ppm PCNB,

 

yet Fusarium was able to grow nearly as well at 100 and
1000 ppm as at 1 ppm. Rhizoctonia was completely inhibited
at higher concentrations. Germination of Fusarium conidia
was completely inhibited at all four concentrations. Pythium
was partially inhibited at all four concentrations but was
able to grow about equally well at each of the concentra-
tions. Growth inhibition by PCNB appears to be of a fungis—
tatic nature.

Olin—Mathieson 1921 was broadly toxic to all three
test fungi, including the conidial and sclerotial forms

of Fusarium and Rhizoctonia. OM—1921 was fungistatic to

 

Rhizoctonia R-54 mycelium and sclerotia at all four concen~

 

trations, and to Fusarium F~83 conidia and mycelium, except
at the 1000 ppm concentration where the mycelium did not
respond when transferred to fresh medium. This compound
was fungistatic to Pythium at 1 ppm but at higher concen-

trations appeared to be fungicidal.

45

Treatment of Mineral Soil

 

PCNB Treatment

 

Unsterilized mineral soil, artificially infested

with Rhizoctonia, was treated with PCNB and sampled, 0, 11,

 

33 and 44 days after fungicide treatment. Colony counts
of fungi, bacteria and actinomycetes were made at each samp»

ling and soils were assayed periodically for Rhizoctonia

 

by the buckwheat—stem baiting technique. Cucumber seeds
were planted in these soils as a test crop for evaluation
of damping-off disease.

Because of the fungistatic nature of PCNB, a comm
parison of colony counts from the various soils was made
on standard medium and on medium containing 50 ppm PCNB,
which is approximately the same concentration as was present

in the soil.

Assays for Fungi--Colony counts of fungi were re-

 

duced markedly by washing the soil samples (Fig. 1, Table
3 A & B). Comparison of the average counts of the three
soils at 0, 11, 33 and 44 days showed a reduction of counts
in washed soils of 65%, 85.8%, 63.8% and 86.5% respectively.

Results of colony counts of fungi from Rhizoctonia»

 

amended vs. Natural soils were different for washed and
non-washed soil samples. For washed samples, colony counts

were greater in Rhizoctonia—amended soil; 19.6, 112.9, 68.8%

 

and 20.8% greater at 0, 11, 33 and 44 days respectively.

Considering the non-washed soil samples, counts in Rhizoctoniaw

 

 

46

 

 

 

 

Fig. l. Fungus colonies assayed per gram (air dry equiv-
alent) of washed and non-washed soil samples.
Natural, Rhizoctonia-amended and Rhizoctonia +
PCNB mineral soils at 0, ll, 33 and 44 days on
standard medium without PCNB.

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Treatment

After PCNB Soil

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47

IUHpmHDMDm Hocno comm Eoum

unease no: 06

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Hoppma been mcp an pmonHow mquEScuIO.Q.m

 

 

 

u e.m n m.mm e m.eeH m m.eH as m26d + mneoeuowaem
e m.m e H.mm e o.mHH n m.sm es esconuousrm
d e.m u m.mm u o.mma e H.Hm es amassez
n e.e n H.mm e o.mmH n o.em mm mzua + encouuounrm
n H.@ e H.mm n m.ma n m.mm mm uncouuouerm
e m.e e m.oe u o.eeH e e.em mm Heusnez
a: an e m.mea a: as mZOd + uncouoounem

I: a: u m.mom a: as dacouuouarm

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u m.a e m.e e o.mm e o.mN o Heusuez
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e o.mH u o.mm u m.eom o e.mm ed mzud + uncouuouscm
u e.ma n H.ee n 0.6mm n m.mm ed eneonuouflrm
u m.HH e m.mm e m.mea e H.mm as Humanez
u o.mm n m.em e o.mma n o.em mm mzod + eaconuossem
n e.ma m m.em n m.mm n m.mm mm usuopuonaem
u o.eH e H.0m e m.mma u m.om mm Huusnuz
u m.am e o.mm e m.ema n m.HN HA mZOd + unconuouecm
n m.mH e m.om e m.ema n e.em Ha euecueenueconuouaem
m m.ma e m.mm u m.eHH e e.HH Ha Heusnez
n e.em n e.em u o.mm u o.em o umucuEeueaeouuousrm
e m.aH u m.om u m.em e H.0m o Hussuuz
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AmCOHHHHEN AmQOHHHHEV AmUCMmSOQWC Amendmsocfiv
mmuou E maumpumm HHom pmcmmzlcoz HHom comma: pcmsummua HHom poNMmm<
nocenu< Hausa Hausa umbu< mama snow,
.mame ed ecu mm .HH .0 be umaaamu maaou

 

 

mzum + macowuouflcm can masonoouflcm .Hmudpmz
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48

amended soil were .5% greater, 17.2% greater, 44.5% less,
and 25.9% greater at 0, ll, 33 and 44 days respectively.
Only the latter two percentages were considered statistic—
ally significant.

Colony counts of fungi in Rhizoctonia—amended vs.

Rhizoctonia—amended-PCNB treated soil were also different

 

for washed and non-washed soil samples. Colony counts from
washed soils indicated an 11.3% reduction, 7.3% increase
and a 39.8% reduction by PCNB soil treatment at 11, 33 and
44 days. Only the last was significant. But in non~washed
soil samples, PCNB soil treatment increased colony counts
14.7%, 62.4% and 35.7% at 11, 33 and 44 days. The last

two were considered significant increases.

Soil treatment with PCNB had a differential effect
on fungus colony counts. It did not appreciably alter counts
of fungi in the washed soil fraction which presumably con“
tained a high proportion of mycelial fragments, debris par»
ticles and the heavier spores. The only significant effect
was a reduction in colony counts in PCNB treated soil at 44
days. But in non-washed soil colony counts of fungi were
increased by PCNB treatment.

Similar results were obtained when soil dilutions
were plated on medium containing PCNB (Table 3 B, Fig. 2).
PCNB in the medium did not appear to change the relative
counts of fungi in the three soils, except in nonmwashed
soil at 44 days when colony counts in the treated and nonm

treated RhizoctoniaJSOils were lower in relation to Natural

 

 

49

Fig. 2. Fungus colonies assayed per gram (air-dry equiv~
alent) of washed and non—washed soil samples.
Natural, Rhizoctonia—amended and Rhizoctonia +
PCNB mineral soils at 0, ll, 33 and 44 days on
standard medium containing 50 ppm PCNB.

 

 

 

 

 

 

 

 

 

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50

soil (Fig. 5). PCNB in the medium also did not reduce total
counts of fungi until the 44 day sample. The reduction was

particularly evident in the non—washed soil sample.

Assgys for Bacteria--Colony counts of bacteria from

 

PCNB treated Rhizoctonia-amended soils are shown in Table 3

 

and Fig. 3.

Adding Rhizoctonia to Natural soil increased bac—

 

terial counts 33.7, 2.5%, 8.4% and 60.7% at 0, ll, 33 and
44 days. Only the counts at 0 and 44 days were considered
significantly higher. The same sample dilutions on medium
containing 50 ppm PCNB showed no significant increases in

colony counts for Rhizoctonia—amended soil.

 

Comparison of PCNB-treated Rhizoctonia-amended soil

with Rhizoctonia control soil showed a 56.2% increase in

 

bacterial colony counts from PCNB treated soil at 33 days
and a 16.8% increase at 44 days after treatment. The same
sample dilutions on PCNB medium showed an increase of 52.7%
in colony counts from PCNB treated soil at 33 days, and an
83.0% increase at 44 days. In addition to the changes in
relative numbers of bacteria in the treated soils, there
was a general reduction in the total counts of bacteria

on PCNB medium as shown in Figs. 4, 6.

Assays for Actinomycetes—-Colony counts of actino-

 

mycetes from PCNBwtreated Rhizoctonia—amended soils are

 

also shown in Table 3 and Fig. 3.

Adding Rhizoctonia to Natural soil increased

 

51

Fig. 3. Bacterial and actinomyCete colonies assayed per
gram (air-dry equivalent) of Natural, RhiZoctonia—
amended and Rhizoctonia + PCNB mineral soils at
0, ll, 33 and 44 days on standard medium without

 

 

   
    

    

Natural soil.

 

 

 

PCNB.
// “ii
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/
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/
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< 423%
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(SUOIIIIw)
1108 30 memo lad saruotoo

52

Fig. 4. Bacterial and actinomycete colonies assayed per
gram (air-dry equivalent) of Natural, Rhizoctonia-
amended and Rhizoctonia + PCNB mineral soils at

0, ll, 33 and 44 days on standard medium contain-
ing 50 ppm PCNB.

 

   
    
 
 

 

 

 

 

 

 

 

F:
I f .3
I l
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I
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l E +3
I 53
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ere \ I Q
8‘” | 83
5% .
.2135. I a:
one 4:
Cf: <11
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+3de I—I I m
an: a
2mm E |
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I I
II I
I
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I I I I l l I I
O O O O
(I) \O .3 CU
(SUOIIIIw)

{103 go mega Jed saiuotoo

OHIO medium
+ SOppm PCNB

W.

N = Natural Soil.

OHIO
medium

I:I

Fig.

= Rhizoctonia-Amended PCNB-treated.
Rhizoctonia-Amended Untreated.

= Rhizoctonia-Amended Soil.

R
T
U

5.

0
LA
(\I

53

Fungus colonies from washed and non-washed samples
of Natural, Rhizoctonia-amended and Rhizoctonia +
PCNB mineral soils at 33 and 44 days after treat—
ment. Comparison of the number of colonies result—
ing from plating on standard medium vs. medium
containing 50 ppm PCNB.

   
     
    

H
0H
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Fig.

Soil Extract or Water Agar
+ SOppm PCNB.

’4

7

Soil Extract
or Water Agar.

[j

6.

54

Bacterial and actinomycete colonies from samples

of Natural, Rhizoctonia—amended and Rhizoctonia +
PCNB mineral soils at 33 and 44 days after treat—
ment. Comparison of the number of colonies result~
ing from plating on soil extract or water agar vso
the same.medium with 50 ppm PCNB.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

8
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TIOS JO mexs Jed sa1uoIoo

 

55

actinomycete colony counts at 0 days, reduced colony counts
19.0% at 11 days, then increased counts 14.1% and 16.1%

at 33 and 44 days. Only the last difference was not conm
sidered statistically significant. The same sample diluH
tions on PCNB medium only showed a significant increase

in actinomycete counts of 80.0% at 33 days. This is much
larger than the 14.1% increase on medium without PCNB.

PCNB soil treatment also increased actinomycete
colony counts 43.2%, 18.6% and 9.5% at 11, 33 and 44 days.
The last was not statistically significant. In PCNB medium,
the same soil dilutions produced no significant changes
in relative numbers of actinomycetes in these soils, but
as in the bacterial counts, the total number of actinomym

cetes was considerably reduced (Fig. 6)°

Rhizoctonia Recovery from PCNB Treated Soilm-Attempts

 

were made to assay for the presence of Rhizoctonia at 0 and

 

11 days after fungicide treatment. At the time of treating

(one week after Rhizoctonia infestation) 16 week old buckm

 

wheat stem segments were buried in Rhizoctonia and Natural

 

soils and recovered after four days. Rhizoctonia was recov~

 

ered from 14.9% of the stem segments from Rhizoctonia soil

 

and from 7.0% of those from Natural soil. Pythium was also
recovered from many of the same stem segments (Natural 31.6%;

Rhizoctonia soil 12.3%).

 

For the assay made 11 days after PCNB treatment,

an attempt was made to minimize the possible competitive

S6

influence of Pythium on Rhizoctonia recovery by using stem
segments which had been vacuum infiltrated with 50 ppm Dexon

solution. Rhizoctonia recovery using Dexon-treated seven

 

and 17 week old buckwheat stem segments buried in the various
soils is given in Table 4. Recovery from Rhizoctonia soil

using Dexon-treated stem segments was less than with non—

 

treated stem segments, for both seven and 17 week old stems.
Similar results were obtained in Natural soil except for the

unusually high recovery of Rhizoctonia from Dexon-treated

 

 

seven week old stem segments. This was probably not a valid
representation of the level of Rhizoctonia in this soil.
Table 4. Recovery of Rhizoctonia from Dexon-treated and

non-treated buckwheat stems buried in Natural,
Rhizoctonia-amended and Rhizoctonia + PCNB soils

 

 

 

 

Percentage Rhizoctonia Recovery from £2,

 

 

Buckwheat Stems Control Soil Rhizoctonia Rhizoctonia
Soil + PCNB Soil

Age 7 weeks

Dexon-treated 7]. 09 4O 00 O

No Dexon 33.3 46.0 0
Age 17 weeks

Dexonwtreated 6.5 6.2

No Dexon 21.9 30.0 0

12_Percentage recovery based on 32 stem segments
buried in each soil sample.

 

Although estimation of the level of Rhizoctonia in
these soils was not conclusive, a higher percentage of re-

coveries was obtained from Rhizoctonia-amended soils, dis~

 

regarding the Dexon—treated segments. No Rhizoctonia was

 

57

recovered from PCNB—treated soils.

Disease—Control-—The effectiveness of PCNB for con-

 

trolling damping—off disease was evaluated by observations
of cucumber seedlings planted in the same soils that were

sampled for the Dilution plate assay and Rhizoctonia recovery

 

experiments. Post—emergence damping—off was recorded at
approximately daily intervals and isolations were made from
the diseased seedlings. Pythium and Fusarium were commonly
recovered from the seedlings in each of the soils but Rh}:

zoctonia recovery was very low. Results of disease observam

 

tions were as follows:

 

Estimated
Pre-emergence Post-emergence Surviving
Damping-off Damping—off Seedlings
Natural soil 49.5% 10.0% 40.5%
Rhizoctonia-
amended 47.0 8.0 45.0
R—amended—-
PCNB—treated 32.0 29.0 39.0

Pre~emergence damping-off was partly controlled by PCNB
soil treatment (Fig. 7). The postnemergence damping-off
shown for the PCNB treatment (curve B) closely paralleled
that of Natural soil (curve A), indicating a lack of conm
trol of pre~ or post-emergence damping-off by Pythium.
The absence of Pythium control obscured any evidence of

Rhizoctonia control. In later experiments with Rhizoctoniam

 

amended Natural soil, this factor was counteracted by adding

Dexon fungicide for control of Pythium.

Seedlings.

of Surviving

Number

 

58

Fig. 7. Survival of cucumber seedlings in unsteamed min-
eral soil amended with Rhizoctonia (R~54) and
treated with PCNB.

 

    

   
  
   

 

 

8C)H (A) Control (untreated)
x Pythium Soil.
70-.
(B) PCNBetreated
R-Sh + Pythium
6 Soil
01
50 — \\\
(C) Rhizoctonia'RHSh + Pythium
hO-— (untreated) Soil.
3O.~
20——
10-1
0 *I I I 7 I 1‘
5 10 15 20 25 30

Days After First Seedling Emergence

59

Dexon Treatment

 

Several experiments were made to determine the ef~
fect of Dexon soil treatment on actinomycetes, bacteria
and fungi in a mineral soil naturally infested with Pythium.
Granular Dexon was mixed with the soil at the rate of 20
lbs. of active ingredient per acre. After treatment, the
soil was stored at 70—80° F. in polyethylene bags and mois-
ture was held at approximately 50% of water—holding capacity.
Colony count assays were made at treatment time and one,
two, three and four weeks thereafter. An assay for Pythium
recovery by the potato cube baiting method was also made
at the same intervals.

In one experiment, the soil was accidentally overm
watered after the first soil sample had been removed and
it had to be placed in open flats to dry. The soil was
later returned to polyethylene bags and the experiment was
completed with one, two, three and four week samples. Since
the soil structure was altered somewhat by stirring and
drying, the experiment was repeated. The results of the
latter experiment generally confirmed those of the first,

so the results of the latter experiment are given in Table 5.

Assays for Fungi; Bacteria and Actinomycetes—HObserw
vations from washed soil samples showed no significant dif»
ferences in colony counts of fungi from Dexon—treated vs.
non-treated soils. Among non-washed soil samples, there

was a significant increase (21.1%) in fungus colony counts

60

Table 5. Colonies of fungi, bacteria, and actinomycetes
assayed per gram of mineral soil (air-dry equiv—
alent). Control and Dexon-treated soils sampled
O, 1, 2, 3 and 4 weeks after soil treatment.

 

 

Weeks
After Fungi (thousands) Actino-
Soil Soil Washed Non-washed Bacteria mycetes
Assayed Treatment Soil Soil (millions) (millions)
Control 0 43.5 149.5 26.9 6.8
Control 1 61.4 a 149.5 a 12.8 a 77.2 a
Dexon 1 70.8 a 118.0 a 17.0 b 9.1 b
Control 2 61.2 a 128.0 a 21.4 a 14.1 a
Dexon 2 66.1 a 155.0 b 21.8 a 10.1 b
Control 3 59.0 a 133.0 a 21.6 a 13.4 a
Dexon 3 58.7 a 149.5 a 33.3 b 13.0 a
Control 4 -- 132.0 a 23.6 a 12.8 a
Dexon 4 —— 112.0 a 20.3 b 13.4 a

a,b--numbers followed by the same letter do not
differ from each other significantly at the
5% level.

 

in Dexon—treated soil at two weeks after treatment, but no
differences were apparent at one, three and four weeks (Table
5, Fig. 8)°

Bacterial colony counts were significantly higher
for Dexon—treated soil at the first (32.8%) and third (54.1%)
week after treatment and significantly below the control
at the fourth week (14.0%) (Table 5, Fig. 9). Also, there
were no consistent differences between colony counts of
actinomycetes in Dexon—treated and non—treated soils. At
one week after treatment, there was a significant increase
(26.4%) in actinomycete counts and a 28.5% decrease at the

second week. There were no differences at three and four

61

Fig. 8. Fungus colonies assayed per gram (air-dry equiv-
alent) of washed and non-washed soil samples.
Control and Dexon treated mineral soils sampled
at O, l, 2, 3 and 4 weeks after fungicide treatH

 

   

 

 

 

 

 

 

ment.
_z-
m f 53
3 / H
I / I
m / H
Ed ”8“
IO at?)
am I
z 3
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I 8
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a n I g
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0 o I
Q o H
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a
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x I a h
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0001 X
1103 JO WEJD lad saxuotoo

62

 
        

 

 

 

 

Fig. 9. Bacterial and actinomycete colonies assayed per
gram (air—dry equivalent) of Control and Dexon
treated mineral soils sampled at O, 1, 3 and
4 weeks after fungicide treatment.

/» -4-
/
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/
/
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/
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IIOS JO maza 18d saruotog

63
weeks after treatment (Table 5, Fig. 9).

Bythium Recovery—~The effectiveness of Dexon as a
soil treatment for control of Pythium sp. was tested by the
potato cube baiting method:

Percentage Recovery of Pythium from Potato Cubes Ag

 

 

 

Weeks After Treatment Natural Soil - Dexon Soil
1 93.3 0.8
2 98.2 18.3
3 79.1 16.7

4 44.2 8.3
La Percentage based on 120 cubes buried in each
soil sample.

Pythium was greatly inhibited in the period just after treatw
ment with Dexon, but it appears that it may survive or par-
tially recover from the effect of Dexon treatment over a
period of time. The assay at four weeks after treatment
showed proportionately lower recoveries from both soils.
This was due to the fact that an unidentified fungus colw
onized potato cubes from both treated and non-treated soils,
introducing not only the factor of competition but also

making identification of Pythium more difficult.

Bayer 29957 Treatment

 

Bayer 29957, a fungicide-nematocide reported to

have activity against Fusarium spp., was examined for its

 

effect on fungi, bacteria and actinomycetes when used as

a mineral soil treatment. Soil samples were assayed at

 

 

64

one, two, three and four weeks after treatment.

Assays for Fungi, Bacteria and Actinomycetes--Soils
were assayed as in previous trials and the counts summarized
(Table 6 and Figs. 10 and 11). From washed soil samples,
there were no differences in colony counts due to B-29957
soil treatment. From the non-washed soil samples, fungus
colony counts from treated soil were 43.0% greater at three
weeks and 16.2% less than from untreated soil at four weeks
after treatment.

Table 6. Colonies of fungi, bacteria, and actinomycetes
assayed per gram of mineral soil (air-dry equiv-

alent). Control and B-29957 treated soils sampled
1, 2, 3 and 4 weeks after soil treatment.

 

 

Weeks .
After Fungi (thousands) Actino-
Soil Soil Washed Non—washed Bacteria mycetes

Assayed Treatment Soil Soil (millions) (millions)
Control 1 42.5 a 133.5 a —- ~~
B-29957 1 37.5 a 139.0 a 28.2 26.4
Control 2 48.5 a 119.0 a 16.8 a 17.1 a
B-29957 2 4006 a 10805 a. 1702 a 1.40]. b
Control 3 61.7 a 164.0 a 25.5 a 14.7 a
B-29957 3 55.0 a 234.5 b 31.5 b 16.1 a
Control 4 56.8 a 172.0 a 24.2 a 17.4 a
B-29957 4 51.1 a 144.0 b 16.7 b 14.5 a

a,b-—numbers followed by the same letter do not
differ from each other statistically at the
5% level.

 

Colony counts of bacteria followed a pattern sim-
ilar to the above at two, three and four weeks after fungi-

cide treatment, with counts 23.5% above the control at three

65

Fig. 10. Fungus colonies assayed per gram (air—dry equiv-
alent) of washed and non-washed soil samples.
Control and B-29957 treated mineral soils sampled
at 1, 2, 3 and 4 weeks after fungicide treatment.

 
      
   

 

 

 

é a
m o
E? E;
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b
‘ a
5: I ox
O\ I (II
0\ m
01 F4 I
I O
m \ H
7 4: I -m $4
$3 a)
0 +3
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.<
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C) d) C) c> c> <3
U\ 0 U\ 0 Ln
0: GI PI .4

0001 X
{108 JO wads Jag sexuotoo

66

Fig. 11. Bacterial and actinomycete colonies assayed per
gram (air—dry equivalent) of Control and B~29957
treated mineral soils sampled at 1, 2, 3 and 4
weeks after fungicide treatment.

  
   

Treatment

  

Control
Control

BACTERIA
Soil

ACTINOMYCETES

 

\.
B-29957
I
2
B-29957

After

\
\
Weeks

 

 

30“
20 —
10"

(suorttrm)
{103 JO WBJD 18& setuotoo

67

weeks and 31.0% below the control at four weeks (Table 6,
Fig. 11). Week one control samples for the bacterial and
actinomycete assays were omitted so no comparison could be
made for that time. Colony counts of actinomycetes were
17.7% lower in fungicide treated soil than in Natural soil
at the second week but differences at the third and fourth
week were not apparent. I

The initial drop and subsequent rise to values above
the control as noted for Dexon soil treatment was apparent
from the counts of fungi (non—washed samples), bacteria

and actinomycetes from B-29957 treated soil (Figs. 10, 11).

Pythium, Fusarium, and Nematode Recovery-—The standw
ard potato cube bait—recovery technique was used to assay

for Pythium, Fusarium, and nematodes in treated and nonm

 

treated soils. Although this technique was not intended

for recovery of Fusarium, the fungus often appeared after

 

Pythium and was distinct enough that its recovery could be
recorded. Nematodes were also frequently present on bait

buried in the soil. Since B-29957 was characterized as a

nematocide, recovery based on the number of blocks having

nematodes was recorded for both soils. At one week after

soil treatment the following recoveries were obtained.

Percentage recovery from potato cubes buried in soil ia_

 

 

Pythium Fusarium Nematodes
Control 98.0 6.3 10.4
Bayer 29957 89.5 3.1 5.2

1§_Percentage based on 120 cubes buried in each
soil sample.

 

68

A potato cube assay at four weeks after treatment gave very
poor results. Cubes were rotted very quickly by bacteria
and Pythium was recovered in only 11.5% of the cubes from
control soil and 28.1% of cubes from B-29957 treated soil.
In an assay at five weeks, Pythium was recovered from 76.0%
of the cubes from control soil and 94.8% of cubes from B—29957
treated soil. In summary, Pythium recoveries from treated
soil were 8.7% less than control at one week, 144.3% above
control at four weeks and 23.8% above control at five weeks.
Although not much significance can be attached to the very
high recovery at four weeks, the higher recovery at five
weeks is another indication that Pythium is affected by

this fungicide and may even be stimulated by it.

Nematodes were recovered from 66.7% of the cubes
from control soil and 28.1% of the cubes from treated soil
at five weeks. It is apparent that this chemical has nema~
tocidal activity, with a reduction in nematode recovery of
50% and 58% respectively at one and five weeks after treat»
ment.

From the single observation above, Fusarium appears

 

to be inhibited by B-29957, but the low percentage of rem

covery precludes any definite conclusion.

Rhizoctonia, Pythium, Fusarium and Nematode Recoverye-

 

Buckwheat stems were used to measure recovery of Rhizoctonia,

 

Pythium, Fusarium and nematodes in B~29957 treated and non-

 

 

treated soils. Although this method was primarily for

69

Rhizoctonia recovery, the other organisms were recovered

 

frequently and the percentage of blocks containing each
organism was recorded:

Percentage recovegy from Buckwheat stems buried in soil La

 

'9

Rhizoctonia Pythium Fusarium Nematodes

 

 

Control 4.2 90.5 15.0 66.7
Bayer 29957 5.2 95.0 53.2 44.8
1§_Percentage based on 96 stem segments buried in
each soil sample.
There were only slight differences in recovery of

Pythium and Rhizoctonia between treated and non—treated

 

soils. But Fusarium was recovered from 53.2% of segments

 

from treated soil as compared to only 15.0% from the non~
treated soil. This is the opposite result from that ob-
tained in the potato cube assay. Nematocidal activity of
B-29957 is shown by the lower percentage of nematode recov—

ery from treated soil.

Difolatan Treatment

 

Assays for Fungi, Bacteria and Actinomycetes-~Soil
treatment with the broad spectrum fungicide Difolatan clearly
reduced populations of fungi, bacteria and actinomycetes
for at least 4 weeks after soil treatment (Table 7 and Figs.
12 and 13). A11 differences between counts of fungi in
treated vs. non—treated soils were statistically significant
and most of them highly significant during the four week

assay period. Washed soil samples showed an average

Fig.

70

 

 

 

 

 

 

 

 

 

 

 

 

12. Fungus colonies assayed per gram (air—dry equiv~
alent) of washed and non-washed soil samples.
Control and Difolatan treated mineral soils sam~
pled l, 2, 3, and 4 weeks after fungicide treatw
ment.

E

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F .e

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5" "H \o 3
H B o

I E

E] I o

l 1 I I I I I I I

s s s e a .

0001 X

IIOS JO WBJD Jed seruotoo

71

Fig. 13. Bacterial and actinomycete colonies assayed per
gram (air—dry equivalent) of Control and Difola—
tan treated mineral soils sampled at 1, 2, 3 and
4 weeks after fungicide treatment.

 

 

 

 

 

 

 

 

 

 

 

 

 

B
o
— :-
| B
r o p
I
. w
m
,4
'E-I B
M0
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8 I o
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g. «I
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8. I 3
3:1 I I. 1.,
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I I I I I I
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(suottttm)

IIOS go weds 18d satuotoo

72

Table 7. Colonies of fungi, bacteria and actinomycetes
assayed per gram of mineral soil (air-dry equiv~
alent). Control and Difolatan-treated soils.

 

 

Weeks
After Fungi (thousands) Actino~
Soil Soil Washed Non-washed Bacteria mycetes

Assayed Treatment Soil Soil (millions) (millions)
Control 1 42.5 a 133.5 a -- ~-
Difolatan 1 26.0 b 38.0 b 8.2 8.9
Control 2 48.5 a 119.0 a 16.8 a 8.5 a
Difolatan 2 17.5 b 43.0 b 12.7 a 4.3 b
Control 3 61.7 a 164.0 a 25.5 a 14.7 a
Difolatan 3 24.4 b 67.5 b 19.0 b 6.9 b
Control 4 56.9 a 172.0 a 24.2 a 17.4 a
Difolatan 4 20.0 b 43.0 b 14.5 b 7.0 b

a,b--numbers followed by the same letter do not
differ from each other statistically at the
1% level.

 

reduction in counts by the Difolatan treatment of approxi~
mately 56% and in non—washed soil approximately 66% (Table
7, Fig. 12).
There were also highly significant reductions in
colony counts of bacteria and actinomycetes (30% and 54.1%
respectively) in Difolatan treated soil (Table 7, Fig. 13).
Control samples for bacteria and actinomycetes sampled one
week after soil treatment were omitted by error, so no comm
parison with non-treated soil could be made for that time.
If counts for fungi, bacteria and actinomycetes
are expressed as a percentage below control, the continued

toxicity over the four week period is evident:

73

Colony counts from treated soil expressed
as percentage below control

 

 

 

 

Weeks After flung;
Soil Treatment Washed Non—washed Bacteria Actinomycetes
1 38.8% 71.5% -- ~-
2 63.9 63.9 24.4 49.4
3 60.5 58.8 25.5 53.1
4 64.9 75.0 40.1 59.8

Pythium, Fusarium and Nematode Recovery--Soils were

 

assayed by the potato cube method at one, four and five weeks
after Difolatan treatment:

Percentage recovery_from Potato cubes La

 

 

 

Soil
Assayed Week Pythium Fusarium Nematodes
Control 1 98.0 6.3 10.4
Difolatan 1 8.4 2.1 0
Control 4 .
Difolatan 4 .
Control 5 76.0 66.7
Difolatan 5 39.6 12.5

12 Percentage recovery based on 96 potato cubes
buried in each soil sample.
Percentage recovery of Pythium from Difolatan treated soil
was very low. Also, based on the limited data above, Di-
folatan soil treatment reduced the percentage recovery of

Fusarium and nematodes.

 

Rhizoctonia, Pythium, Fusarium and Nematode Recoveryem

Results of the buckwheat stem-segment assay also indicated

 

74

that Difolatan was inhibitory to Pythium and Fusarium.
Although the percentages were rather low, recovery 0f.§fl$‘
zoctonia and nematodes was also reduced by Difolatan soil
treatment at one week after soil treatment:

Percentage recovery from buckwheat stem segments 13

Rhizoctonia Pythium Fusarium Nematodes

 

Control 4.2 90.5 66.7 15.6
Difolatan 2.0 27.1 17.6 6.2
13_Percentage based on 96 stem segments buried in

each soil sample.

Treatment of Organic Muck Soil-

Dexon + DAC-469 Treatment

Dexon + Diamond Alkali Co. 469 (octachloro tetra~
hydro thiophene) were chosen as a combination treatment of
two selectively active fungicides for control of dampingmoff
disease caused by Pythium and Rhizoctonia. ~An attempt was
made to evaluate the effect of this combination on a disease
complex under reasonably natural conditions using red beet
seedlings as a test crop. The Dexon + DAC—469 mixture was
applied to muck soil at 20+30 lbs. active per acre, respec~
tively. Soil samples from various combinations of fungicidem
treated, non-treated, planted and non—planted soils were
assayed at time of fungicide treatment, seedling emergence,
and seedling harvest (0, l and 3 weeks). Data on the effect
of soil treatment on numbers of fungi, bacteria and actinomyw

cetes was analyzed by the CDC 3600 computer (Tables 8,9,10,11).

7S

 

 

 

 

Table 8. Colonies of fungi, bacteria and actinomycetes
assayed per gram of muck soil (air—dry equivalent).
Natural, Ehizoctonia-amended and Rhizoctonia +
(Dexon + DAC 469) soils sampled at 0, 1 and 3
weeks (time of treatment, emergence, harvest).
Muck Fungi Actino—
"Soil Washed (thousands) Bacteria mycetes
Assayed (a; Week Soil Non-washed (millions) (millions)
Natural NP 0 47.2 a 126.6 a 38.9 a 38.3 a
Rhizoctonia
Control NP 0 55.2 b 119.4 a 67.2 b 38.2 a
Natural NP 1 56.3 164.3 28.9 36.8
Natural P 1 49.3 202.6 23.0 39.7
Treated P 1 47.5 217.8 44.1 36.2
Rhizoctonia
Control NP 1 53.7 193.7 35.1 35.3
Rhizoctonia
Control P 1 63.5 182.3 48.8 39.2
Rhizoctonia
Treated P 1 46.0 217.1 54.0 37.3
Natural NP 3 54.8 93.7 27.2 20.8
Treated NP 3 45.4 75.9 54.1 22.1
Natural P 3 62.4 145.1 30.7 17.8
Treated P 3 54.8 127.8 51.8 16.1
Rhizoctonia
Control NP 3 60.6 60.7 45.7 25.8
Rhizoctonia
Treated NP 3 64.4 52.3 123.2 17.5
Rhizoctonia
Control P 3 54.8 75.4 49.2 19.7
Rhizoctonia
Treated P 3 54.6 97.4 107.2 15.6
‘12 P Planted (Red beets).

NP

Non-planted.

 

76

Table 9. Overall means of fungus colony counts for various

soil treatments and sub-treatments at the time
of harvest (3 weeks after treatment with DAC 469
+ Dexon). Analysis of variance.

 

E.

Main Factor-~Soil Treatment 43

 

a 84.5 Natural soil untreated

a b 75.2 Natural soil fungicide-treated
b 66.7 Rhizoctonia soil fungicide-treated
b 60.4 Rhizoctonia soil untreated

 

Subfactor B-—Planting

a 79.7 Planted (Beets)
b 63.6 Non-planted

Subfactor C--Soil Washing

a 88.2 Non—washed soil samples
b 55.2 Washed soil samples

Soil Treatment——Soil Washing Interaction

a 113.3 Ck-NT NW 12'
a 100.4 Ck T NW
73.75 R T NW
65.4 R NT NW
59.6 R T w
55.8 Ck NT w
55.4 R NT w
49.9 Ck T w

C'U'O‘U‘U‘
00000

Planting—Washing Interaction

a 105.6 P NW
b 70.8 NP NW

b c 56.5 P W

c 53.8 NP W

12 a,b,c-—numbers followed by the same letter do
not differ from each other statistically
at the 1% level.

 

£2.R = Rhizoctonia soil Ck = Natural soil
T = Treated (fungicide) NT = Non-treated
W = Washed soil sample NW = Non—washed
P = Planted (Beets) NP = Non-planted

 

77

Table 10. Overall means of bacterial colony counts for

various soil treatments and sub-treatments at
the time of harvest (3 weeks after treatment
with DAC 469 + Dexon). Analysis of variance.

 

Main Factor--Soil Treatment

‘La a 160.8 Rhizoctonia soil
b 80.5 Natural soil

Subfactor B-—Fungicide Treatment

a 168.3 Dexon+DAC 469 treatment
b 73.0 Non-treated

Subfactor C—-Planting
5% level
a 127.6 Planted
b 113.7 Non—planted

Soils vs. Fungicide Treatment Interaction

12’ a 230.4 R T
b 106.2 Ck T
b 91.2 R NT

c 54.9 Ck NT

Fungicide-Treatment vs. Planting Interaction

a 185.33 T NP
b 151.3 T P

C 76.2 NT P

C 69.9 NT NP

‘12 a,b,c——numbers followed by the same letter do
not differ from each other statistically
at the 1% level.

 

éb'R = Rhizoctonia soil Ck = Natural soil
T = Treated (fungicide) NT 2 Non-treated
P = Planted (Beets) NP = Non—planted

 

 

78

Table 11. Overall means of actinomycete colony counts for

various soil treatments at the time of harvest
(3 weeks after treatment with Dexon+DAC 469).
Analysis of variance.

 

Main Factor--Soil Treatment

a 39.2 Natural soil
a 40.3 Rhizoctonia soil

Subfactor B-—Fungicide Treatment

a 43.9 Non-treated
b 35.5 Dexon—DAC 469 treated

Subfactor C--Planting

a 43.1 Planted
b 36.3 Non-planted

Soil vs. Fungicide—Treatment Interaction

a 47.3 R NT
b 40.4 Ck NT

c 37.9 Ck T

d 33.3 R T

Soil vs. Fungicide—Treatment vs. Planting Interaction

a 53.7 R NT NP 5% level
b 43.2 Ck NT NP
b 41.7 Ck T NP

bc 41.0 R NT P
d 37.7 Ck NT P
d 34.2 Ck T P
d 34.0 R T NP
d 32.5 R T P

13 a,b,c,d--numbers followed by the same letter
do not differ from each other statis-
tically at the 1% level (or 5% level
as indicated).

 

£b_R = Rhizoctonia soil Ck = Natural soil
T = Treated (fungicide) NT = Non-treated
P = Planted (Beets) NP = Non-planted

 

79

The effect of the fungicide combination on Pythium and Rhi—

zoctonia by baiting techniques and its effect on damping—off

 

disease was also studied.

Assays for Fungi-—Assays for fungi in fungicide
treated soils should be considered in relation to the fac-

tors associated with fungicide treatment.

1) Differences between Rhizoctonia-Amended and Nat»

ural Muck Soils-~a) Assays from non-washed-Soil samples——

 

There were no differences between colony counts of fungi
in Natural muck vs. Rhizoctonia—amended muck at the time
of treating and planting or at the time of seedling emer—
gence (one week). At the time of seedling harvest counts

of fungi in Rhizoctoniaramended soils averaged 34.9% less

 

than in Natural soil (Table 9 D). b) Assays from washed
soil samples-~At the time of treating and planting colony

counts of fungi were 17% greater in Rhizoctonia—amended

 

muck than in Natural muck but this difference was not ap-
parent at the time of seedling emergence (one week) or at

seedling harvest (three weeks).

2) Differences between Fungicide—Treated and Non—

Treated Soils—~a) Assays from non-washed soil samples-~At

 

the time of seedling emergence, colony counts of fungi were
higher in fungicide—treated soil but the differences were
not considered significant. Fungus colony counts at seed~

ling harvest (three weeks) also showed no differences due

80

to fungicide treatment. b) Assays from washed soil samples-—
No differences in colony counts of fungi were noted for
fungicide treated vs. non-treated soils, at either the time

of seedling emergence or seedling harvest.

3) Differences between Planted (Beets) and Non—Planted

 

Soils-~Ana1ysis of fungus colony counts from washed soil
samples at the time of seedling harvest showed no differ—
ences due to planting, but in non—washed soil samples, col—
ony counts in planted soils averaged 49.2% greater overall
than in non-planted soils (Table 9 E). An increase was
also noted in non-washed soils from samples at emergence
(one week) (Fig. 14), but data from washed soil samples
showed only a small increase in fungus counts for planted

soils, over the four week period (Fig. 15).

Assays for Bacteria—-Bacteria1 colony counts from

 

0, 1 and 3 week samples of fungicide treated Rhizoctonia~

 

amended and Natural soils are given in Table 8 and Fig. 16.

1) Differences between Rhizoctonia~Amended and Nat~

ural Muck Soils-—Considering all treatments within these

 

soils as a group, the bacterial colony counts in Rhizoctonia~
amended soils were 72.8%, 54.6% and 99.8% greater than those

in natural muck soil at 0, 1 and 3 weeks.

2) Differences between Fungicide-Treated and Non-

Treated Soils—~Bacterial colony counts in Dexon+DAC-469

 

treated soils were 32.0% greater than in non-treated soils

81

' . 14. 'Fungus colonies assayed per gram (air-dry equiv-

alent) of non—washed soil samples. Natural, Eh}:
zoctonia-amended and Rhizoctonia + (Dexon+DAC-
469) muck soils at 0, l and 3 weeks after fungi-
cide treatment.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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82

' . 15. Fungus colonies assayed per gram (air-dry equiv—

N = Natural Soil.

alent) of washed soil samples. Natural, Rhizoc—
tonia-amended and Rhizoctonia + (Dexon+DAC—469)
muck soils at 0, 1 and 3 weeks after fungicide

 

 

 

 

 

 

 

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83

Fig. 16. Bacterial colonies assayed per gram (air-dry equiv-
alent) of Natural, Rhizoctonia-amended and Rhizoc—
tonia + (Dexon+DAC-469) muck soils at 0, l and 3
weeks after fungicide treatment.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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84

kxmaweek)and 130.6% greater at three weeks after treatment.
Considering the various treatment—soil—planting interactions,
bacterial colony counts in Natural soil were 93.5% greater

in fungicide treated soil than in non—treated soil. In

Rhizoctonia-amended soil, colony counts were 152.6% greater

 

in fungicide-treated soil than in non-treated soil (Table

10 D).

3) Differences between Planted and Non-Planted Soils~~

In non-treated soils counts of bacteria were slightly higher

 

in planted soils than in non—planted, but not all the dif-
ferences were significant. In treated soils, assayed at
three weeks, the reverse occurred with bacterial counts

18.4% less in planted soils (Table 10 E, Fig. 16).

Assays for Actinomycetes-—Results of assays at 0, 1

 

and 3 weeks after treatment are given in Table 8 and Fig. 17.

1) Differences between Rhizoctonia-amended Muck and

 

Natural Muck Soils—~No differences in colony counts were

 

apparent from the data observed at 0 and 1 week (Fig. 16)
or from the analysis of actinomycete colony counts at three

weeks (Table 11 A).

2) Differences between Fungicide-Treated and Non~
Treated Soils--No differences due to fungicide treatment
were found at emergence (one week). At the time of seed—

ling harvest, counts in both Rhizoctonia-amended and Natural

 

soils were significantly lower (29.6% and 6.3% respectively)

 

 

 

Fig.

17.

85

Actinomycete colonies assayed per gram (airwdry
equivalent) of Natural, Rhizoctonia—amended and
Rhizoctonia + (Dexon + DAC-469) muck soils at-

 

 

0, l and 3 weeks after fungicide treatment.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Treatment

Soil

Dexon + DAC~M69

After

Weeks

 

86

in fungicide treated soil than in non—treated soils (Table

11 D and Fig. 17).

3) Differences between Planted (Beets) and Non-

Planted Soils—-In both Rhizoctonia-amended and Natural soils,

 

actinomycete counts were less in soils planted with beets,
but the only significant reductions in planted soil were
18.0% in fungicide—treated Natural soil and 23.7% in non—

treated Rhizoctonia soils (Table 11 E and Fig. 17).

 

Rhizoctonia and Pythium Recoveryf-Treated and conm
trol soils were assayed for recovery of Rhizoctonia and
Pythium by previously described baiting methods. Percent-
age recovery was recorded at the time of fungicide treat~
ment (0 weeks), the time of seedlflNJemergence (one week),
and the time of seedling harvest (three weeks):

% Recovery of Rhizoctonia from Buckwheat
stem segments buried in soil

 

 

 

 

 

 

Muck Soil Weeks After No. of Segments

Assayed Soil Treatment per soil sample Percent
Natural 0 120 0.8
Rhizoctonia—amended 0 120 20.0
Rhizoctonia Control 1 60 21.6
Rhizoctonia-Treated

(Dexon+DAC-469) 1 64 21.9
Rhizoctonia Control 3 96 23.9

 

Rhizoctonia-Treated 3 60 26.6

 

87

% Recovery of Pythium from Potato cubes buried in soil

 

Weeks After No. of Cubes recovered Percent
Soil Treatment yper soil sample Pythium
Natural Muck 0 120 97.5
Natural (control) 1 56 96.5
Treated (Dexon+
DAC—469) 1 64 96.9
Natural (control) 3 74 31.0

Treated (Dexon+
DAC-469) 3 57 3.5

Rhizoctonia recovery from Rhizoctoniamamended soil

 

ranged from 20—26.6% as compared to less than 1.0% for Nat—
ural muck soil. Soil treatment with Dexon+DAC—469 had no

apparent effect on Rhizoctonia in the soil.

 

At the time of emergence Pythium was recovered from

96.0% of the potato cubes buried in Dexon+DAC-469 treated
soil. At seedling harvest (three weeks), there was a rem
duction in recovery of Pythium in the fungicide treated

soil, but the low percentage recovery and the conditions

of the experiment make interpretation of this result difm
ficult. The potato cubes from the three week assay dried
considerably during the 15 hour incubation period on agar

blocks and Pythium was recovered from only a few blocks.

Disease Control-~Observations on damping-off disease

 

of red beet seedlings are shown graphically in Fig. 18.
In non—treated soil post—emergence damping—off was greatm

est in soil naturally infested with Pythium. In the same

Seedlings

Surviving

Of

Nmflmr

88

Fig. 18. Survival of red beet seedlings in Natural and
Rhizoctonia-amended muck soils treated with Dexon
+ DAC-469 fungicides.

 

 

 

   
 

 

 

 

 

180‘
Natural Soil (Treated)
NV ' i ‘ A A A
160— ' ‘ ' '

h Rhizoctonia-Amended Soil
1 O7 (Treated)
120—

100“

80-

Natural Soil —_‘\\\\\\\\

60* (Non-treated)

40—

Rhizoctonia R-Sb.’ Amanded Soil

20‘ (Non-treated)

0 v 2 r A * ‘ fl
I I I

5 10 15

Days After First Emergence of Seedlings

89

soil artificially infested with Rhizoctonia, pre—emergence
damping—off was especially severe and the remaining number
of seedlings was quickly reduced by post—emergence damping—
off. In Dexon+DAC—469 treated soil, both pre- and post~
emergence damping—off were controlled adequately in Natural
soil but post—emergence damping-off by Rhizoctonia was not

controlled adequately by this fungicide-combination.

Rhizoctonia Isolates

 

Pathogenicity in Muck Soil—~Pathogenic capabilities

 

of various isolates on red beet seedlings is shown in Table

12. Rhizoctonia R—53 and R—54 were most pathogenic. Rm46,

 

R-47 and R—52 isolates also reduced stands and yields apprem
ciably but reduction was due largely to seedling loss after

emergence. Rhizoctonia R-53 and R—54 also produced consid—

 

erable pre—emergence damping-off (Table 12). Several of

the Rhizoctonia isolates in Dexon-treated non—steamed muck

 

soil are shown in Fig. 19.

A single replicate test in natural soil infested
with Rhizoctonia showed the need for Dexon soil treatment
to control damping-off (Table 13 A). Pythium killed so
many of the seedlings early in the experiment that too few

were left for evaluation of Rhizoctonia damping-off. A

 

single replicate experiment was also done in conjunction
with the Dexon soil treatment experiment using a mixture
of Dexon and Difolatan (30 + 30 1bs./A) as a soil treatment

to determine the response of the isolates to Difolatan soil

90

Table 12. Stand, survival and yield of red table beet seed—
lings in Natural muck soil amended with Rhizoc—
tonia isolates and treated with Dexon.

 

 

 

Post Total
Total Emergence Weight Weight
Emergence Damping~off Survivors Per Plot Per Plant

Ck 131.2 ab 5.0 c 126.2 a 37.3 ab .295 a
R~42 161.2 a 31.0 bc 130.2 a 44.8 a .345 a
R-44 141.0 ab 4.8 c 136.2 a 44.0 a .321 a
R-45 147.0 ab 9.0 bc 138.0 a 48.0 a .343 a
R-46 137.8 ab 86.5 a 50.0 bc 16.9 cd .345 a
R-47 100.5 b 89.8 a 10.8 cd 3.5 d .326 a
R-49 155.0 a 5.5 c 149.5 a 48.1 a .319 a
R-51 139.2 ab 13.5 bc 125.8 a 36.9 ab .294 a
R-52 117.8 ab 40.0 b 77.8 b 23.5 bc .304 a
R—53 5.8 c 5.8 c 0 d 0 d O b
R-54 17.0 c 17.0 bc 0 d 0 d 0 b
R-55 166.5 a 14.0 bc 152.5 a 47.4 a .303 a

a,b,c,d means followed by the same letter do not
differ significantly at the 5% level.

 

Seedlings

of Surviving

Number

91

Fig. 19. Progress of post-emergence damping—off of red

160H

 

beet seedlings in Dexon—treated muck soil natur—
ally infested with Pythium or additionally in-
fested with various Rhizoctonia isolates.

R-u2

 

 

 

 

 

 

 

 

 

 

 

140—
- \\ v~——‘__________ _ 1, - ___q
120‘ Natural Soil
- R-Sh (Dexon + Difolatan)
100‘
80‘ 44.11
R-52
60‘
? M
R-h6
ho-
R-Sh
20— R-h7
0\-._-- ..1.1
I I ‘ I
5 10 15

Days After First Emergence Of Seedlings

92

Table 13. Stand, survival and yield of red beet seedlings
in natural muck soil amended with various Rhi-
zoctonia isolates.

 

 

Post Total
Total Emergence Weight Weight
Emergence Dampingroff Survivors Per Plot Per Plant

Part A. No chemical soil treatment (non—replicated)

Ck 21 12 9 2.4 .266
R-42 31 28 3 .9 .306
R-44 24 18 6 .9 .153
R~45 18 17 l .3 .280
R-46 47 36 11 3.6 .329
R-47 110 48 62 20.3 .327
R-49 22 18 4 .8 .202
R—Sl 15 12 3 1.3 .443
R—52 35 26 9 2.7 .300
R-53 0 0 0 0 0
R-54 9 9 0 0 0
R-55 22 17 5 1.84 .083
Part B. Dexon and Difolatan treatment

Ck 184 7 177 51.0 .288
R—42 130 9 121 30.5 .251
R-44 152 4 148 35.2 .238
R—45 126 9 117 25.9 .220
R—46 112 36 76 13.9 .182
R-47 115 54 61 14.1 .230
R—49 167 6 161 45.4 .281
R-51 158 15 143 35.8 .250
R~52 150 13 137 32.4 .236
R—53 73 31 42 10.8 .256
R-54 126 34 92 22.6 .245
R—55 158 6 152 40.2 .264

 

93

treatment (Table 13 B).

Since differences among isolates in response to
the fungicides were apparent, a further investigation was
made using representative isolates R—45, R-51 and R—54.
Each isolate was subjected to four chemical soil treatments:
Dexon (30 1bs./A), Difolatan (30 lbs./A), Dexon+Difolatan
(30+30 lbs./A) and Dexon+PCNB (30+30 lbs./A). The latter
mixture was included as a standard for comparison of 323*

zoctonia control, and to supplement data on in—vitro response

 

of the isolates to PCNB. The progress of damping-off disease
was followed daily (Figs. 20, 21, 22, 23).

The effectiveness of chemicals for disease control
was different for each isolate. In natural Pythiumwinfested
soil, Dexon + Difolatan and Dexon + PCNB were equally efm
fective in disease control (Fig. 20). Dexon effectively
controlled post-emergence damping—off but was somewhat less
effective in control of pre-emergence damping—off. Difolatan
was even less effective in controlling pre-emergence damping»
off and was not effective in controlling post-emergence
damping—off.

In the same soil infested with Rhizoctonia R-45,(Fig. 21)
a relatively non—pathogenic isolate, Dexon+Difolatan, Dexon+
PCNB, and Dexon alone controlled post—emergence dampingmoff.
In this case, pre—emergence damping—off in the Dexon soil
treatment was even less than that of the control. Pre-
emergence damping—off followed approximately the same pat-

tern as in the control (Fig. 20).

Seedlings

Surviving

Of

Number

94

Fig. 20. Survival of red beet seedlings in Natural muck
soil (Control) treated with-Dexon, Difolatan and

 

 

 

 

 

 

 

   

 

PCNB.
Natural Soil
1ho _ Dexon + PCNB
- Dexon + Difolatan ~5— 77" ‘ ‘ ?:;—.
120-H 1___l___?__ - ‘ -
“A
Dexon
loo-—
80—
Difolatan
6o—
ho-
20‘4
O

 

 

I I l
5 10 15

Days After First Emergence Of Seedlings

 

 

 

 

Seatumgs

Surviving

Of

Nmmmr

95

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fig. 21. Survival of red beet seedlings in Natural muck
soil amended with Rhizoctonia R—45 and treated
with Dexon, Difolatan and PCNB.

R46
Dexon + Difolatan
140— I- _ N
. .___1__..___1___. . Dexon + PCNB
-fi. w “.—- f : ._. I
120‘“ Dexon
100-
80—
60-
Difolatan
ho-
20~
O . 1
I | I
S 10 15
Days After First Emergence Of Seedlings

Seedlings

Surviving

Of

Number

 

96

 

 

 

 

 

 

 

 

Fig. 22. Survival of red beet seedlings in Natural muck
soil amended with Rhizoctogia R—51 and treated
with Dexon, Difolatan and PCNB.

R-Sl
llI-O—w
,____ Dexon + PCNB
.\‘N“‘.
\\*\. ‘ ‘u ‘
120- - 1, ::::-——-——-4———1___1__..___.__..
1__gg-___*fi-
Dexon + Difolatan
100- \\
Dexon
80*
60%
1+0 — 1.
Difolatan
20‘
O
I l I
5 10 15

Days After First Emergence Of Seedlings

Seedlings

Surviving

Of

Mmmer

97

Fig. 23. Survival of red beet seedlings in Natural muck
soil amended with Rhizoctonia R—54 and treated
with Dexon, Difolatan and PCNB.

 

 

 

 

 

   
  

 

 

 

     
 

 

R-Sh
1&0 —
~_—_*“P“\\‘___1 Dexon + Difolatan
120—— - _ _fig\\“‘¥g
loo-—
&3—
Difolatan
6o - . ~~.
Dexon + PCNB
V M
to.— Dexon
20‘-
O .
I I I
5 10 15

Days After First Emergence Of Seedlings

98

In soil infested with R-51, neither Dexon nor Di-
folatan alone controlled post-emergence damping—off. Dexon
controlled pre-emergence damping-off better than Difolatan,
but Dexon combined with Difolatan or with PCNB controlled
both pre- and post-emergence damping-off. Dexon-PCNB was
slightly more effective in control of pre-emergence damping—
off than Dexon+Difolatan (Fig. 22).

For the R-54 isolate, a number of differences were
apparent (Fig. 23). Dexon + Difolatan controlled both pre-
and post-emergence damping-off but Dexon+PCNB controlled
neither. Pre— and post-emergence damping-off were even
greater in soil treated with Dexon alone. Difolatan alone
did not control pre-emergence damping-off as well as it
did in the control soil (Fig. 20) but was more effective
in control of post—emergence damping-off than in the con—
trol soil.

The response of R-54 to Difolatan and PCNB was quite
different from that of R-51. R-54 was more sensitive to
Dexon + Difolatan than to Dexon + PCNB (Fig. 23) and the
opposite occurred for Rhizoctonia R—Sl (Fig. 22) which was
slightly more sensitive to Dexon + PCNB. This was noted
when damping—off observations were made, and supports earlier

observations on the insensitivity of Rhizoctonia R—54 to

 

PCNB soil treatment.
The data on damping-off disease, root rot rating,
and seedling weights for the various chemicals within each

Rhizoctonia isolate are shown in Table 14. A split~plot

 

 

 

 

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pm QCTQTMMHU MHQCMUHMHcmHm Doc mum QOQQmH menu mQ pmonHom mcmmzllmkc.o.Q.m

99

 

 

 

 

 

 

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H.mH 0mm. TUUQ m.Hm Q m.mm m m.o¢ o.mm 0.0m CMQMHOMHQ
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m.m mmw. m m.sm m m.mHH Q o.oH m.om m.oHH coxmo
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m.m emu. we m.mm Q m.mm m o.mw m.mm m.wm cmumHOMHQ
m.H mmm. Qm mem. m m.va Q m.¢ m.mm m.mmH coxma
mvlm
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m.v wow. Qm m.Hm m m.mmH Q o.H m.om m.omH QMQmHOMHQ+Qome
w.HH omw. m m.mm. Q o.mm m m.em m.Hm m.m0H CMQMHOMHQ
m.m Hmm. UUQm v.m¢ m m.mHH Q m.v o.Hv o.omH coxmo
Houpcou
Qon umm .m,ucmHm mm QOHm muo>H>usm .mumEMIQmom .mumEmtmum mocmmumam
xmucH \uz com: \ps Hmpoe mmoumcaaemo Hmuos
pom boom cama» academmm

.mCOHchHQEoo HmUHEonImeHomH Mo COmHumm

IEOU .mHMUHEmQUImUHUHmcsw QQHB UTQMTHQ cam memHOmH MHCOQUONHQm mmHQQ QQHS
pmtcmem HHom MUSE HmuSch CH mmcHHpmmm Dme ton mo mpHmH> can Hm>H>u3m .mpfiMQm .wH mHQme

100

analysis of variance showed differences in overall chemical

response of the Rhizoctonia isolates (Table 15 A) and dif—

 

ferences in overall Rhizoctonia control by the chemical

 

soil treatments (Table 15 B).

Rhizoctonia R-54 reduced total emergence most, had

 

the highest pre- and post-emergence damping-off, the lowest
number of surviving seedlings and lowest seedling weight
per plot. This isolate also had the highest root rot rating
(Table 15 A). With the R-45 isolate, the situation was re»
versed indicating that the isolate was practically non=
pathogenic. R—Sl was intermediate in action with respect
to total emergence, pre— and post-emergence damping—off

and final stands. Seedling weight per plot and mean weight
per seedling were not significantly different from the con-
trol except for R—54 with a higher mean seedling weight.
The latter may be an artifact (Table 15 A).

Significant differences were also found between
chemical soil treatments. Dexon+Difolatan treatment allowed
the highest total emergence, lowest pre— and post—emergence
damping—off, largest number of survivors and greatest seedm
ling weight per plot (Table 15 B). The opposite occurred
with Difolatan treatment alone and the differences between
Dexon+Difolatan and Difolatan alone were significant in
every case. In several measurements (post—emergence damp-

ing—off, number of surviving seedlings, and seedling weight

per plot» Difolatan + Dexon was significantly different

 

 

 

101

 

 

 

 

 

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TUCMUHMHCmHm
Q 0.0 N00. 0 0.s¢ Qm 0.00H UQ m.vH Q 0.00 m 0.mmH mzum+coxma
Q 0.0 va. m m.mm m N.NNH o 0.0 Q 0.mm m m.0mH cmumHOMHQ+coxmo
m m.mH 0mg. Q 0.0m U H.Hm m 0.00 m 0.00 Q e.e0 stmHOMHo
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Q N.¢ Q 0H0. m 0.00 m s.mHH Q 0.mH m 0.0m m 0.0NH 001m
Q m.m Q mmm. Qm 0.Hv m 0.00H Q m.SH m H.sm m m.mmH Houpcou
memHomH Mo comHQMQEOU .4 puma
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xmecH \uz cam: \Qs Hmuoe mmonmcadsma Qmpoe
pom boom nHmHs mcflflemmm
.mCOHpMCHQEOU HMUHEmnoIQOHomH mo comHHmQ
IEOU .mHMUHEmQUITUHUH0CSm QQHB Umbmmup cam memHOmH MHCOQUONHQm mmMQQ QQHB

notcmem HHom MUSE Handpmc CH m0CHHUTmm Qme Umu mo mUHmH> paw Hm>H>MSm .mUCMQm .mH mHQme

102

from Dexon alone. None of the Dexon + PCNB yield and surw
vival data differed significantly from Dexon alone. Dexon+
PCNB treatment was not significantly different from Dexon+
Difolatan at the 1% level. At the 5% level of significance
there was greater post-emergence damping-off and fewer seed"
ling survivors. Dexon + PCNB controlled damping-off as

well as the Dexon + Difolatan mixture in soil infested with
R-51, but it failed to control the disease in R—54 infested
soil as discussed previously. Also, from Table 14 and 15 B,
Difolatan alone did not control damping-off by Pythium sp.

as well as did Dexon alone.

Sensitivity to PCNB In—Vitro—-A comparison of lin-

 

ear growth of the isolates under the influence of 100 ppm
PCNB in the medium may be made from Table 16. PCNB had
the least effect on R-41, R-43, R-47 and R—56 and was most
inhibitory to R-44, R-51, R—52 and R-55. The remaining
isolates were intermediate, ranging from 49.0 to 70.0% of
control growth. Isolate R-54, used in the soil treatment
experiments, was intermediate (49.0% of control growth)

in response to PCNB.

Sensitivity to Difolatan In—Vitro-—The general culu

tural characteristics of the Rhizoctonia isolates on Potato-

 

dextrose agar and some typical reactions of these isolates

to Difolatan (50 ppm) in the culture medium are shown in

Figs. 24 and 25. Several of the isolates, such as RH43,

 

103

 

 

oH®>®H *m

 

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HHCUHEmCU ozv
Cusouo HouQCou

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0Q meoawmm Cmmz

lead om cmpmaomHav “and OOH mzumv
Aesasmz cH HmUHemrov

abacus

MQQ um QCTQTMMHC >HQCCUHMHCmHm DOC mum QOQMmH TEMm >Q CmsoHHow mCmmz II him

m.me m.vm H.me m.mm cam:
0.0 m 0.0 H 0.0 H o.NH mmum
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0.¢m 8 0.0 H N.m0 norm e.mm Helm
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m.mv con m.0 HH m.Hm m 4.0m Helm
H.v¢ con m.mm comm N.ms mums m.mm emnm
m.mq sun n.0m n m.oa ohm m.mm Helm
m.mm on. H.ms mmm m.Hm umeun m.mm mmnm
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0.a0 m 0.0m anon v.0H Cm! v.mH smum

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.HOHQCOU mo QCmuumm
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Eda 00H 0Q pmmoaxm memHomH mHCOQUONHQm mSOHMm> mo szonm mo COHDHQHQCH .0H mHQmB

104

 

Figure 24. Cultural characteristics of Rhizoctonia
isolates R-41 to R—47 and R—49, R—53, R—54
and R—56, on potato dextrose agar medium

105

 

  

Difolatan Difolatan

 

 

Difolatan I Difolatan

Figure 25. Growth response of Rhizoctonia isolates
R-41, R—43, R-47 and R-56 to 50 ppm
Difolatan fungicide incorporated in
the culture medium

106

failed to grow in medium containing Difolatan. Growth of
R-51 and R—52 was limited to the mycelial inoculum disc,
but the isolates evidently produced an exoenzyme that difu
fused out into the agar and produced a clear zone (Fig. 26).
No mycelium was found in the clear zone.

Difolatan was considerably more toxic on the aver-

age to Rhizoctonia isolates than PCNB (Table 16). The growth

 

of R—43, R-47, R-Sl and R-55 was almost cOmpletely inhibited

by 50 ppm Difolatan. R—46, R-53, R—56 were the only iso-

 

1ates that reached as much as 50% of the control growth.
The growth of the remaining six isolates ranged from 23%

to 40% of control growth. Rhizoctonia R-54 growth was in-

 

termediate (38.9% of control) on Difolatan medium. There—
fore, this isolate was in approximately the midrange of
sensitivity to both PCNB and Difolatan in-vitro.
Differential sensitivity of several Rhizoctonia
isolates to PCNB and Difolatan appears definite. Isolates
R-4l, R-43 and R-47 may be classified as insensitive to
PCNB but sensitive to Difolatan and R-45 as moderately
sensitive to PCNB but very sensitive to Difolatan. All

of the Rhizoctonia isolates used were more sensitive to

 

Difolatan than to PCNB.

107

 

Figure 26. Growth response of Rhizoctonia
isolates R—51 and R-52 to 50 ppm
Difolatan fungicide incorporated
in the culture medium

DISCUSSION

Many of the studies of the effects of fungi-

cides on soil microflora have been on volatile—type fungi-
cides, some of which were slightly selective, but many were
broadly toxic to soil organisms (57). For the present study,

comparisons were made of the effects on soil microflora

 

of three limited spectrum fungicides (Dexon, PCNB, Bayer
29957) and a broad spectrum fungicide (Difolatan). The
in~vitro fungitoxic activity of Dexon, PCNB and Olin—Mathie—
son 1921 (an experimental fungicide) was examined prior

to soil treatments. The first two fungicides were most

effective against Pythium and Rhizoctonia, respectively,

 

but OM-1921, which was thought to be most active against

Fusarium, was broadly inhibitory to Rhizoctonia and Pythium

 

as well. For the soil treatments the latter compound was
replaced by Bayer 29957, which was reported by the manufac-

turer to be active against Fusarium.

 

Washing soil samples to determine whether each
fungicide affected the total fungus fraction (mycelium
and spores) or the mycelial and debris fraction in the
soil was partially successful. Washing reduced assay counts
of fungi by 58-79%. The differences in the effect of fungi-
cides on the total fungus fraction vs. the mycelial debris

fraction were not conclusive but there is some evidence

108

109

of a differential action. From non-washed samples, counts

of fungi from Rhizoctonia-amended soil were increased 56.2%

 

by PCNB soil treatment at 33 days and 16.8% at 44 days,
but counts of fungi from washed soil were reduced 38.4%
at 44 days. B—29957 also reduced fungus colony counts from
washed soil as opposed to an increase in colony counts in
the treated non-washed soil. In general, counts of fungi
were less variable from the washed soil samples (Figs. 1,
8, 10, 12, 14, 15). Results might have been improved by
removing a larger percentage of spores, either by a more
rigorous washing procedure, a period of magnetic stirring
before washing, or perhaps use of a soap solution (10) to
aid in release of spores from the soil particles and debris.
Introducing a pathogenic Rhizoctonia isolate into
a non-steamed soil to evaluate the effect of PCNB soil treat»
ment on damping—off fungi, as well as on the general soil
microflora, caused an increase in numbers of actinomycetes,
bacteria and fungi in mineral soils. In muck soils, only
the bacterial counts were increased by addition of Rhizoc~
tonia. The reason for these increases was not investigated
since they did not interfere with the assay of treated vs.
non—treated soils of the same type. The increases should
be taken into account, however, when comparing results of

assays from Natural vs. Rhizoctonia-amended soils. Some

 

of the increases in mineral soil may have been caused by

the small amount of nutrients added to the soil along with

 

110

the Rhizoctonia mycelium. This may have had a rather large

 

effect in mineral soil where the available nutrients are
likely to be at a low level. On the other hand, addition

of a small amount of nutrient to a highly organic muck soil
would be less likely to affect the nutrient situation appre—
ciably and therefore not cause any substantial increases.
Greatly increased numbers of soil microflora with addition
of organic amendments to mineral soils have been observed

by Papavizas and Davey (83).

In Rhizoctonia-amended soil, PCNB soil treatment

 

increased total counts of fungi 56.2% at 33 days and 16.8%
at 44 days as assayed by non-washed soil samples. In washed
soil samples, however, PCNB decreased counts of fungi 38.8%
at 44 days in the mycelial-debris fraction. A slight re~
duction was also noted at 11 days but the difference was

not statistically significant. Perhaps it would be neces-
sary to remove even more of the spore fraction of the soil
to determine the effectiveness of PCNB on Rhizoctonia myu
celium in soil. PCNB increased bacterial and actinomycete
counts in Rhizoctonia soil over the 44 day period. In=vitro,
PCNB seems to have behaved differently. There the total
colony counts of actinomycetes and bacteria were markedly
reduced by plating the dilutions on medium containing 50

ppm PCNB. This indicated that at least some of the soil

bacteria and actinomycetes are sensitive when exposed to

PCNB. Rhizoctonia was not recovered from PCNB-treated soils

11 days after treatment and data indicated partial control

 

111

of pre—emergence damping—off by Rhizoctonia. Rhizoctonia
was probably partially controlled by PCNB but damping-off
by Pythium obscured seedling count differences.

The Rhizoctonia isolate R-54, used as an amendment

 

to natural soil for the PCNB experiments, showed some sensi—
tivity to PCNB in agar-tests at concentrations as low as
1 ppm but appeared to be much less sensitive to PCNB in

soil. This had also been noted in previous greenhouse soil

 

studies, in which this isolate was used (14, 15). Differ—

ential sensitivity of four Rhizoctonia isolates to captan+PCNB

 

and nabam+PCNB was also reported by Sinclair (93). In a
later field test, as well as in agar plate tests (94) he
found that five Rhizoctonia isolates varied significantly
in sensitivity to various chemicals including PCNB, captan
and dichlone, alone and in combination. Thomas (97) re—
ported similar results from field and in—vitro tests with
six biotypes from a single stock culture of Rhizoctonia
solani and Maier (64) found differential responses to PCNB,
Dexon and thiram among 12 isolates of Rhizoctonia solani.
In view of these reports, this study included a test to
determine the pathogenicity and sensitivity to fungicides

of a number of Rhizoctonia isolates in both in—vitro and

 

greenhouse tests. The results showed a broad range of
response in pathogenicity (Table 12) and sensitivity to
PCNB and Difolatan (Tables 14, 15).
With respect to PCNB tolerance, all isolates used

in the present study were inhibited from 20—100% by 100 ppm

112

PCNB in agar plate tests (Table 18). In similar in~vitro
tests Shatla and Sinclair (91, 92) reported that some of
the 36 isolates from cotton tolerated PCNB concentrations
up to 10,000 ppm before growth was inhibited completely,
and the more sensitive ones tolerated 600 ppm. In green~
house tests with nine of the isolates, he showed a positive
correlation between in—vitro sensitivity and sensitivity

to PCNB in soil in all but two isolates. One isolate was

more sensitive to PCNB soil treatment than inuvitro and

 

another gave the opposite reaction. The latter observation

corresponds to the reaction of the R-54 isolate of the pres= II
ent study. The R-54 isolate was clearly more sensitive to

PCNB in—vitro than in greenhouse soil treatment tests. The

in-vitro response of R-45 was very similar to that of R~54

but in soil it was much more inhibited than R—54. R=51

was inhibited considerably by PCNB both in soil and inn

Vitro. The fact remains that Rhizoctonia isolates used

 

in the present study are basically more sensitive to PCNB
than those reported by Shatla and Sinclair. In soil, how~
ever, they appear to respond differently to the chemical.
Some recent work (3) has shown that PCNB may be transformed
into another compound or compounds which appear to retain
fungitoxic properties. If such a process does occur with
PCNB, this could account for the change in response of the
isolates in soil as compared to that inmvitro.

The variability in pathogenicity and sensitivity

to fungicides among Rhizoctonia isolates may help to explain

 

 

 

113

the variability of results of fungicide tests and will be
an important factor in selecting chemicals for control of
diseases caused by Rhizoctonia.

Dexon, at 20 lbs./acre, had no persistent effects
on numbers of fungi, bacteria or actinomycetes. Dexon had
no effect on counts of fungi in the soil, using a washed soil
for assay sample, but a non—washed sample at two weeks showed
a 21.1% increase in counts. Counts of bacteria were genera

ally greater in Dexon treated soil than in natural soil.

 

Although the initial recovery of Pythium one week after
Dexon treatment was less than 1%, subsequent weekly recov» .
eries were 18.6, 21.1 and 18.9% of control, indicating some
survival or recovery from the effects of Dexon in the soil.
Although the soil was protected from direct light, some
Dexon may have been inactivated by reduction—oxidation as
described by Hills and Leach (42), who showed that aqueous
Dexon was unstable in soil when exposed to air and light.
When it was mixed with dry soil at 25 and 50 ppm, it pro~
vided good control of damping—off of sugar beet seedlings.
It also seems possible that Dexon might be inactivated by
other soil microorganisms since it was found that both 3215

zoctonia and sugar beet seedlings are able to decompose

 

Dexon (100, 101).
Similarly, assays from washed soil samples showed

that B-29957 had no effect on fungus counts of the mycelial-

debris fraction. Assays of non—washed soil samples showed

that B-29957 increased counts of fungi 43.0% at three weeks

114

and reduced counts of fungi 16.3% at four weeks. Bacterial
colony counts followed a similar pattern, but actinomycete
counts were not affected by this soil treatment.

There was inconclusive evidence of Fusarium inhibi-
tion in soil by B-29957 using the potato cube recovery as-

say. Some reduction of Fusarium seemed to have been pro-

 

duced but the percentages were small (page £37). On the
other hand, recoveries from buckwheat stems gave a higher
percentage recovery in B-29957 treated soils than in non-
treated soil. There were no differences in recovery of
Rhizoctonia or Pythium from these soils and both assay
methods indicated that B-29957 had nematocidal activity
in soil.

Dexon + DAC-469, a combination of two selective
fungicides, had no persistent effect on fungus populations

in muck soil. Addition of Rhizoctonia did not increase

 

counts of fungi in muck soil as it did in mineral soil.

But assays from washed soil samples showed nearly a 50%
increase in fungus counts in soil in which beet seedlings
were grown. This may have been an increase in mycelial
quantity as a result of the stimulation of growth of spores
under the influence of exudates from plant roots. Bacterial
counts were increased by addition of Rhizoctonia to soil

and also by the presence of beet seedlings. Soil treatment

with Dexon + DAC-469 also increased bacterial counts in

both natural and Rhizoctonia-amended soils. Actinomycetes

were not clearly affected by addition of Rhizoctonia to

 

 

 

 

115

muck soil but there was some reduction of counts effected

by planting beets. Reduction of counts did not appear to
occur consistently. The amount of this reduction in planted
soils was significant only in fungicide-treated Natural

soil and non-treated Rhizoctonia soil at the time of har-

 

vest. Numbers of actinomycetes were reduced by fungicide
treatment only at the time of seedling harvest, and the
reason for this is not immediately apparent. The effects
of fungicide treatment might have been more explainable
if a parallel in—vitro test of these same soil dilutions
had been carried out using the same fungicides in the ap-
propriate culture medium. Information on the toxicity of
the fungicides to these organisms would have been useful
in separating the effects of fungicide treatment from the
other factors affecting these organisms in the soil.
Difolatan, a broad spectrum fungicide, gave a cone
trasting picture to the limited spectrum materials. Treat»
ment of mineral soil at 30 lbs./acre greatly reduced colony
counts of fungi, bacteria and actinomycetes for at least
four weeks after soil treatment. The average reduction
in counts caused by Difolatan was 59.5% as determined from
washed soil samples and 67.3% as determined from nonmwashed
soil samples. These results agree with those of Domsch
(22) who reported that normal application rates of captan
(closely related) and thiram reduced numbers of the major
microfloral components in soil. Other results have been

reported for captan used as a weekly soil drench of 10=1000

 

Illhu

 

116

1bs./acre (11). In this case captan caused a very large
increase in surface-soil bacterial numbers accompanied by

a reduction in numbers of actinomycetes and fungi. Folpet,
also closely related to captan and Difolatan, has a broader
fungitoxic spectrum than captan due to the configuration

of hydrogen atoms near the carbonyl groups (62). This may
be similarly true for Difolatan since in-vitro tests showed
Difolatan to be Considerably more toxic to fungi than cap~

tan (Manufacturer's Data sheet).

 

The toxicity and persistence of Difolatan effects
in soil warrant further work over a longer time and should
be investigated if possible. Time did not permit this for
the present. One of the aims of the present research was
to determine whether the fungicides chosen had harmful efu
fects on the soil microflora, and if so, to determine which
organisms or groups of organisms were affected. Further
research on the effect of this fungicide on soil micro-
organisms might be to determine which of the organisms
within each group: fungi, bacteria and actinomycetes;
are being inhibited by Difolatan in the soil. Perhaps a
combined colony count assay and soil respiration study for
4-8 weeks would delimit the extent and duration of effects
on soil microflora. This could also be accompanied by a
parallel study of effect of Difolatan on nitrification and
ammonification. For nitrification studies, an ammonium
compound could be added to Difolatan-treated soil and nonm

treated soil and the amount of nitrates present in the two

117

soils could be compared periodically for the effects on
nitrification. A study of effect on legume nodulation might
be accomplished by planting a legume crop in Difolatan treated
soil and non-treated soil for comparison of number of root
nodules or the health and vigor of the plants and roots.

Other soil processes, such as cellulose or lignin decomposi~
tion, fat and protein breakdown, mineral transformations

and other reactions could be selected for study as new in—

formation is accumulated. Recently, Domsch (24) carried

 

out an investigation on the effects of captan on decomposi-
tion of glucose, esculin and tannin in soil. From respira-
tory studies he concluded that cellulose and cutin break—
down are the most sensitive of the processes studied in
relation to captan soil treatment. These processes might
be even more severely affected by Difolatan, which has been
found to be much more toxic to fungi than captan. A log-
ical starting point for study of this compound might be

a comparative study of the effects of captan and Difolatan

on soil microflora, in—vitro and in soil.

lOo

LITERATURE CITED

Allen, 0. N. 1957. Experiments in soil bacteriology.
Burgess Publishing Co., Minneapolis. 117 pp.

Aldrich, D. G., & J. P. Martin. 1951. Effect of fumiu
gation on some chemical properties of soils. Soil
Science 73: 149-159.

Ashworth, L. J., Jr., J. D. Price, and B. C. Langley.
1965. A comparison of biological and chemical as-
says for pentachloronitrobenzene in a field soil.
Phytopathology 55: 1051 abst.

 

Bird, L. S., C. D. Ranney and G. M. Watkins. 1957. I
Evaluation of fungicides mixed with the covering— .
soil at planting as a control measure for the cot»
ton seedling—disease complex. Plant Disease Re-
porter 41 (3): 165-173.

Brown, A. L. 1954. Effect of several insecticides
on ammonification and nitrification in two neutral
alluvial soils. Proc. American Soc. Soil Sci. 18:
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