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DECAY

MICROORGANISMS

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THE UNIVERSITY OP ALBERTA

ICE CRO ORGANISMS IN SEED DECAY

A DISSERTATION

SUBMITTED TO THE SCHOOL OP GRADUATE STUDIES
IN PARTIAL FULFILMENT OP THE REQUIREMENTS
FOR THE DEGREE OP
MASTER OF SCIENCE

FACULTY OF AGRICULTURE

J. S. HORRICKS

EDMONTON, ALBERTA
OCTOBER 1952

, • •

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ABSTRACT

This study was undertaken to compare the tendency
of wheat, flax and pea seed to decay in the soil, to deter¬
mine the ability of the seed-decay organisms encountered to
act under different conditions, and to ascertain the relative
persistence of different chemical seed protectants on these
seeds in the soil*

Under all soil environmental conditions tested,
pea seed showed the greatest tendency to decay, wheat the
least, while flax was intermediate* The majority of the
seed decay organisms were active over a fairly wide range of
soil temperature and moisture conditions. Many of the fungal
isolates were capable of causing seed decay of both wheat and
flax in the soil, but not of peas.

Orthocide 1|06 was the only fungicide which per¬
sisted on wheat, flax or pea seed for more than two days in
the soil. Except for this fungicide, the mercurial pre¬
parations persisted longer than the non-mercurial prepara¬
tions on these seed types in the soil. The majority of the
six fungicides tested were more persistent on wheat and pea
seed than on flax seed in the soil.

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TABLE OP CONTENTS

Page

INTRODUCTION . . . . . . . . . . 1

OBJECT OP THE INVESTIGATION . . 2

EFFECT OF ENVIRONMENTAL CONDITIONS ON EMERGENCE . . 3

Introduction . . . 3

Methods . . ............... 3

Effect of different levels of soil moisture
on emergence . . Ij.

Di scussion . . •«»•.••••• 6

Summary ................................ 7

Effect of different soil temperature levels
on emergence . . 8

Discussion . . 9

Summary ................................ 11

Effect of date of seeding on emergence ...... 12

Discussion . . 13

Summary , • 1$

Summary of the effect of environmental con¬
ditions on emergence . . l6

ISOLATIONS FROM ROTTED SEEDS . . . . . . 17

Introduction . . 17

Methods ..................................... 18

(a) Obtaining partially rotted seeds. 18

(b) Isolation of microorganisms from

the partially rotted seeds . 1-9

TABLE OP CONTENTS (Continued)

Page

td) Seed inoculations ••..»••••••••• 20

Results . 21

Summary . . . . 2 4

SEED ROTTING CAPACITY OP PUNC-AL ISOLATES UNDER VARIOUS
CONDITIONS . . 25

Introduction . 25

Methods . . 25

Effect of soil temperature • • . . ...•••• 25

Discussion . . 29

Summary . . . 30

Effect of soil moisture 30

Discussion . . 33

Summary . . 33

Reaction of different types of seed to the
same fungi . . 34-

Discussion . . 37

Summary . . 38

Effect of miscellaneous factors on seed
decay . 39

Discussion 44

Summary • . . 45

Summary of the seed-rotting capacity of the
different fungal isolates under various
conditions ...... . . 4°

TABLE OP CONTENTS (Continued)

Page

PERSISTENCE OP CHEMICAL SEED PROTECTANTS ON SEED
IN THE SOIL . . . . . . . 4-8

Introduction . lj.8

Methods . I]_9

Relative persistence of Ceresan M on viable

and non-viable wheat, flax and pea seed in

the soil under greenhouse conditions . . . $0

Discussion . . $2.

Summary . 54-

Relative persistence of various seed protectants
on viable wheat, flax and pea seed in the soil
under greenhouse conditions . 54

Discussion . 60

Summary . . 6l

Summary of the persistence of chemical seed
protectants on seed in the soil . . . 62

GENERAL DISCUSSION . . 63

SUMMARY . . . . . . . . . . . 66

ACKNOWLEDGMENTS . . 68

LITERATURE CITED . . . &9

APPENDIX I . 73

MICROORGANISMS IN SEED DECAY

INTRODUCTION

Each year large quantities of seeds are sown, of
which many fail to produce plants. Microorganisms causing
seed decay are largely responsible for seedling mortality.
Severity of such damage is determined by several factors,
among which, quantity of inoculum, host susceptibility, soil
temperature and moisture, are among the most important. In
general, the soil inhabiting organisms which cause seedling
blights, usually become aggressive when the environment is
unfavorable for the growth of the seedling.

In the prevention of seed decay, one must endeavor
to render the environment unfavorable for the causal micro¬
organisms, and favorable for the host. Chemicals may be
used to advantage, but only those which combine germicidal
and non- phytotoxic properties with low cost, are likely to

be used

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OBJECTS OP THE INVESTIGATION

This investigation is centered around three distinctly
different types of seed, wheat, flax and peas; the object
being to determine:

(a) their relative susceptibility to decay by the
common soil microflora under different soil
environmental conditions;

(b) the types of microorganisms responsible for
their decay;

(c) the effect of different factors, including
soil moisture and temperature, upon the acti¬
vity of microorganisms responsible for seed
decay;

(d) the ability of the microorganisms that rot one
type of seed to rot the other two types of
seed;

(e) the relative persistence of commercial seed
protectants on seeds in the soil®

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EFFECT OF ENVIRONMENTAL
CONDITIONS ON EMERGENCE

INTRODUCTION

It Is known that seedlings as well as microorganisms
respond differently to different soil environmental conditions*
The object of the following experiments was therefore to
determine the influence of two soil environmental factors,
the soil moisture and the soil temperature at which wheat,
flax and pea seed are most, and least, susceptible to decay
by the common soil microflora. Dickson (9) found that wheat
and corn seedlings were only predisposed to seedling blight
when they were grown at temperatures unfavorable to their
growth. Wilson (i|4* k-5) studied the rate and amount of
germination and its relation to mold attack on wheat, on
blocks of plaster of Paris. The effect of soil moisture and
temperature on pea ©mergence has been studied by Ledingham
(23), Baylis (If), Hull (l£) and Wallace (IfO); Baylis (3)
gave an account of the fungi concerned.

METHODS

The tendency of wheat, flax and pea seed to decay
was compared by exposing these seeds in non- sterile soil to
various moisture and temperature conditions during their

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germination and emergence* The amount of decay was calculated
by comparing the emergence of untreated seed with that of
seed treated with Geresan M* Ceresan M is one of the best
known organic mercurial seed protectants* Ceresan M was
applied in dust form at the rate of jg, 1 and 1 oz* per bushel
for wheat, flax and peas, respectively* The seeds used were
I9I4.9 Red Bobs wheat, I9I4.8 Redwing flax and Homesteader peas*
These seeds germinated 95 percent and over in a blotter test.

Non-sterile, 3 si Edmonton soil- sand mixture was
used in all the experiments* Edmonton soil is described as
a heavy clay; the soil used in these experiments was high in
organic matter* The seeds were sown at a depth of 1 inch.

All the pot experiments were carried out in the greenhouse
in which the air temperature was maintained at l5°C* Signifi¬
cant differences in emergence were calculated by analysis of
variance*

EFFECT OF DIFFERENT LEVELS OF SOIL MOISTURE

ON EMERGENCE

The moisture experiments were carried out in 5i
inch pots, using Ij. replicates and 25 seeds per pot. The
moisture content of the soil was determined every 2 or 3
days and maintained at the designated level* The results
are presented in Table I*

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TABLE I

EFFECT OF DIFFERENT LEVELS OF SOIL MOISTURE ON
THE EMERGENCE OF WHEAT, FLAX AND PEAS IN SOIL

Treatment

Number

of Seedlings Emerged

Low Soil
Moisture

Optimum Soil
Moisture

High Soil
Moisture

Wheat

22*5

21.5

Wheat Ceresan M i oz.

23.0

23.5

22.3

Flax

8.0

8.8

* XX

9.5

7 ^xx

Flax Ceresan M 1 oz.

12.0

15.5

17.5

Peas

1.8

1.5

o.ox

Peas Ceresan M 1 oz.

5.3

l+.o

3.8

x significant between treated and untreated
xx very significant between treated and untreated

When a difference is referred to as being
significant, it means that a difference as great
would occur 5 less times out of 100 by chance
alone, and If highly significant, a difference as
great would only occur 1 or less times out of 100
by chance alone.

Under low, optimum and high soil moisture conditions,
peas showed the greatest tendency to decay, flax the next
greatest tendency, and wheat the least tendency to decay*

Wheat and flax were more subject to decay at low soil moisture,
while peas were more subject to decay at high soil moisture*
Ceresan M significantly Increased the emergence over non-
treated seed to the 1 percent level In wheat under low soil
moisture conditions, and flax under optimum and high soil
moisture conditions. The emergence of peas was only signifi-
cantly increased to the 5 percent level under high soil

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moisture conditions.

Discussion

These results are very similar to those obtained
by other workers. Wilson (44) found that wheat showed a
wide adaptation to varying moisture conditions. A soil
moisture content of 5>0 percent of saturation resulted in high
germination with all the spring and winter wheat varieties
he studied.

That flax when treated with Ceresan M should give
the highest emergence in high soil moisture and the lowest
in low soil moisture, suggests that soil moisture may play a
part in fixing the mercurial dust to the seed. Muskett (36)
found that the effectiveness of mercurial dusts is related to
their ability to adhere to the seed, by obtaining better
results when the dusts are applied and afterwards fixed to the
flax seed by the use of water or separated milk.

The relatively low emergence in the peas was
probably due to watering them immediately after sowing.

Baylis (If.) found that watering peas immediately after sowing
markedly depressed emergence, and a steady improvement was
shown as the date of watering was postponed. A safe practice
is to sow the seed in slightly moistened soil and not to water
it until at least three days after sowing. There appears to
be a critical period during the course of germination when
high soil moisture is very deleterious. The duration of this

. .

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period depends upon the rate of germination, which is related
to soil temperature* Baylis (3) stated that the most important
fungi concerned in preemergence blight of peas are Pythium
and Fu sari uni species. Hull ( 1 5) concluded that the severity
of preemergence blight of peas is greatly accentuated by high
soil moisture and that a certain measure of control could be
obtained by dusting the seed with a commercial mercurial
preparation. He found that the mercurial compound resulted
in the greatest increase in stand when the soil was wet and
when wrinkled- seeded peas were used. On the other hand,
there is a risk of phytocidal damage when the treatments are
applied under dry soil conditions. It was also found in the
present studies that only under high soil moisture conditions
was a significant difference between the emergence of treated
and untreated pea seed obtained.

Under all the soil moisture conditions tested, peas
showed the greatest tendency to seed decay, followed by flax
and then wheat. Flax and wheat were most subject to decay
under low soil moisture, while peas were most subject to de¬
cay under conditions of high soil moisture.

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EFFECT OF DIFFERENT SOIL TEMPERATURE LEVELS

ON EMERGENCE

The temperature tests were carried out in temperature
control tanks in the greenhouse, in which the soil temperature
was maintained at 1$° , 20°, 25° and 30°C and the air tempera¬
ture at l5°C. Each seed type was tested separately, using ip
replicates and 25 seeds per crock. That is, there were if.
crocks with untreated seed and 4 crocks with seed treated with
Ceresan M to act as checks, at each temperature level. The
soil moisture was maintained at an optimum level for plant
growth. The results are presented in Table II.

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TABLE II

EFFECT OF DIFFERENT SOIL TEMPERATURE
LEVELS ON THE EMERGENCE OF WHEAT,
FLAX AND PEAS

Treatment

Number

of Seedlings

Emerged

Wheat

Flax

Peas

i5°c

21.5

!3-3xx

9-°xx

i5°c

Ceresan

22.8

21.3

16.8

20°C

22.3

19.3

20°C

Ceresan

24.0

19.8

25°C

20.5

19.5

13»3xx

25°C

Ceresan

23.8

21.5

20.0

30°C

22.0

18-3XX

.Ojyj

30°c

Ceresan

23.3

22.8

22.5

x significant between treated and untreated
xx very significant between treated and untreated

The emergence of wheat was not significantly
reduced over that of the controls at any of the temperatures
from 15° to 30° C* Flax emergence was very significantly
reduced at l£° and 30° C* The lowest emergence in the pea
test occurred at 15>°C and the highest at 30°C, while the
emergence at 20° and 25>°C was intermediate and nearly equal.
The emergence of peas was very significantly reduced over
their controls at all the temperatures used.

Discussion

Wilson ([jl}.) carried out germination tests on blocks

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of plaster of Paris, and found that l5°C was the optimum
germination temperature of wheat. Higher temperatures of
20° and 30°C gave more rapid but not so complete germination.
He found that mold attack on germinating wheat seed was of
little importance at l£°C, but at 30°C the endosperm of the
kernel is so affected that there is outward leaching of
soluble food materials which are readily utilized by ever¬
present fungi. Fungal growth was favored at 30 °C. He noticed
an inverse relationship between the total percentage of
germination and the percentage of moldy kernels.

Leach (21) states that the relative growth rates
of the host and pathogen determine to a considerable degree
the severity of preemergence infection at different tempera¬
tures. This growth rate effect would largely explain the
fairly uniform emergence throughout the different temperature
levels in the wheat experiment.

If the emergence at each temperature is totalled
in the flax experiment, there is a slight increase in emer¬
gence with increasing temperature, with the lowest emergence
at 15°C and the highest at 30°C. Thus, under these conditions
15>°C appears to be the most unfavorable temperature at which
to sow flax. There was considerable post- emergence blighting
at 30°C so the most favorable temperature appears to be
between 20° and 25°C.

The most unfavorable soil temperature for pea

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©mergence also appears to be 15>°C, although the pea- decaying
organisms seemed extremely active at all the temperatures
used. They were likely more active at the higher temperatures,
since for example, the plants at 30°C took five days to
emerge while those at 15°C took nine days to emerge. If the
time to emerge had been the same at all the temperatures,
then it would be reasonable to assume that the greatest de¬
crease in emergence would have occurred at the two higher
temperatures* The effectiveness of chemical treatment seemed
to increase with increasing temperature. Leach (21) found
that seed decay and preemergence infection of garden peas
were most severe between 12° and 2£°C.

Wallace (lj.Q), Baylis (ij.) and Hull (15) concluded
that soil temperature was less important than soil moisture
in its effect upon pea seed decay and preemergence blight.

In general, preemergence blight is most severe at tempera¬
tures that are relatively less favorable to the host than to
the pathogen.

Under all soil temperatures used, peas showed the
greatest tendency to decay, followed by flax and then wheat.
The emergence of wheat was not significantly reduced at any
of the temperatures from 1$° to 30°C. Its optimum temperature
would likely be about 20°C. The lowest emergence in flax and
peas was at l5°C and the highest emergence was at 30°C.

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EFFECT OF DATE OF SEEDING ON EMERGENCE

This experiment was carried out in the field using
single row plots consisting of 12 foot rows, 1 foot apart;

100 seeds were sown in each row at a depth of lj inches.

Each treatment was replicated four times in randomized blocks.
Soil moisture analysis was carried out every other day and
the soil temperature was taken every noon at seed level from
the time of sowing until the emergence was recorded. Daily
overnight low temperatures were also recorded. Soil moisture
and temperature levels are listed in Table III.

TABLE III

SOIL MOISTURE AND TEMPERATURE LEVELS

Early Late
Seeding Seeding

Average Soil Moisture 17* 2$ 20.3$

Average Noon Soil Temperature 20.6°C 2Q.5°C

’“'Average Overnight Low Soil Temperature 7«$°C 10.2°C

The average overnight low soil temperature was
taken at a depth of Ij. inches, about £0 yards from
the test plots.

The soil was rather dry for the first four days
after seeding the first test, whereas the soil moisture was
optimum throughout the second test. In general, the seed-
lings in the early test were exposed to a slightly lower soil
temperature and to drier conditions than those in the late

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seeding test. The results are presented in Table IV.

TABLE IV

EFFECT OF DATE OF SEEDING ON THE
EMERGENCE OF WHEAT, FLAX AND PEAS IN
THE FIELD

Tr eatment Number of Seedlings Emerged

Early Seeding Late Seeding

Wheat

85.ox

75.5

Wheat Ceresan M | oz.

91.3

86.5

Flax

66*8xx

67.5:

Flax Ceresan M 1 oz.

80.0

76.5

Peas

27.8^

61.5.

Peas Ceresan M 1 os.

65.0

70.0

x significant between treated and untreated
xx very significant betv/een treated and untreated

Early seeding improved the emergence of wheat,
little affected the emergence of flax, but was detrimental
to the emergence of peas. In all cases Ceresan M showed
the greatest Improvement in emergence in the early seeding.
As in the previous experiments, wheat showed the least
tendency to seed decay, followed by flax and then peas.

Pi scussion

The results of this date of seeding experiment
illustrate well the value of seed treatment especially when

the seeds are sown under conditions unfavorable for their

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growth. Evidence of this is shown in the early seeding of
peas and the late seeding of wheat. These results substanti¬
ate those of the previous moisture and temperature green¬
house experiments in that wheat proved the most tolerant of
the various environmental conditions and peas the least
tolerant.

The particularly low emergence from the untreated
pea seed in the early seeding test could possibly be accounted
for by the greater activity of Pythium species at the lower
soil temperature. Ledingham (23) at Saskatoon obtained an
abrupt decrease in emergence between the first and second pea
sowing dates* while there was very little change in soil
temperature# This worker suggested that P. ultimum and other
organisms concerned in preemergence blighting of peas required
a period in the spring to initiate active vegetative growth
and that the early sown peas may have passed their suscep¬
tible stage before the fungi had increased sufficiently to
cause much seed decay. McLaughlin (32) found that the per¬
centage of Pythium isolates from the soil were generally
high in the winter* spring and fall seasons and low in the
summer. He showed that a combination of high soil tempera¬
ture with low soil moisture generally resulted in a reduction
in the percentage of Pythium isolates, if the first seeding
had been two weeks earlier the results presented here may have
been quite different.

The cultural study of peas is not a simple problem.

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but the findings are mainly in agreement that seed protectants
are beneficial, their value depending on seed and environ¬
mental conditions* In England and the United States of America,
the cardinal factor in preemergence blighting has been shown
to be excessive soil moisture and, perhaps more particularly,
rain soon after planting in its tendency to compact the soil
around the seed, providing moist, poorly aerated conditions*

Soil temperature has been given a minor role by the English
workers but in general they consider higher temperatures as
being conducive to maximum emergence in garden peas* In the
United States, McNew (33) found progressively less seed
piece decay with each increase in temperature from 15° through
20° and 25° to 32°C. Ledingham (23), as well as Hutton (1?)
of Australia, found a tendency toward poorer emergence as the
season advanced from cool spring to hot summer* Ledingham
was able to correlate emergence with temperature but not
with moisture*

Summary

Early seeding increased the emergence of wheat,
little effected the emergence of flax and reduced the emer¬
gence of peas* Regardless of the time of seeding, wheat
showed the least tendency to decay, followed by flax and then

peas*

- x6 -

SUMMARY OF THE EFFECT OF ENVIRONMENTAL CONDITIONS

ON MERGENCE

Under all environmental conditions tested, peas
showed the greatest tendency to decay and wheat the least,
while flax was intermediate o

Wheat showed remarkable tolerance to its environ¬
ment and especially to soil temperatures during its germina¬
tion and emergence period. High emergence was discouraged
by suboptimum soil moisture and was encouraged by optimum
soil moisture, a temperature of about 20°C and early spring
field sowing,

Loxv soil temperature and moisture favored pre emer¬
gence blight of flax, while higher soil temperatures and
moisture levels increased emergence. Field flax may be sown
early in the spring if a seed protectant is used.

Peas, the most sensitive of these three grains to
environmental conditions, readily succumb to high soil moisture
(especially for the first three days after sowing) and to low
soil temperatures. Good pea emergence was favored by low
soil moisture and high soil temperature. Since the soil
moisture-temperature balance appears to be a very delicate
one, seed protectants should always be used on garden peas.

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- 17 -

ISOLATIONS PROM ROTTED SEEDS

INTRODUCTION

In the previous section the influence of soil
environmental conditions on the decay of wheat, flax and
pea seed by microorganisms of the common soil microflora
was considered* Isolation of some of these microorganisms
from partially rotted seeds was attempted* This is
discussed in the present section*

Many workers have isolated organisms carried by
the seed. Out of 6,201]. surface-sterilized samples of cereal
seed, Machacek (28) recovered lp3 genera and 102 species of
fungi, the major portion appearing to be innocuous inhabitants
of the seed. Alternaria was the most common genus and
Helmintho sporium the most common pathogen isolated. Greaney
(13) associated wheat seed infected with J|. sativum with low
germination. Fungal hyphae may always be found between the
epidermis and the inner pericarp of normal wheat seed (18, 37) •
Large numbers of certain species of epiphytic bacteria are
commonly found on Y/heat (20, 2ip, IjJL).

Padwick (38) made isolations from surf ace- sterilized
cotyledons of several varieties of peas grown in sterilized
and unsterilized soil. He obtained an abundance of common
molds and several pathogenic fungi, namely: a Fusarium

- 18 -

of the section Roseum, Fusarium culmorum and Botrytis cinerea,

He concluded that the rotting of pea seeds in soil may be
due to various fungi, and in nature it presumably would fre¬
quently be due to the combined effects of several, although
the greater pathogenicity of the above mentioned pathogenic
fungi in conjunction with their frequency of isolation and well-
known wide distribution in soil would suggest their importance*
Bavlis (3) concluded, that species of pythium were more im¬
portant than those of Fusarium in causing pea seed decay*

METHODS

( a ) Obtaining Partially Rotted Seeds

Non-treated seeds^’of Red Bobs wheat. Redwing flax
and Homesteader peas were sown in Edmonton black soil at a
depth of 1 inch* The soil temperature was maintained in
controlled temperature tanks at 1$° , 20°, 2$° and 30°C for
wheat and at 20°C for flax and peas* The moisture content of
the soil was kept at optimum or slightly above for plant
growth* Seeds showing signs of decay or delayed germination
were removed from the soil after the following intervals of
times ij. days from the 30°0 tank, 7 days from the 2$°C tank,
llf days from all the tanks and 28 days from the 1$°C tank*

* The wheat and flax seed was harvested in 194-9 an^

194® » respectively.

i '• ‘j, • ;.c- ;hr : vv o

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- 19 -

(b ) Isolation of Microorganisms From the Partially Rotted Seeds

Upon removal from the soil the seeds were surface-
sterilized by submerging them for 1 minute in 70 percent
ethyl alcohol and for 2 minutes in 1:1000 mercuric chloride,
after which they were washed three times for a period of 5
minutes in sterile distilled water*

By means of sterile forceps the surface-sterilized
seeds were placed in Petri plates containing potato dextrose
agar at the rate of four seeds per plate. The plating pro¬
cedure was varied in the following ways:, by placing the seeds
on hardened acidified medium, by completely submerging the
seeds in acidified medium before it hardened, by placing the
seeds on a hardened medium containing crystal violet, by
completely submerging the seeds in a medium containing crystal
violet before it hardened, and by placing the seeds on just-
hardened medium. The acidification of the medium to pH Ij. with
2^ percent lactic acid inhibits the growth of bacteria, while
a medium containing 1:125,000 crystal violet commonly in¬
hibits fungal growth and has a bacteriostatic effect upon
gram-positive bacteria.

The plated seeds were incubated at the same tempera¬
ture as they had been kept at In the soil. Organisms growing
from the seeds were transferred to potato dextrose agar slants
and a record was kept of them*

. . . . . ' _ . . . , . . .

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( c) Testing the Isolates

A preliminary method using one or two replicates,
was employed to test the seed-rotting capacity of these
isolates* Surface-sterilized seed was inoculated with them,
then sown in a sterile 3:1 Edmonton soil-sand mixture in 5>i
inch pots, at a depth of if- to lj inches, and at the rate of
25 seeds per pot* The pots for a given organism were held
at the temperature at which that organism had been isolated.
Organisms showing seed-rotting ability were retested using
four replicates and a randomized block design*

(d) Seed Inoculations

The following methods of seed inoculation were
employed in all the seed inoculation tests undertaken in
the present investigation. The seed was always surface-
sterilized, as previously described, before being inoculated.

For bacterial seed inoculation a 10 ml. water
suspension of a 2l|_ hour culture was poured on the seed.

The seed was left to soak at room temperature for 2 hours,
partially vacuumed for 1> minutes, then left to soak again
for at least 2 more hours before sowing* The liquid inoculum
was decanted off the seed on the sterile soil at seed level
before the seeds were sown. This method is similar to that
employed by Wallin (Ij.2) when working with Xanthomonas translucens
var. cerealis, only he applied the vacuum pump immediately
after the inoculum was added to the seed.

, ' •
r . - * : . ‘ :

■ i ■ ‘ - ’.a • .< ■

» . . .

►

, • ■ .

The fungal isolates were grown in $0 gm. of a
sterile 10 percent cornmeal- soil medium in 2^0 ml# Erlenmeyer
flasks at room temperature for a period of two to three weeks.
The inoculum was then cut up on the flasks by means of a
sterile spatula, poured out on sterile soil in pots and mixed
with the top 3/l| inches of this soil. The seeds were sown
on this mixture and then covered by more sterile soil. The
contents of one Erlenmeyer flask v/ere used to inoculate the
seeds sown in one pot. The medium in the flasks and the soil
in the pots were steam- sterilized at about l£ pounds pressure
for three and eight hours, respectively. A 10 percent corn-
meal medium had previously been used by Henry (IIl).

RESULTS*

A summary of the number of seed-decay organisms
isolated from non- surf ace-sterilized wheat, flax and pea
seed after varying periods of time in the soil at different
temperatures is presented in Table V.

* The seed decay organisms, numerically designated,
are listed in Appendix I.

■

. .

22 -

TABLE V

NUMBER OP SEED-DECAY MICROORGANISMS ISOLATED PROM
NON- SURFACE- STERILIZED WHEAT, FLAX AND PEA
SEED ’"AFTER VARYING PERIODS OP TIME IN
THE SOIL AT DIFFERENT TEMPERATURES

Type

Type of

Temperature Number

Number of

Organism

of Soil in

of Days

Organ! sms

Seed

Isolated

°C in Which

Seeds

Isolated

Able to

Used

Seeds Were

Were in

Significantly

Rotted

the Soil

Decrease

Emergence

Wheat Bacteria

15°

11+

15°

20°

C\J

25°

30°

Wheat Fungi

15°

150

20°

11+

25°

2^°

11+

30°

Flax Bacteria

20°

11+

Flax Fungi

20°

ll+

1+2

Pea Bacteria

20°

11+

21+

Pea Fungi

20°

11+

w The seed was

not surface-

sterilized

before

it was sown.

... . . ; . .. ; - , : . ' ■

. <

■

; n/j

'v . 8- :

mi .

i ...

23 -

According to these results, more fungi than bacteria
were isolated from the rotted seeds and more of the former
than the latter were able to reduce emergence significantly.

Not only were more organisms isolated at the lower soil
temperatures, but more of the organisms isolated at these
temperatures appeared to possess seed-rotting ability; this
could, of course, be due to the slower growth rate of the
host plants at the lower soil temperatures.

There doesn’t appear to be any correlation between
the number of days wheat seed was in the soil and the number
of bacteria isolated from the seeds. Seed kept at a soil
temperature of 20°C for a period of fourteen days produced
the greatest number of bacterial isolates and yielded the
only bacterial isolate that was able to reduce the emergence
of wheat significantly.

In general, at all the soil temperatures used, the
number of fungal saprophytes isolafc ed from wheat seed
increased directly with the period of time the seed was in
the soil. Fungi capable of causing severe preemergence
blighting of wheat were isolated from rotting seed at all the
soil temperatures tested, although the number Isolated from
soil at 30°G was rather low.

The number of fungi and bacteria isolated from
rotting pea seed was nearly equal. There were over three
times as many fungi as bacteria isolated from rotting flax

„

- 2k -

seed. Although peas proved the most susceptible to seed
decay, a very low portion of the fungi isolated from them
were capable of rotting their seeds. None of the bacteria
isolated from flax or pea seed rotted these seeds under the
conditions tested.

The above discussion is based solely on the results
obtained and should be evaluated as such. It is realised that
any variation in materials or methods, such as in chemicals
used for surface sterilizing the seeds, or media employed,
would possibly have a marked effect on the results obtained.

SUMMARY

The fungi surpass the bacteria in the number
isolated from the rotted seeds and in the number able to
reduce significantly the emergence of wheat, flax and peas.

The majority of the seed-decay organisms were isolated at
the lower soil temperatures. The number of saprophytic fungi
isolated from wheat seed increased directly with the period
of time the seed was in the soil. Wheat seed-decay fungi
were isolated at all the soil temperatures tested. A very
lov/ percentage of the fungi isolated from decaying pea seed
were able to reduce the emergence of peas significantly*

■

- 25 -

SEED ROTTING CAPACITY OP FUNGAL
ISOLATES UNDER VARIOUS CONDITIONS

INTRODUCTION

In the previous section fungi capable of signifi¬
cantly reducing the emergence of their host when applied to
seed were isolated from rotting wheat, flax and pea seed®

The purpose of this section Is to discuss the ability of
these fungi to rot seeds of the crop plants in question under
different conditions®

METHODS

The experimental methods used in this series of
tests were the same as those described in the previous section®

EFFECT OF SOIL TEMPERATURE

The fungi that were isolated from decaying wheat
seed at the various soil temperatures and found pathogenic,
were tested for seed-rotting ability at different soil tempera¬
tures® Temperature may alter the susceptibility of the host
or the pathogenicity of the microorganism® Dickson (9)
found that the seedling blight organism. Gibber ell a saubinetii,
would only produce severe infection of corn at low tempera¬
tures and of wheat at high temperatures* In this case

. t

. .

, i /.:• ' : A

■

- 26 -

temperature influenced the susceptibility of the host by
altering it chemically* Low temperatures are known to in¬
crease the disease-producing capacity of several fungi in¬
cluding Rhizoctonia solani, Fythium ultimum and Tilletia
caries, while higher temperatures increase the pathogenicity
of Helmintho sporium sativum and Clado sporium fulvum* Leach
(21) found preemergence infection most severe at temperatures
that were relatively less favorable to the host than to the
pathogen as measured by the ratio of their growth rates*

A comparison was made of the ability of the fungi
that were isolated in the present studies from wheat seed
held at soil temperatures of l5°> 20° and 30°G, to reduce
the emergence of wheat at these soil temperatures*

After the emergence data was analyzed by the analy¬
sis of variance, the mean of each treatment was converted to
a percentage preemergence blighting on the basis of its
check* The following formula was used:

x ™ J X 100 = percent blighting
x

where x 22 the percent of emergence in the check

y =s the percent of emergence in the treatment*

This method was introduced by Abbott (1) and it seems to
offer a reliable means for comparing results when several
series of experiments have been carried on, each based on a
different check* A summary of the results is presented in
Table VI.

•. ' . .. . ■ ■ • •

. .

* . ... .

. . . : I A:'. \ J

„ .

■: t ’ : ;

- .. ' . .

27 -

TABLE VI

ABILITY OF FUNGI ISOLATED FROM ROTTING WHEAT SEED AT
SOIL TEMPERATURES OF 1$° , 20 and 30°C, TO PRODUCE
PREEMERGENCE BLIGHT OF WHEAT AT THESE SOIL TEMPERATURES

Soil Temperature
at Which the
Organisms Were

Isolated Organism

Percentage of Preemergence
Blighting in Relation to
Uninoculated Checks

Soil Temperature

1 5°c

20 °c*

30°c

15°G

135

34.kxx

36 .5>XX

55 .6xx

13&

4O . lj.xx

36 .5xx

o2. 2xx

137

l?*^

32.6 XX

67 .8xx

138

17*ipc

2l|.2x

53-9xx

139

19. 7x

33 »7xx

37»2xx

310

30. 9x

0.0

332

86 .2xx

0.0

11.1

3 45

75.7xx

11.2

30 .6xx

20°C

ilk

39*5xx

Ij2«3xx

22.5

127

30 .2xx

[|.2.3xx

22.5

143

35*8xx

3I1. 2xx

16.7

145

40 .5xx

1$ .8x

41.7

llj.6

38 .Ixx

2q..7xx

16.7

14.9

30 .2xx

19*5xx

10.0

264.

31*2xx

28 *9xx

47 .3

268

38 .Ixx

30 .Oxx

14.2

281

28 mQxx

l6 .8x

26.7

286

I|2.8xx

23 • 7 xx

14.2

30°C

159

12.3

7.1

24 »5

172

13.8

4.4

24.5

240

12.3

0.0

6o.9xx

247

17*2

11.5

29. lx

x significant
xx very significant

* The 20°C soil temperature level in the experiments
where the organisms isolated at 15° and 30° were
employed was not constant.

- 28 -

The majority of the fungi that were isolated at a
soil temperature of l5°C were quite active at all soil
temperatures tested. In general, the ability of the isolates
135 to 139 9 inclusive, to produce preemergence blight of
wheat increased with increasing soil temperature, while the
310 and 332 isolates appeared relatively ineffective at the
two higher temperatures* The 34-5 isolate significantly re¬
duced the emergence only at 1$° and 30°C, being over twice
as effective at the lower temperature* The following iso¬
lates, in decreasing order, caused the most preemergence
killing: 332, at a soil temperature of 15°C, 137# at a soil
temperature of 30°C, and 135 and 13&, at a soil temperature
of 20 °C* Six out of these eight isolates showed a wide
adaptability to the various soil temperatures*

All the isolates that were obtained at a soil
temperature of 20°C signif icantlv reduced the emergence of
wheat at a soil temperature of l5°0 but not at a soil tempera
ture of 30°C* Only isolates lllj. and 127 of these ten isolate
were more effective in causing preemergence blighting at 20°C
than at a soil temperature of 1>°C * There might have been
significant reductions in emergence at a soil temperature of
30°C if there had been greater uniformity within the treat¬
ments and if the emergence of the checks had not been so low*
On a percentage basis of the means, isolates ll\$ and 261j.
caused more preemergence blighting at a soil temperature of
30°C than at the other soil temperatures*

—

• r-' <

-..is, . ' r - -■ ■ • . . • .

t ; <

;■

. •

- 29 -

The organisms isolated at a soil temperature of
30 °C were incapable of causing significant reductions in
emergence at soil temperatures of 15° and 20°C. The emer¬
gence in all the treatments at 20°C was low and nearly equal.

In comparing the organisms isolated at the various
soil temperatures, those isolated at 15°C contain the out¬
standing ones so far as seed-rotting ability is concerned.
While isolates 332 and 34-5 don't appear to possess so wide a
tolerance to the various soil temperatures as some of the
other organisms, they were capable of causing the greatest
decrease in emergence* Isolates 135 and 136 possess a high
degree of pathogenicity and a wide tolerance to the various
soil temperatures, since they caused the greatest average
decrease in emergence, taking into consideration all of the
temperatures tested. The majority of the organisms isolated
at 15°C were more adaptable to the different soil tempera¬
tures than those isolated at 20°G, while the 30 °G isolates
appeared to be active only at or near that temperature.

Discussion

These preemergence-blighting fungi of wheat, appear
to have a sufficiently wide temperature range to enable them
to operate quite effectively at the soil temperatures in
which wheat is generally sown. Sowing wheat when the soil
temperature was at a particular level would therefore not be
a very satisfactory means of avoiding damage completely, but

' or. ■: . •

“■

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- !

■ . - ■ •_ . : f: . . ■ .1 \ . . . .. : . ’/ . • ' . . " ; j

. ? o: . •.

. Fi'U.'iv.v 5,' :;-x; w ;d

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. . r . .... ■' ' :. ■ • ~ •: •••■. ; v C. , l .. /

■

. . .

- . r.. ■ V ;

- 30 -

it might be used to reduce damage.

Summary

Five out of the eight pathogenic organisms that
were isolated from rotting wheat seed at a soil temperature
of 15>°C significantly reduced the emergence of wheat at soil
temperatures of 15°, 20° and 30°G. The pathogenicity of
these five organisms increased with increasing soil tempera*
ture. The organisms isolated at 20°C proved most pathogenic
at 15°C and relatively non-pathogenic at a soil temperature
of 30°Ce The seed-rotting ability of the fungi isolated at

a soil temperature of 30°C was quite specific for that
temperature. In general the organisms isolated at a soil

temperature of ±$°C were least specific in their reactions
to soil temperature and those isolated at 30°G were most
specific to soil temperature while the 20°C isolates were
intermediate.

EFFECT OF SOIL MOISTURE

Although all fungi are classified as being aerobic,
some are known to thrive under near- anaerobic conditions®

It is the purpose of this experiment to find the level of
soil moisture most conducive to preemergence blighting®

Machacek (26), while experimenting with wheat
seedlings in non- sterile soil, found that if the soil was
kept too moist the seedlings were frequently attacked by

... .. ‘ . - ■ ; ■ I i'-I: ; r\I - £■**;.'

o* ■ • -

. ;« • • . • • • ■

~ -

I uf.r jc £/>.. I o i'l" XI';. ;C C ' I II

• :

V ,

- 31 -

species of Pythium and damped off, and if the soil was kept
too dry, the seedlings were occasionally attacked by Rhizoctonia
solan i. His results show that with a decrease in the amount
of water given a seed bed, there was an increase in the amount
of blighted seedlings. Although G-ibb erella saubinetii and
some Fusaria are favored by low soil moisture, other Fusaria,
Helmintho spor ium sativum and Pythimn species are favored by
high soil moisture (11).

The test organisms used in this experiment were
fungi that had been isolated from rotting wheat seed at a
soil temperature of 2Q°C. Red Bobs wheat was sown at the
rate of 25 seeds per pot. The soil moisture was maintained
at three different levels. Soil moisture analyses were made
every other day and water was added as necessary to maintain
the desired level. The mean moisture levels were ll|..5#,

21.3# and 26.3# for low, optimum and high, respectively.

The air temperature of the greenhouse varied from a daily
mean high of 26.£°C to a daily mean low of 11.9°C. The three
moisture tests were run concurrently on on© bench in the
greenhouse. Significant differences in the emergence were
obtained by means of a split-plot analysis. The results are
presented in Table VII.

\ ... ■ ; , ' . • .. ' .! -'a . . ;v .0 - ■ • ' ■ ' ' ' ''V ^ *

J J S • : . ■ .• • • ' V '• 5 ' -■

; ■ :

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; ii ■

* * .

... . . : $qo ,

n .. ' • t .

r,:: i, ■■ u t-fi., v !;» n.s 7lxiro»:te:'

' •

.! -LLi :

. .

TABLE VII

COMPARISON OP THE ABILITY OP THE FUNGI ISOLATED PROM
ROTTING WHEAT SEED AT A SOIL TEMPERATURE OP 20°C TO
CAUSE PREEMERGENCE BLIGHT OP WHEAT UNDER LOW, OPTIMUM
AND HIGH SOIL MOISTURE CONDITIONS

Average Number of Seedlings Emerged**

Low Optimum High

Organism Soil Moisture Soil Moisture Soil Moisture

Hit-

12.8

11.3

13.0

127

18.5

11.5

13.3

1^3

12.3

13.5

10.8

145

13.3

12.3

14.0

146

14..0

13.3

11.3

149

12.8

11.8

264.

i4*5

13.0

12.0

268

14.. 8

11.5

13.3

281

16.3

13.3

11.8

286

14-.3

14*3

11.3

Check

14.5

16.8

9-3

Based on I4. replicates, 25 seeds per replicate#

1 ' - o • / ..

■

- 33 -

There was a highly significant difference in the
emergence of the wheat seedlings at the three moisture
levels* The greatest preemergence blighting occurred under
high soil moisture conditions and the lowest under low soil
moisture conditions, while the preemergence blighting under
optimum soil moisture conditions was intermediate* There were
no significant reductions in emergence by any of the organisms,
due possibly to the low emergence in the checks* The lo?/
emergence in the checks was probably due to early contamination
after seeding by a multitude of microorganisms which flour¬
ished in the sterile soil and c ornmeal -soil medium at the
relatively high greenhouse temperatures*

v»

Discussion

These results are quite opposite to those obtained
by Machacek (26), and to the results of an earlier experi¬
ment reported in this paper, when the greatest decrease in
the emergence of wheat was obtained under low soil moisture
conditions. It should be noted though, that the above con¬
trary results were obtained when using non-sterile soil. It
is not reasonable to expect the same results in non-sterile
soil with its compliment of complex ml cr of lor a, as in steri¬
lized soil containing a single parasitic fungus*

Summary

There was a highly significant difference in the
emergence of the wheat seedlings at the three moisture levels.

. • . : o... ,

. ■

' . - ' ' • ■ '

■

. ■' ;J • . . - • ; ■ ' . * to ' 'ill

. : ... . . . . ••

- 34 -

The lowest emergence occurred under high soil moisture con¬
ditions and the highest under low soil moisture conditions,
while the emergence under optimum soil moisture conditions
was slightly higher than intermediate* No other significant
differences were observed*

REACTION OF DIFFERENT TYPES OF SEED TO THE SAME FUNGI

We have seen how these isolates from rotting seed
react to different soil temperature and moisture levels* A
major aim of this study was to ascertain their relative
reactions on different types of seed* That is considered in
this section. Would these fungal isolates, for instance,
have the necessary enzyme compliments to enable them to
attack a seed that was high in starch like wheat as aggres¬
sively as ones that were high in lipoids like flax, or high
in proteins like peas? In an attempt to answer this question,
preemergence-blighting fungi isolated from each type of
rotting seed at a soil temperature of 20 °G were tested
against the two other types of seed* Padwick (38) found
several fungi pathogenic to pea cotyledons that were not
isolated from peas* Greaney (13) isolated Helmintho sp opium
sativum from wheat, barley and rye seed, and found that it
was only associated with low germination in naturally in¬
fested wheat seed. The same seed and methods were used in
this experiment as were employed in the previous experiments*
Tests involving the isolates from flax and pea seed were

.. * . •

. .. .

* 1

. i.

•

' ' -

: •• •; re ■■ i, , /..• rj .. ■ .L; ■

- 35 -

carried out concurrently against the three types of seed as
two separate experiments, whereas the isolates from wheat
seed were tested against the three types of seed in individual
experiments. In order to make an overall comparison of the
emergence it was therefore necessary to convert the number of
seedlings emerged to the percentage of preemergence blighting
on the basis of their individual checks. This method was
previously described on page 26, The results are presented
in Table VIII.

- 36 -

TABLE VIII

COMPARISON OF THE PREEMERGENCE BLIGHTING OF WHEAT, FLAX
AND PEA SEED BY FUNGI ISOLATED FROM DECAYING WHEAT,
FLAX AND PEA SEED AT A SOIL TEMPERATURE OF 20°C

Seed Type From Percentage of Preemergence

Which Organisms Blighting in Relation to

Were Isolated Organisms Uninoculated Checks

Wheat Flax Peas

Wheat

114

l|.2.3xx

30 .8x

31 *9xx

127

lj.2 . 3xx

9.2

12.9

143

3 4- • 2xx

8l .Xxx

[j.2 .9xx

i45

1$. 8x

7l}.*lxx

20.0

146

2[}..7xx

Ij.5*9xx

20.0

19 *5 xx

57*8xx

264

28 *9xx

63 .2xx

214

268

30 .Oxx

32.14.x

7.1

281

l6 .8x

22<>7

0.0

286

23 * 7xx

16.2

16.7

Flax

F120

9.9

934xx

0.0

FI 24

7 4-

87 ,8xx

0.0

F2!3r

Sb* 2xx

97 «oxx

. 5*9

F213g

18. 7x

85.lpcx

12.9

F236r

50 .7xx

I|i!-.7xx

1.2

F236q

11.3

83®7xx

38 « 2xx

Pea

P268

0.0

21. 7x

10.0

P270

4.0

90 «9xx

36 .Oxx

x significant
xx very significant

- 37 -

The wheat preemergenee blighting organisms were
able to rot the seeds of flax much more easily than the seeds
of peas* In fact, the isolates from II4.3 to 26I4,, inclusive,
which comprise $0 percent of the wheat isolates tested,
caused more severe preemergence killing of flax than of wheat.
Only isolates ill), and ll|3 were able to signif icantly reduce
the emergence of peas and these were the only isolates from
wheat that were capable of significantly reducing the emer¬
gence of the three types of seed. Organisms 127, 28l and 286
appear to be specific wheat seed decay organisms in that
they were unable to significantly reduce the emergence of
flax or peas.

Out of the six preemergence-blighting organisms
isolated from flax seed, only three: F213r,F213q and F236R
were capable of significantly reducing the emergence of wheat
and only one, F236^, was able to significantly reduce the
emergence of peas. Isolates F120 and Fl2lj. appeared specific
for flax. Hone of the flax isolates were able to significantly
reduce the emergence of the three types of seed.

Neither of the isolates obtained from pea seed
were capable of preemergence killing of wheat, only one
significantly reduced the emergence of peas, while they both
significantly reduced the emergence of flax.

Discussion

From these results it would appear that many similar

■ .. ' ‘ • ■

. < \ t

f .... ' .. ' . . . i '..V : ■’ • • < • ■ • ■

-I..’’ - . '

: • .. , :

, - ' o- •. ■

- •'

- 38 -

fungi are capable of causing significant preemergence killing
of wheat and flax, and that only a few of these fungi are
able to cause a significant reduction in the emergence of
peas# This seems a little irregular, since earlier tests
showed peas the most susceptible of these three seed types to
seed decay. Padwick (3^) looked upon the rotting of pea
seed in the soil as a complex disease in which more than one
distinctly parasitic fungus may be playing a part. He v/as
able to show, however, that the following fungi from wheat
were highly pathogenic to pea cotyledons t Fusarium avenaceum,
F. culmorum, F. gr amine arum and Helmintho spor ium sativum.

Of the three types of seed exposed, flax appears to
be the most susceptible to preemergence blighting by the
various fungal isolates. It is interesting to note that a
fungus which was isolated from decaying pea seed and was
relatively unable to rot that seed, caused significant pre¬
emergence blighting of flax.

It a rotation plan to minimize pre emergence blighting
was based on these results, it would not seem advisable to
sow flax in infested soil after wheat or peas. It would also
not be advisable to rotate wheat with flax, but peas would
be fairly safe on infested soil upon which flax or wheat had
grown.

Summary

Out of the ten organisms which were isolated from

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- 39 -

decaying wheat seed and found to be able to significantly
reduce the emergence of wheat, seven were able to reduce the
emergence of flax and only two were able to reduce the emer¬
gence of peas* Three organisms were found specific to wheat,
while two were able to cause significant preemergence blight¬
ing in wheat, flax and peas* Of the six preemergence-blight¬
ing organisms isolated from flax, three were found to signi¬
ficantly reduce the emergence of wheat and only one the
emergence of peas* Two were specific to flax and none were
able to significantly reduce the emergence of the three
types of seed* Neither of the isolates obtained from pea
seed were able to cause pre emergence killing of wheat, only
one significantly reduced, the emergence of peas, while both
significantly reduced the emergence of flax* Flax appeared
to be the most susceptible to the various isolates* Pre¬
emergence blighting of flax and wheat may be caused by a
fairly similar group of fungi* Only two out of the eighteen
isolates tested were capable of causing a significant reduction
in the emergence of the three types of seed, ten caused seed
decay of two types of seed and five were specific to the
type of seed from which each was isolated*

EFFECT OF MISCELLANEOUS FACTORS ON SEEP DECAY

All the previous tests with the isolates were carried
out using sterile soil and normal seed* The following ex¬
periments were designed to determine how one of the pre-

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- 40 -

emergence-blighting fungi would act in non- sterile soil, on
seed treated with a mercurial dust, on injured seed, and on
normal seed after it had been sown in a soil medium contain¬
ing ground seed of the same type.

Machacek (26) found that the amount of soil-borne
infection of cereal seedlings by soil-borne organisms was
negligible when friable, non- sterile soil was kept moist and
at 20°C. Steam sterilizing changes the physical condition
of the soil and if it is prolonged, toxic amounts of ammonia
may be released.

Hurd (l6) found that if wheat is injured over the
endosperm, 100 percent fatal infection results when the
spores of Penicillium or Rhizopus are present; but if the
injury is over the embryo, the seeds remain practically
immune. Field and greenhouse tests made at the University
of Alberta, using non- inoculated seed in non-sterile soil,
have shown that the reduction in emergence is greater when
the injury is over the embryo than over the endosperm.
Previous work at this laboratory has also shown that an in¬
crease in emergence due to seed treatment with a mercurial
fungicide, is greatest with embryo- injured seed, least with
normal seed and intermediate with endosperm- injured seed.

The medium upon which the parasite has grown may
have quite an effect upon it. In the laboratory a fungus
may be forced to live on artificial substrates for long

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- 41 -

periods. Its diet is often unbalanced there since it is
frequently overfed with carbohydrate and nitrogen compounds.
In the sudden transference onto a formerly suitable host
plant, the conversion to the original parasitic mode of life
cannot always be accomplished rapidly enough. Adaptive
enzymes, which some fungi possess, must therefore be brought
into play. Fomes igniarius, the false tinder fungus of
apple trees, is a more successful parasite when it has pre¬
viously been grown on wood from suitable trees than if it
had been cultured on bread or agar. Gibberella saubinetii,
after previous culture on oatmeal, killed only 10 percent of
Pinus seedlings, whereas after culture on steamed rice under
the same external conditions, it killed f>0 percent of the
seedlings (12).

Only wheat was used in this experiment, the endo¬
sperm of which was injured by cutting a niche with a scalpel
approximately J mm. wide, ij- mm. long and i mm. in depth,
midway on the side of the kernel. Injury to the embryo was
accomplished by severing the testa over it longitudinally
with a dissecting needle. All the other seeds used in this
experiment were hand-picked to ensure their soundness.
Ceresan M, an organic mercurial fungicide, was used to treat
the seed. It was applied at the rate of •§■ oz. per bushel.
The wheatmeal-soil medium contained 10 percent ground wheat
seed. The test organism chosen was one that had been iso¬
lated from rotting wheat seed at a soil temperature of 20°C .

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- 42 -

During the course of this experiment the greenhouse tempera¬
ture varied from a daily high mean of 26.1°C to a daily low
mean of 11*8°C. A summary of the results is presented in
Table IX.

- 43 -

TABLE IX

ABILITY OP A FUNGUS ISOLATED PROM A ROTTING WHEAT SEED
AT A SOIL TEMPERATURE OP 20°C TO PRODUCE PREEMERGENCE
BLIGHT OP WHEAT UNDER DIFFERENT SOIL, SEED AND
MEDIA CONDITIONS

Treatment

Average
Number of
Seedlings
Emerged'"'

Isolate llj.3 Against Normal Seed in Sterilized

Soil 14.0

Check Normal Seed in Sterilized Soil l4*5

Isolate l43 Against Normal Seed in Non-Steri-

lized Soil 15*0

Check Normal Seed in Non-Sterilized Soil 13 *0

Isolate l43 Against Ceresan M Treated Normal

Seed in Non-Sterilized Soil lf>*3

Check Ceresan M Treated Normal Seed in Non-

Sterilized Soil 18 *0

Isolate 143 Against Embryo- Injured Seed In

Sterilized Soil 4»5

Check Embryo- Injured Seed in Sterilized Soil 6,5

Isolate 143 Against Endosperm- Injured Seed in

Sterilized Soil 11*8

Check Endosperm- Injured Seed in Sterilized Soil l£*5

Isolate l43* Previously Grown on Wheatrneal-

Soil Medium, Against Normal Seed, in Steri¬
lized Soil l4»3

Check Wheatmeal-Soil Medium and Normal Seed in

Sterilized Soil 18*8

x significant
xx very significant

'"' Based on 4 replicates, 25 seeds per replicate

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- kh -

The low emergence in the sterile soil check plots
was probably due to early post- seeding contamination of the
sterile cornmeal- soil medium, since a similar decrease in
emergence was obtained when this inoculum was placed in non-
sterile soil. Further evidence of this is that a highly
significant increase in emergence was obtained when Ceresan
M treated seed was sown in non- sterile soil. Although these
contaminating organisms were very effective in reducing
emergence, the seed treatment appeared quite effective against
them and relatively ineffective aga5_nst isolate ll|3»

Whether isolate ll}3 was present or not, the emergence
of endosperm- injured seed was very significantly better than
that of embryo- injured seed, and nearly equal to that of
normal seed. Isolate 1I4.3 significantly reduced the emer¬
gence of endosperm- injured seed over its check, but not that
of embryo-in jured seed.

After previous growth on a wheatmeal-soil medium,
isolate li|3 significantly reduced the emergence of wheat
over its check. This could either mean an increase in its
pathogenicity due to the medium or that the wheatmeal-soil
medium proved less favorable to contaminants than did the
cornmeal- soil medium.

Discussion

These results stress that one cannot be too
cautious in avoiding contamination when using sterile soil

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- 45 -

under relatively high temperature conditions* Similar methods
proved satisfactory under moderate temperature conditions*

The relative predisposition of seed to decay by mechanical
injury, was found to be similar to that obtained by earlier
experiments in this laboratory, and different from the results
obtained by Hurd (l6), in that the embryo- in jured seed
appeared to be much more liable to preemergence blighting
than endosperm- injured seed* Since the Ceresan M seed treat¬
ment was ineffective in preventing preemergence blight by
isolate ll|3* it would suggest that this isolate attacked the
seedling rather than the seed itself* It is of practical
significance that the normal soil microflora did not appear
to nullify the preemergence blighting ability of isolate llj_3 •

Summary

Although Ceresan M was non-effective against iso¬
late llp3 > it significantly increased the emergence of normal
wheat in non- sterilized soil* Regardless of treatment, embryo-
injured seed showed the lowest emergence, \fhile the emergence
of endosperm- injured seed was nearly equal to that of normal
seed* The fungus isolate ll|3 significantly reduced the
emergence of endosperm- Injured seed, and of normal seed
after it was grown on a 10 percent wheatmeal-soil medium,
over their checks. This fungus also significantly reduced
the emergence of embryo- injured seed over endosperm- Injured
seed that had been inoculated with it.

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- 46 -

SUMMARY OF THE SEED-ROTTING CAPACITY OF THE DIFFERENT

FUN SAL ISOLATES UNDER VARIOUS CONDITIONS

Preemergence blighting fungi that were isolated
from decaying wheat seed at soil temperatures of l£0, 20°
and 30°C were tested for their ability to rot wheat seed
over this range of temperatures. In general these isolates
proved quite active at all the soil temperatures tested.

Those isolated at l£°C were the most active and those iso¬
lated at 30°C the least active.

The wheat seed-decaying fungi that were isolated
from rotting wheat seed at a soil temperature of 20°G were
tested against wheat seed at low* optimum and high soil
moisture levels. Highly significant differences in emergence
were obtained between each of the moisture levels, with the
lowest emergence in high soil moisture and the highest emer¬
gence in low soil moisture, while the emergence under optimum
soil moisture conditions was intermediate.

The fungi that were isolated from decaying wheat,
flax and pea seed at a soil temperature of 20°C were tested
against each of these seeds. The majority of the fungi iso¬
lated from either flax or wheat seed were able to cause
significant preemergence blighting of flax and wheat but not
of peas. Of the eighteen isolates tested, the following
were capable of causing a significant reduction in emergences
two from wheat of the three seed types, ten of two seed

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types, and five were specific to the seed types from which
they were isolated* Flax appeared to be the most susceptible
to the various isolates and peas the least susceptible. The
majority of these fungi were non-specific to one seed type.

When one 20°C wheat isolate was tested against
wheat seed under various conditions, it was found to signifi¬
cantly reduce the emergence of endosperm-in jured seed, and of
normal seed after it was grown on a 10 percent wheatmeal-
soil medium, over their checks. It also significantly re¬
duced the emergence of embryo-injured seed over endosperm-
injured seed. Regardless of treatment, embryo- injured seed
showed the lowest emergence, while the emergence of endo¬
sperm-injured seed was nearly equal to that of normal seed.
Although Ceres an M was non-effective against isolate lip3 , it
increased the emergence of normal wheat to a highly signifi¬
cant degree in non- sterile soil.

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- 1+8 -

PERSISTENCE OP CHEMICAL SEED
PROTECTANTS ON SEED IN
THE SOIL

INTRODUCTION

The foregoing work demonstrated the value, and
strongly suggested the need, of seed protectants when certain
plants are grown from seed in soil. Much work has been done
on the testing of various substances as seed protectants (6,
8, 19, 31) 9 on determining suitable rates of application and
wider uses of substances which show promise as seed dressings
(8, 30); also on their relative toxicity to pathogens (1+6,

35) j plants (8), and animals. Work has also been done on
their seed- adhering properties when used with inert dusts
(10, 1+3)5 with water (10, 22), or with oil (10); on their
decomposition in soil (5, 7)? and on their effect on the
soil flora (5) I on the accuracy and uniformity of application
by means of commercial seed treating machines (27), and on
methods of assaying them (2, 22, 25, 27, 29, 3I+, 39) • To
the writer ! s knowledge, no work has been published on the
persistence of these seed fungicides on seed in the soil.
Therefore such studies were undertaken and are discussed in

the present section

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- 49 -

METHODS

Wheat, flax and pea seeds were sown within three days
after treatment with commercial seed protectants, at a depth
of lj- inches in a 3:1 Edmonton soil-sand mixture, in 5 inch
pots at the rate of at least 25 seeds per pot* The seed
used was Red Bobs wheat. Redwing flax and Homesteader peas.

The pots were placed on an open bench in the greenhouse and
watered daily.

At daily intervals four seeds were removed from
the soil of each seed treatment and freed of growth and ad¬
hering soil. These were placed in a Petri dish containing a
suspension of potato dextrose agar and the test fungus, just
before the agar hardened. The plates were incubated at about
25°C for 2 4 - 36 hours. The persistence of the fungicides
was determined by inverting the Petri plates and measuring
the average radius of the zones of inhibition of the test
fungus around the seeds. The test fungus had been isolated
from a decaying wheat seed at a soil temperature of 20°C.
Although it proved non- pathogenic to wheat, it is a profuse
producer of spores, grows rapidly and in general serves very
well as a test organism.

About 3 ml. of agar was added to Petri plates used
for wheat and flax, and about 10 ml. to those used for peas.

The wheat and flax seeds were completely submerged in the
agar, whereas the pea seeds were only partially embedded in

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- 50 -

it. A large loopful of spores, (and probably some mycelium)
of the test organism was suspended in 10 ml. of sterile
distilled water, and 1 ml. of the suspension was added to
each Petri plate. Observations were made of the amount of
growth from the seed and the condition of the seed when it
was removed from the soil.

RELATIVE PERSISTENCE OF CERESAN M ON VIABLE AND NON- VIABLE

WHEAT, FLAX AMD PEA SEED IN SOIL UNDER

GREENHOUSE CONDITIONS

This experiment was carried out to compare the re¬
lative persistence of Ceresan M on viable and non-viable
wheat, flax and pea seed in the soil, and to check the
possibility of antifungal substances being produced by ger¬
minating seed. If non-viable seed produced results similar
to viable seed, it was planned to use the former in the sub¬
sequent tests.

The non-viable seeds were prepared by exposing
viable seeds for two minutes to 1 5 pounds steam pressure in
an autoclave, after which they were dried at room temperature.
Ceresan M was applied at the rate of 1 oz. per bushel for
flax and peas, and J- oz. per bushel for wheat. The seeds
were manually shaken for a period of five minutes in stoppered
containers after the fungicide was added, to help ensure an
even distribution. The stoppers were then removed. The
other methods employed are similar to those previously des-

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cribed on page 1+9 • A summary of the results Is presented in
Table X.

TABLE X*

RELATIVE PERSISTENCE OP CERESAN M ON VIABLE AND NON-
VIABLE WHEAT, FLAX AND PEA SEED IN SOIL UNDER
GREENHOUSE CONDITIONS

Radius of the Zone of
Inhibition in mm® After 0-5

Treatment

Days

in the Soil

2345

Wheat Viable Ceresan M | oz»

8.0

3.0

2.3 1.5 1.0 0.0

Wheat Non-viable Ceresan M i 02#

8.5

2.0

0.0

Flax Viable Ceresan M 1 oz#

7.3

2.0

0.0

Flax Non-viable Ceresan Ml oz.

4.5

0.0

Peas Viable Ceresan M 1 oz.

21.0

3.3

0.0

Peas Non-viable Ceresan Ml oz.

21.5

2.3

0.0

w The results for the untreated viable and non-
viable seed, which served as checks, were omitted
from the above table since they caused no inhibi¬
tion of the test organism*

After seed treatment the amount of Ceres an M ad¬
hering to the non- viable seed, as indicated by the size of
the zone of inhibition, was much less on flax, and slightly
more on wheat and peas than that on viable seed. After one
day in the soil the amount of seed dressing persisting on
non-viable seed was less than that on viable seed for all

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- 52 -

the seed types. Viable wheat exhibited the greatest ability
to retain the fungicide, since after four days in the soil
it produced a trace of a halo, whereas all the other treat¬
ments failed to produce any inhibition after the seeds were
two days in the soil.

The wheat seed treated with Ceresan M made slightly
more rapid growth than the untreated seed. Seed treatment
had little effect on the growth of flax and peas. By the
fourth day in the soil, the viable flax seeds were discarding
their seed coats. Much trouble was experienced from soil
adhering to the non-viable, seed treated and non-seed treated
flax and pea seed. By the fifth day in the soil, the non-
viable, non- treated pea seeds were very water-soaked, fell
apart easily, and the seed coat tended to be shed with the
adhering soil. Ceresan M appeared relatively ineffective in
preventing the decay of non-viable pea seed. Such seed
proved more subject to decay than untreated, viable pea seed*

Discussion

Due to the variance in the ability of the fungicide
to adhere to non-viable seed as compared to viable seed, and
to its more rapid deterioration in the soil, it would appear
inadvisable to use non-viable seed in any subsequent tests
of this sort. Hurd (l6) found that death renders previously
immune wheat seeds immediately susceptible to attack by

*• .

„

- 53 -

Penicillium and Rhizopus* It does not appear to be an easy
matter to compare the relative adhesiveness of the fungicide
on the three types of seed by the method employed in this
paper due to the difference in the size of their seed*

This type of bioassay, though simple, is not with¬
out faults. Some contamination occurred in the Petri plates
from soil adhering to the seed and likely from the seed it¬
self* The frequency of contamination increased with increasing
time the seed was in the soil* Probably the easiest way to
avoid this contamination would be to employ a test organism
that would grow rapidly in a very acidic medium at low tempera¬
tures* In preliminary tests the size of the zone of inhibi¬
tion was found to vary with the concentration of the test
organism and its concentration would vary with the amount of
agar added to the plate. The radius of the halos was also
found to decrease in size, to the average extent of 1 mm.,
during the period of incubation from 2lj. to hours* Thorn-
berry (39) * when using paper disks on the surface of the
agar to test toxicants, found that with a relatively thick
layer of ” seeded” agar, the zones of inhibition would have a
cone-shaped, poorly defined margin which was difficult to
measure* Other factors affecting the size of the halo would
be: uniformity of coverage of the fungicide (27* 43)* the
type of filler, its state of subdivision and whether or not
substances of an adhesive nature had been added (10, l|3)l
the interval between treating and seeding (27)* also the

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amount of agitation the treated seed was exposed to before
seeding (10). Pitzgibbon (10) demonstrated that the general
adhesive characteristics of seed dressings are fairly well
maintained from one variety of seed to another, so that a
dressing satisfactory for any one cereal tested will be
satisfactory for all in this respect.

Summary

Ceresan M did not persist as well in the soil on
non- viable wheat, flax and pea seed as it did on the same
types of viable seed. Regardless of treatment the non-
viable seed was more subject to decomposition in the soil
than the viable seed. Of the seed types tested, viable wheat
exhibited the greatest ability to retain the fungicide, since
after four days in the soil it produced a trace of a halo,
whereas all the other treatments failed to produce any
inhibition of the test organism after two days in the soil.
The untreated viable and non-viable seed failed to inhibit
the growth of the test organism.

RELATIVE PERSISTENCE OF VARIOUS SEED PROTECTANTS ON

VIABLE WHEAT s FLAX AND PEA SEED IN THE SOIL UNDER

GREENHOUSE CONDITIONS

This experiment was designed to enable a comparison
between commercial seed dressings which contain mercury and
non-mercurial preparations, and between dry and wet methods

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- 55 -

of treatment; as measured by the ability of these seed dres¬
sings to persist on viable wheat, flax and pea seed in the
soil under greenhouse conditions*

Manufacturers are endeavoring to produce fungicides
which possess high toxicity to microorganisms and low toxicity
to higher animals* This quality is found to a greater ex¬
tent in many of the non-mercurial than in the mercurial pre¬
parations* The hazard to humans of inhaling poisonous dusts
while treating grain has been overcome by using some of the
preparations in liquid form.

The methods employed in this experiment were similar
to those described on page l| 9* and seed from the same stock
was used* The trade names of the fungicides used, along with
their active ingredients and rates of application, are listed
in Table XI*

, . , ■' . ■

■

- .

TRADE NAMES, ACTIVE INGREDIENTS, AND RATES OF APPLICATION

OF THE FUNGICIDES

- 56 -

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- 57 -

The other rates of application are either those
recommended by the manufacturer, or those that were found to
be satisfactory and are in common use. The fungicides were
vigorously shaken with the seed for a period of five minutes
in stoppered Erlenmeyer flasks, after which the stoppers
were removed, except in the case of panogen lip which remained
in a stoppered Erlenmeyer for a period of l\B hours. Only
Geresan M slurry and Panogen lip were applied in liquid form.

A summary of the results is presented in Table XII.

RELATIVE PERSISTENCE OF VARIOUS SEED PROTECTANTS ON VIABLE WHEAT, FLAX
AND PEA SEED IN THE SOIL UNDER GREENHOUSE CONDITIONS

erf i — I
•H
d O
<D CO
P

Crf 0
0 ,C|
Sh -P

3 01
O d
p. o

<rf iH

5h
Sh 0
O PL.

•H

•H PJ
A *H
H H
A ^
S3 erf
H >

a <

o
tSJ 0
d

<H 0

O 0
CO

S d

a ©

p
PI erf
•H 0
U
m p
d .Pi
•H
d
erf
Prf

erf

-d

•

“LA

“1A

•

C^~

erf

•

r—

-d-

r—

CVi

-d-

•

erf

•

“LA

“A

•

erf

•

!>>

-d*

erf

•H

erf

feO

_ d

W P

erf

EH Jh

a o

- 59 -

The fungicides appeared more persistent on wheat
and pea seed them on flax seed in the soil. Orthocide l\.Ob
proved far superior to the other seed dressings in its ability
to persist on each of the seed types in the soil. Even after
remaining I4.9 days on wheat seed in the soil, it was capable
of producing some inhibition of the growth of the test organism.
Of the other seed dressings, Panogen 1)4 persisted longest on
wheat (two days), Ceresan M dust, the longest on peas (three
days), while none of the other fungicides persisted on flax
seed even after one day in the soil. The hexachlorobenzene
formulation on seed failed to show any fungistatic effect
against the test organism. The wet seed treatments did not
appear superior to the dry seed treatments, and except for
Orthocide lj.06, the non-mercurial preparations proved inferior
to the mercurial seed dressings in their ability to persist
on seed in the soil. On wheat and peas, but not on flax,
the Orthocide L4O6 preparation appeared to increase in fungi¬
cidal activity after one day in the soil. The ability of
the various fungicides to adhere to wheat and flax seed when
not exposed to the soil, exhibited the same trend, the order
in decreasing adhesiveness being Orthocide I4O6, Ceresan M
slurry, Panogen 1I4., Ceresan M dust, MTH, and C.X.L. Bunt
Cure. While on pea seed Ceresan M slurry was the most ad¬
herent, Orthocide lj.06, Ceresan M dust and MTH were nearly
equal; while Panogen 1J4 showed quite poor adhering qualities.

Ho pronounced phytocidal effect from any of the treatments
was noticed. Fungicide persistence tests on flax were dis-

tv-ti -ctj .. hi

* -

■

?. ! - /; ■ ■ ; . , •

* .

••

■■■••■

• •

• lT. '* , .

- 6o -

continued after four days in the soil since the seeds had lost
their seed coats by that time. They were discontinued on
wheat and peas after i.j-9 and 3>k- days in the soil, respectively,
due to lack of treated seed material.

discussion

Although the majority of the fungicides did not appear
to persist on the seed for a prolonged period of time in the
soil, it is possible that their protective effect may be
exerted in the soil surrounding the seed for a longer period
of time. Machacek (27) tested solutions of fungicides of
various concentrations by absorbing them on paper disks and
plating them on M seeded” agar. He found that the amount of
diffusion of two of the fungicides was related to concentration,
but that the diffusion of three fungicides was not so related.
Thornberry (39) found that this method could not be used with
cationic toxicants since they are adsorbed by the agar. Fung¬
icides would also vary in their ability to act at a distance.
Caution should therefore be exercised when evaluating the
persistence of different fungicide preparations by the method
employed in this paper*

Baines (7) found that in soils where mercurials are
effective as fungicides, that the mercury compounds are reduced
by the soil to metallic mercury which migrates in the soil as
mercury vapors. Any factor that prevents the conversion of
a mercury salt to metallic mercury destroys the fungicidal

effects of the mercury. Some of these limiting factors
are : The presence of mercury-precipitating ions; a

- 6l -

soil with a high mercury-binding capacity; also a soil having
a high oxidation, and conversely, a low reducing potential*
Under moist aerobic conditions, Booer (5>) found that the
addition of 0*2 percent powdered sulfur to the soil, whilst
producing no immediate effect, completely eliminated in four
to seven days the retardation of plant growth resulting from
the addition of 0.02 percent mercury. The formation of
mercuric sulfide is the means by which the toxic effect of
mercury is eliminated from soil under field conditions from
season to season (5)«

Summary

Orthocide i|06 proved far superior to the other seed
dressings tested in its ability to persist on wheat, flax
and pea seed in the soil. Of the other seed dressings,
Panogen lip persisted the longest on wheat, Ceresan M in dust
form the longest on peas, while none persisted on flax seed
in the soil long enough to make a comparison. Bunt Cure on
seed proved inactive against the test organism used and
hence its persistence on seed in the soil could not be
measured. The wet seed treatments did not appear superior
to the dry seed treatments, and except for Orthocide Ip06> ,
the non-mercurial preparations proved inferior in their
ability to persist on seed in the soil to the mercurial seed
dressings. Although the ability of the various fungicides
to adhere to wheat and flax seed v/hen not exposed to the
soil exhibited the same trend, they appeared more persistent

J * ' * : ‘D .■

<> 1

■ "

. ■ •

■«

- 62 -

on wheat and pea seed than on flax seed in the soil.

SUMMARY OF THE PERSISTENCE OF CHEMICAL SEED

PROTECTANTS ON SEED IN THE SOIL

Ceresan M, used as a dust, did not persist as well
in the soil on non- viable wheat, flax and pea seed, as it did
on the same types of viable seed. Regardless of treatment,
the non-viable seed was more subject to decomposition in the
soil than the viable seed. Of the six fungicide preparations
tested, Orthocide 1|06 proved superior to the others in its
ability to persist on viable wheat, flax and pea seed in the
soil. The dry seed treatments were about equal to the wet
seed treatments in their ability to persist on seed in the
soil, and except for Orthocide 1|06, the non-mercurial pre¬
parations proved inferior to the mercurial seed dressings
in this respect. According to this method of testing, the
fungicides were more persistent on wheat and pea seed than on
flax seed in the soil®

- 63 -

GENERAL DISCUSSION

The differences in the susceptibility to decay of
the three kinds, of seed included in this study, are probably
explainable on the basis of the availability of their food
supply to microorganisms of the soil. The garden pea seed
which proved most susceptible to decay is known to have a
readily available supply of simple carbohydrates together
with a high protein content. This combined with its slower
growth rate and its more vulnerable seed coat renders it
very susceptible to decay.

More fungi than bacteria were found capable of
causing seed decay of wheat, flax and pea seed in the soil.
The fungi are likely capable of a faster entry into the seed
whereas the bacteria, would be more dependent on natural
openings in the seed coat for their entry.

The methods employed for the isolation of organisms
responsible for seed decay were satisfactory for obtaining
these organisms from wheat and flax seed. They were
unsatisfactory for obtaining them from pea seed, since a
very low number of organisms isolated from decaying pea seed
was separately capable of decaying them.

Soil environmental conditions exert a marked influ¬
ence on the amount of seed decay, but usually only conditions

■ . ; ,J • 1 ; . */;X , •

. . .v ' . 1 ; . ,

. : .

. ‘ ! •

- (>k -

which are relatively less favorable to the host than to the
pathogen result in much of this decay. The soil moisture
and temperature when not at an optimum level for the host
will have a strong bearing on the amount of seed decay. Soil
low in moisture was found to favor the decay of wheat and
flax seed, while high soil moisture favored that of pea seed.
Soil temperature exerted its influence by encouraging the
decay of flax and pea seed when low, and that of wheat seed
when high.

In nature the amount of seed decay would vary with
the changes in the soil environment. The critical period
would be the first two weeks following seeding. Some control
of the soil environmental factors may be obtained through
cultural practices such as varying the date of seeding,
rotating the crops and regulating the water supply. Wheat
sown late in the spring was found to be much more susceptible
to seed decay than that which was sown early. Flax seed is
relatively susceptible to decay by fungi capable of decaying
wheat or pea seed. This finding could apply in a crop
rotation by avoiding the sowing of flax on land where much
decay of wheat or pea seed had occurred.

Since the environmental conditions of the soil are
relatively unpredictable, it is important to have at least
the most susceptible seeds protected by chemicals. To be
effective, the chemicals must inhibit the growth of the seed
decay organisms on or near the seed in the soil. In addition.

* ■ .r. '

* ■

< ■

* ■

« . V • '

' ' ■ • ' ■ ■ a< u ■ ' ■■■ :,eo

- 65 -

ability to persist on the seed should increase their
protective value. Of the six fungicides tested, only Ortho-
cide 1|.06 was outstanding in its ability to persist on seed
in the soil, but aside from this fungicide, the non-mercurial
preparations were inferior to the mercurial seed dressings
in this respect. The majority of the fungicides only persisted
on the seed in the soil for a day or two. Since many of them
are known to be effective in reducing the incidence of seed
decay, it may be that they exert their effect in the soil
surrounding the seed for a longer period of time.

•>

‘ •

- 66 -

SUMMARY

1* Under all environmental conditions tested, pea seed

showed the greatest tendency to decay in the soil, wheat
the least, while flax was intermediate*

2* Decay of pea seeds was encouraged by high soil

moisture (especially for the first three days after
sowing) and by low soil temperatures*

3* High temperature and low moisture encouraged the

decay of wheat seeds in the soil*

1^. Low soil temperature and moisture favored preemer¬

gence blight of flax*

5* Fungi were found to be the most important seed-

rotting microorganisms*

6* Seed held at low soil temperatures yielded most of

the seed-rotting fungi which were isolated*

7* The number of fungal saprophytes isolated from

decaying wheat seed increased directly with the period
of time the seed was in the soil*

8* In general, the preemergence blighting fungi that

were isolated from decaying wheat seed at soil tempera¬
tures of 15°, 20° and 30°C proved quite adaptable in

: ■ ' c •'

.... - . ' . .

, .

, •

- 67 -

their ability to rot wheat seed over this range of temp¬
eratures.

9. Sterile soil with a high moisture content proved

more conducive to wheat seed decay by fungi that had
been isolated from rotting wheat seed than sterile soil
with a lower moisture content.

10. Flax appeared to be more susceptible than wheat
or peas to preemergence blighting by fungi that were
isolated from decaying wheat, flax and pea seed.

11. Preemergence blighting of wheat by a fungus isolate
was most severe when embryo- injured seed was used.
Endosperm- injured seed proved only slightly more sus¬
ceptible to it than normal seed.

12. The seed fungicide, Ceresan M, used as a dust, did
not persist as well in the soil on non- viable wheat, flax
and pea. seed, as it did on viable seed of the same types.

13. Orthocide Ipo6 proved much superior to the other
fungicides tested in its ability to persist on viable
wheat, flax and pea seed in the soil.

lit. Except for Orthocide i|06, the non-mercurial

fungicides proved inferior to the mercurial seed dressings
in their ability to persist on seed in the soil.

Most of the fungicides tested were more persistent
on wheat and pea seed, than on flax seed in the soil.

■

* .

„

.. r"

■

- 68 -

ACKNOWLEDGMENTS

The writer wishes to express his thanks to Dr# A.

W# Henry, under whose supervision the investigation was con¬
ducted, for helpful suggestions and criticisms during the
investigation and in the preparation of this manuscript, and
to the Department of Veterans Affairs of Canada for financial
assistance#

* . ,

•«." ..... . ■ ;. : : • ", y. .* '< i.v-., ::v;c i ' - £:^ ';.;i -...v

- 69 -

LITERATURE CITED

1. ABBOTT, W. S. A method of computing the effectiveness
of an insecticide. J. Econ* Ent. 18 : 265-267.

1925.

2. ARNY, D. C* The bioassay of Ceresan M on treated oat
kernels. Phytopathology 42:222-223. 1952.

3* BAYLIS, G. T. S. Fungi which cause preemergence in¬
jury to garden peas. Ann. Applied Biol.

28:210. 1941,

4. , DESHPANDE, R. S. and STOREY, I. F.

Effect of seed treatment on emergence of peas.
Ann. Applied Biol. 30:19. 1943 •

5« B00ER, J. R. The behavior of mercury compounds in

soil. Ann. Applied Biol. 31:34°“359* 1944*

6. BURNETT, L. 0. and REDDY, C. S. Seed treatment and

date of sowing experiments with six varieties
of flax. Phytopathology 21:985* 1931.

?• DAINSS, R. H. Fungicidal action of mercury in soils.
Phytopathology 26:90. 1936.

8. DS ZEEUW, D. J. and ANDERSEN, A. L. Response of pea
varieties to dry and slurry methods of seed
treatment. Phytopathology 42:52. 1952.

9* DICKSON, J. G. The influence of soil temperature and
moisture on the development of the seedling-
blight of wheat and corn caused by Gibber el I a
saubinetii. J. Agr. Research 23 : 837-870. 1923 •

10. FITZGIBBON, M. Seed disinfection. The determination

of the adhesiveness of seed dressings to
cereal seeds. J. Soc. Chem. Ind. 62:8. 1943 *

11. FOISTER, C. E. The relation of weather to fungous

diseases of plants. Botan. Rev. 12:54^“591*

1946,

12. GAUMANN, E. Principles of plant infection. 194&*

Translation by W. B. BRIERLEY, Crosley
Lockwood and Son Ltd. London. 1950*

- 70 -

13* GREANEY, F. J. and MACHACEK, J. E. The prevalence and
control of seed-borne diseases of cereals*

Sci. Agr. 26:59-78. 19l|i>.

lip • HENRY, A* W. Influence of soil temperature and soil
sterilization on the reaction of wheat seed¬
lings to Ophiobolus gramini s Sacc. Can. J.
Research 7:198 - 203 . 1932.

15 • HULL, R. Effect of environmental conditions and more
particularly of soil moisture upon the emer¬
gence of peas. Ann. Applied Biol. 24:681-689.
1937.

l6. HCJRD, A* M* Seed coat injury and viability of seeds

of wheat and barley as factors in susceptibi¬
lity to molds and fungicides. J. Agr. Research
21:99-122. 1921.

17 • HUTTON, E* M. The field emergence and yield of garden
peas as affected by treatment of the seed
with fungicidal dusts. J. Council Sci. Ind.
Res. Aust. 17:2. 1944*

18. HYDE, M. B. The sub epidermal fungi of cereal grains.

1. A survey of the world distribution of
fungal mycelium in wheat. Ann. Applied Biol.
37:179-186. 1950.

19* JACKS, H. The efficiency of chemical treatments of

vegetable seeds against seed-borne and soil-
borne organisms. Ann. Applied Biol. 38:135“
168. 19^1.

20. JAMES, N., WILSON, J. and STARK, E. The microflora of

stored wheat. Can, J. Research (C) 2l\.i22l\.-
233. 19^6.

21. LEACH, L. D. Growth rates of host and pathogen as

factors determining the severity of preemer¬
gence damping off. J. Agr. Research 75 :l6l-

179. 19^7.

22. LEBEN, C. and KEITT, G. W. A bioassay for tetramethyl-

thiuramdi sulfide. Phytopathology IpO :950“93>4*
1950.

23 o LEDINGHAM, R. J. The effect of seed treatment and

dates of seeding on the emergence and yield
of peas. Sci. Agr. 26 : 2lp8 . 1948*

- 71 -

2 )(.. LEDINGHAM, R. J., SALLANS, B. J. and SIMMONDS, P. M.

The significance of the bacterial flora on
wheat seed in inoculation studies with Helmin-
thosporium sativum. Sci. Agr. 29:253-262.

19 Wt

25. LOCKWOOD, J. L., LEBEN, C. and KEITT, G. W. A cultural

plate for agar diffusion assays. Phytopatho¬
logy Ij.2 s 44-7 . 1952.

26. MACHACEK, J. E. and WALLACE, H. A. H. Non-sterile soil

as a medium for tests of seed germination and
seed-borne diseases in cereals. Can. J.
Research (C) 20:539-5>57® 19^1-2.

27* . An agar- sheet method of testing the

efficiency of seed treating machines. Can.

J. Research (C) 28: 739-744. 1950.

28. 3 CHEREWICK, W. J., MEAD, H. W. and

BR0ADF00T, W. C. A study of some seed-
borne diseases of cereals in Canada. II. Kinds
of fungi and prevalence of disease in cereal
seeds. Sci. Agr. 31:193-206. 1951*

29® McCALLAN, S. E® A. The pea seed treatment of evaluating
fungicides in the greenhouse. Phytopathology

37:15. 1947.

30. . Evaluation of chemicals as seed

protectants by greenhouse tests with peas and
other seeds. Contrib • Boyce Thompson Inst.

15:91-117 • I9I4-8 .

31* . Testing techniques (fungicides).

Contrib. Boyce Thompson Inst. l6:299* 195>1*

32. McLAUGHLIN, j. H. The isolation of Pythium from soil
at various seasons of the year as related to
soil moisture and temperatures. Phytopatho-

logy 37:15. 1947.

33* McNEW, G-. L. Pea seed treatments as crop insurance.
Canner 96:13. 1943.

34* MILLER, H. J. A. A comparison of laboratory and field
retention and protection value of certain
copper fungicides. Phytopathology 33:899“9°9*
1943.

35* MUSKETT, A. E. and C0LH0UN, J. Biological technique
for the evaluation of fungicides. II. The
evaluation of seed disinfectants for the control

. 1. -• ■

- 72 -

of seed-borne diseases of flax. Ann. Botany
6:219. 1942.

36. MUSKETT, A. E. and COLHOUN, J. The prevention of seed-
borne diseases of flax by seed disinfection*
Ann. Applied Biol. 30:?. 1943*

37* OXLEY, T. A. and JONES, J. D. Apparent respiration of
wheat grains and its relation to a fungal
mycelium beneath the epidermis. Nature 1 54:

826. 19!^.

38. PADWICK, G. W. Complex fungal rotting of pea seeds.

Ann. Applied Biol. 25:100-114* 1938.

39* THORNBERRY, H. H. Paper disc plate method for the
quantitative evaluation of fungicides and
bactericides. Phytopathology ij.0 : 419-4^9 .

1950.

4-0. WALLACE, H. A. H. and HENRY, A. W. Soil moisture and
temperature in relation to the value of seed
treatment of peas with organic mercury dusts.
Proco Can. Phyt. Soc* 8, June 28-30. 1937*

IjJL. WALLACE, R. H. and LOGHHEAD, A. G. Bacteria associated

with seeds of various crop plants* Soil Sci.

71:159-166. 1951.

42. WALLIN, J. R. Seed and seedling infection of barley,

bromegrass and wheat by Xanthomonas translucens
var. cerealis. Phytopathology 36:440-457 •

1946.

43. WESTON, W. A. R. D. and BRETT, C. C. Seed disinfection

IV. Loss of vitality during storage of grain
treated with organo-mercury seed disinfectants.
J. Agr. Sci# 31:500. 1941*

44* WILSON, H. K. and HOTTES, C. F. Wheat germination

studies with particular reference to tempera¬
ture and moisture relationships. Agron. J.
19:181-190. 1927*

45. . Wheat, soybean, and oat germination
studies with particular reference to tempera¬
ture relationships. Agron. J. 20:599-619*

1928.

46. WILSON, J. D. Field testing of fungicides for vegetable

disease control. Phytopathology 4l:l°50-

1058. 1951.

\ .•

- 73 -

APPENDIX I

NUMERICAL DESIGNATION OF THE SEED DECAY MICROORGANISMS

ISOLATED FROM ROTTING WHEAT, FLAX AND PEA SEEP IN THE

SOIL

(a) Fungi from Wheat Seed

i$°o*

135>xx>

137x,

1383c I39x» 3iox,

332

XX*

345;

20 °C

■^Jlxx*

“kjpxx*

lk3xx*

A5X,

l46xx.

149;

XX’

264xx»

268 xx^

28^x*

28 8 XX

25°c

175xx>

}^\xx*

192^,

17?xx>

■*-94xx>

17^,

195xx>

l80x,

2®Lcx*

222xx

30°c

159, 172, 240

xx* 2^7

(b) Bacterium from Wheat Seed
20°C 8^xx

20°C F120xx, FX2kxx?/ ^F213rxx* F213gxx* F236rxx*

F236c-xx* P268xw

(d) Fungus from Pea Seed

20°C P27Qxx

x significant seed decay organism
xx highly significant seed decay organism

* temperature of the soil in which seeds were rotted

** Organism P268 was isolated from decaying pea seed, but
was only found capable of significantly reducing the
emergence of flax®

, - - •

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