SOME ASPECTS QP'EHE *DAMPlNG-OFF 0F
BLACK PENE, PiNUS’ MGRA ARJN.

Thais in: 5:213 Degm of M1. 5).
MECHEGALN STAYE JNER’ERSVI'E'Y‘
bemefrios Symecn Kaiiidis
2959

mm

 

 

 

 

 

 

This is to certify that the

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thesis entitled
Some aspects of the dampingqoffi of

black pine, gyms nigra Arn.

 

presented by
Demetrios Symeon Kailidis

has been accepted towards fulfillment
of the requirements for

PhoDo degree in BOtagzy and Plant Pat-helow

Major professor ‘1

 

Date May 22, 1959

 

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SOME ASPECTS OF THE DAMPING—OFF OF
BLACK PINE, PINUS NIGRA ARN.

 

By
Demetrios Symeon Kailidis

AN ABSTRACT

Sulnitted to the School for Advanced Graduate Stacie: of
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of

DOCTOR OF PHILOSOPHY
Department of Botany and Plant Pathology

1959

QM <3 SW7

Demetrios Symeon Kailidis ABSTRACT

The damping-off disease in Michigan nursery soil was studied.
Black pine was used as the main host. The main pathogenic fungi iso-

lated were: Rythium irregulare, E. ultimum, E. debaryanum, Rhizoctonia

 

 

 

solani, Fusarium solani, E. ogysporum, E. moniliforme, and Botgytis

 

cinerea. Among Eythium the most common was 2. irregulare, E. ultimum

 

was less common, and P. debaryanum was the least common. Among Fusaria

 

the most common was F. oiysporum, F. solani was less common, and F.

moniliforme was the least common. Pythium perniciosum was isolated and

 

identified from soil; this report on this species is the second one in
this country. This species was not pathogenic to black pine seedlings.
Eythium grew in cultures better at pH 5.5, and in a temperature
.between 200 and 30° C. Of Rhizoctonia solani four definite strains were
isolated and studied. The different strains showed different pathogen-

icity, and characteristic appearance in cultures. Rhizoctonia solani

 

generally grew faster at pH b.5 to 6.5, and its growth at pH 8.0 was
also satisfactory. This fungus grew also faster between 200 and 300 C.,
and at the lower temperature of 120 C. tested, it grew slower than
Eythium.

Fusaria proved to be "strong" species, since their growth in dif-
ferent environmental conditions was influenced much less than the spec-

ies of the genus Eythium and Rhizoctonia. Several characteristic prop-

 

erties of FUsaria in cultures were also studied. Among other things it
was found that several strains of Fusaria possess the ability to form
zonation under all environments tested, and that at 120 C. constant

temperature all pathogenic Fusaria formed zonation.

Demetrios Symeon Kailidis ABSTRACT
Several of the principal pathogenic damping—off fungi when grown
in mixed cultures were found to possess antibiotic properties of which

the stronger one seems to be possessed by Fusarium solani strain 3.

 

In greenhouse and field isolation during a continuous 12 month
study gythium was more prevalent during winter months in a closed humid

well heated greenhouse, Fusarium was less prevalent, and Rhizoctonia

 

least prevalent. During spring and autumn under a cooler environment

Fusarium was more prevalent, Rhizoctonia less prevalent, and Eythium

 

least prevalent. The same was true for summer in greenhouse and field.
Eythium was not isolated at all in several cases during warm dry periods.
Thus Fusarium was able to dominate the other pathogenic fungi under a
variety of conditions, and it was proved to be the main cause of black
pine damping-off in Michigan.

Germination studies of black pine seed proved that for good germ—
ination a medium moisture is necessary at the beginning, and after a few
days good aeration proved to be the more critical factor. Thus finally
better germination was obtained with the smaller quantity of moisture
tested. In media (soil and plates) at pH h.5 germination of black pine
was twice that at pH 8.5. At constant temperatures two optima of germ-
ination appeared to be at 2&0 and 310 C. Alternating temperatures were
generally favorable for seed germination. Thus under favorable moisture,
pH, and temperature seeds of black pine germinated faster and gave sev-
eral times higher total germination number. Thus in cases of optimum
conditions germinated seeds have more possibilities to escape damping-
off. Black pine seedlings became resistant to disease in 25 to 27 days

after germination.

Demetrios Symeon Kailidis ABSTRACT

During warmer periods the preemergence damping-off was higher than
during cooler periods.

Under a medium soil fertility in which seedlings grew in a medium
way damping-off was lighter, than in sand where seedlings grew faster
but were probably too tender, or in muck soil where seedlings were
heavier in dry organic matter but these seedlings grew too slowly.

In cases in which seedlings of black pine were allowed to grow
under favorable conditions in the field or greenhouse, and these seed-
lings root-rotted, isolations proved that the main pathogenic fungus
was Fusarium. All three species of Eusagia which were able to cause
damping-off, were able also to cause root-rot.

A modification of the older Cholodny and the modified Krinchkova
soil slide technique found by the author was used, and it proved to
possess advantages over the older ones.

In nursery practice soil must be light, slightly acid, and of
medium fertility. Watering after sowing must be medium during the first
days, and quite light later. The time of sowing also must be tested

for each locality and for each species used.

SOME ASPECTS or THE DAMPING-OFF or
BLACK PINE, PINUS NIGRA Ami.

 

By
Demetrios Symeon Kailidis

A THESIS
Submitted to the School for Advanced Graduate Studies of
Michigan State University of Agriculture and
Applied Science in partial fulfillment of

the requirements for the degree of
DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1959

TABLE OF CONTENTS

INTRODUCTION
LITERATURE REVIEW . . . . . . . . . . . . . .
MATERIALS AND METHODS
Plantings and Soils . . . . . . . . . . .
Isolation and Culture of Fungi

Nematode Isolation . . . . . . . . . . . . . . . .

A Modification of Modified Cholodny Soil Slide Technique.

ISOLATION AND IDENTIFICATION OF SPECIES, THEIR IMPORTANCE
AND PATHOGENICITY . . . . . . . . . . . . . . . . . .

OBSERVATIONS ON CULTURAL APPEARANCE, GROWTH .

Eythium .

mlizoctonia O O O O O Q 0 O O O 0 I O I I O O I I O O O

 

Fusarium

Pigmentation of Fusaria . . . . . . . .

Zonation of Fusaria .

Sporodochia .

Nematodes .
SEASONAL DAMPING-OFF OF BLACK PINE .
MODIFIED CHOLODNY SOIL SLIDE TECHNIQUE . . . . . . . . . . .
BLACK PINE SEED<SERMINATION

Black Pine Seed Germination and Moisture .

Black Pine Seed Germination and Temperature .

The Effect of pH on Black Pine Seed Germination .

DAMPING-OFF OF BLACK PINE SEEDLINGS . . . .

Relative Pathogenicity of Different Strains and Species .

Page

12

13

15
22
22
26
28
29
3o
32
32
it
us
b6
A6
A9
52
56
56

TABLE or CONTENTS (Cont.)

Page
Damping-off of Black Pine Seedlings and pH . . . . . . . 60
Black Pine Damping-off and Soil Quality . . . . . . . . . 65

Relationship of Damping-off and Root-rot of Black Pine

Seedlings . . . . . . . . . . . . . . . . . . . 71
DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . 73
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . 85

 

I\I

ACKNOWLEDGMENTS

The author is very grateful to Professors Miriam and Forrest C.
Strong for suggestions and advice given throughout this investigation.
To the above and Drs. D. J. deZeew and E. S. Beneke for assistance
and criticism of this manuscript. To the above and Drs. J. E. Cantlon
and J. W. Butcher for help in author's study in this country.

The author is also very grateful to Dr. E. S. Beneke for help in
identification of Pythium isolates; and to Drs. W. C. Snyder and H. N.
Hansen, Professors at the University of California, who identified the
Fusarium isolates.

Finally the author is very grateful to State Scholarships Founda-
tion of Greece under which scholarship and support these studies in at-

taining this degree were possible.

INTRODUCTION

Damping-off is a universal disease which commonly and regularly
attacks the seedlings of conifer and broad leaved species of trees as
well as most herbaceous plants. This disease occurs in.young tender
seedlings and usually attacks the stem at the soil surface level, al-
though it may affect the cotyledons as well as the entire root system.

In the forest tree nursery this trouble is one of the most import-
ant causes of losses in the seedling population. Severity of the dis-
ease varies from year to year even in the same nursery; in some seasons
losses are negligivle while in other years heavy losses may occur.

Studies concerning the nature and control of damping-off of seed-
lings have been carried on for many years with various crop plants in-
cluding trees. 8

In the work reported here it is the aim of the author to study the
principal fungi which cause damping-off in conifer tree seedlings and

particularly with one species, black pine, Pinus nigra Arnold, to ob—

 

tain pertinent information concerning their habits of growth in rela-
tion to various environmental conditions of the soil, and the plants
which they attack.

This species of pine was chosen because it is one of the most im-
portant species found in south and southeastern Europe where it is used
extensively for reforestation purposes (56).

In Greece after the war an extensive program of reforestation was
started and additional new nurseries were built. Little or no damping-
<3ff occurred in the new nurseries for several years, but, as the years

jpassed damping-off became more and more important.

This study concerns several aspects of the damping-off of black
pine seedlings and the pathogenic organisms which cause it. Naturally
infested soil from the Bogue Nursery, Michigan State University, East
Lansing, Michigan was used mainly in the course of this investigation.
At the Bogue nursery conifer seedlings have suffered for many years
from the damping-off disease (77). Soil from the Chittenden Forest
Service Nursery at Wellston, Michigan was also used.

The causal organisms were identified from the two Michigan nurser-
ies. The relative pathogenicity of each of these organisms, the percent-
age present, and the relationship between them throughout the year was
studied. The possible influence of other soil microflora and micro-
fauna, and particularly the influence of environmental conditions on
damping-off fungi was also studied. Extensive studies were made on
pure cultures of different pathogenic fungi in different environments,
and on the germination of black pine seed under different disease and

environment conditions.

LI TE RATURE REVIEW

The damping-off of forest tree seedlings and several of the causal
fungi were known to European plant pathologists and foresters before
the end of the last century (31). Atkinson (3) was the first to study
the damping-off disease in the United States. Jones (N3) and Spaulding
(75) in 1908 independently published on control measures; both intro-
duced sand as a seed bed.covering; Jones used formalin, and Spaulding
used sulfuric acid for the control of the disease. Hartley (33) in

1910 isolated Rhizoctonia Sp. and Pythium sp. as the causal agents of

 

coniferous damping-off. He began inoculations with these two fungi
and studied their effect against conifers. He found that considerable
damping-off could occur under very dry conditions.

The following year Gifford (21) reported his studies, in which he
found that Fusarium alone was the cause of damping-off. He also stated
that alkaline soil favored the disease. Hartley and Merril (3b) in

191A reported Pythium debaryanum and Fusarium sp. as the causal agents

 

of damping-off of coniferous seedlings and sulfuric acid as the best
control. The same year Spaulding (76) reported that damping-off of
conifers is caused by several widely different fungi and occurs under

a variety of conditions. Graves (2b) in 1915 inoculated healthy pine
seedlings with Cylindrocladium scoparium, but obtained no sign of path-

ogenicity. The same year Hartley and Bruner (35) found that Rhizoctonia

 

was capable of attacking pine seedlings too mature to be killed by

either Pythium debaryanum or Fusarium moniliforme.

 

 

The next important contribution on the damping-off in forest nur-

series was that of Hartley (32) in 1921. He reviewed and discussed the

b
economic significance of damping-off, its importance on conifers, the
relative susceptibility of forest trees, the causal fungi, their viru-
lence and the relation of environmental factors to the disease. In 1923
Hansen et 31 (29) studied several nursery practices in connection with
the damping-off of coniferous seedlings. They concluded that prelimin-
ary soaking of the seeds of white pine and Norway pine did not increase
the rate of germination but increased injury from damping-off. Increases
in depth of cover decreased germination and increased damping-off.
Germination was lower and both damping-off and Chemical injury were
higher in clay than in either peat or sand. Half shade on the seed bed
gave the best results.

Delevoy (1h) in 1926 published his work with coniferous damping-
off in Belgium. Damping-off there was due to Fusarium sp. and Corticium
yagum. He concluded that the best period of sowing was early spring,
and the best covering was sawdust and coarse sand. He worked also on
control methods with different chemicals. A number of articles to which
I refer recorded damping-off counts in various sections of the United
States and the results of using variously applied chemical compounds
(A2, 63, 91, 9S).

Elianson (18) in 1928 showed that of 22 species and strains of
Pythium of different origin all but three caused damping-off in some
Conifer seedlings. However, he obtained negative results with Altern-
Eria and Fusarium. Rathbun-Gravatt (60) in 1931 demonstrated in cul--

tures on agar media that strains of Pythium ultimum and Corticium vagum

 

 

caused more apparent germination loss of certain conifers than any

species of Fusarium tested except 5. sporotrichioides.

 

5
Roth (6b) in 1935 in Switzerland worked with damping—off of Picea
excelsa, utilizing the pathogens Fusarium bulbigenum var. blasticola,

Pythium debaryanum, and Rhizoctonia solani. He found no serious losses

 

at pH values below 5.5. His damping-off fungi were generally most de-
structive at intermediate temperatures. His Fusarium species caused
damage at high temperatures at which Pythium and Rhizoctonia were rel-
atively harmless. Ten Houten (78) in 1939 reported his work on damp-
ing-off of coniferous seedlings in Holland. He found less damping-off
at pH b.5. Early spring sowing gave less damping-off compared with
other periods and he obtained best results when seeds were covered with
coarse sand.

Roth and Riker (65, 66, 67) in 19b3 studied the damping-off of red
pine in Wisconsin nurseries. They succeeded in distinguishing between
the field symptoms of damping—off exhibited by Pythium and Rhizoctonia.
They made careful studies on the temperature and moisture relations of
the two causal agents as they affected red pine. Pythium irregulare was

favored by more moist soil, cool weather and pH 5.2 and above, while

_§hizoctonia solani was encouraged by dry soil, warm weather and a pH

 

5.2 and lower. Their conclusion was that factors favoring one fungus
discouraged the other, thus we are speaking of two different diseases
lflhen we refer to damping-off cauSed by Pythium and Rhizoctonia. They
(Excluded Fusarium from their studies as preliminary tests showed its
Erppearance to be negligible in Wisconsin nursery soil.

Tint (79, 80, 81) in 19h5 used a great number of Fusaria of differ-
euit origin in his study on damping-off of red pine. He found that the
\firulence of the fungi depended to a large extent upon the conditions

14nder which they were tested, and also upon the nature of their

6

nutrition prior to inoculation. The virulent Fusaria generally could
cause relatively equal preemergence and postemergence damping-off. He
found that in liquid cultures where nitrogen was deficient there was
little damping-off, while on the contrary an excess of nitrogen gave
severe damping-off, but with an excess of phOSphorus damping-off was
slight. With an excess of calcium, red pine gave higher losses from
damping-off. As to the pH behavior, he found that postemergence damp-
ing-off of red pine was low in more acid samples, reached a maximum on
the acid side of neutrality, fell off sharply at pH 7.5 in sterilized
soil and at pH 8.1 in unsterilized soil, and then became higher in more
alkaline substrate. Finally while his losses from both preemergence
and postemergence damping-off were higher at higher temperatures, they
were negligible at an average 100 C. using in the last case as host, red

pine and as pathogen, Fusarium oxysporium. Summarizing Tint found that

 

any deficiency or excess of the nutrient, the concentration of the solu—
tion, any unbalanced ratio, the acidity etc. influenced the growth and
health of the plant and pathogen, by influencing the entire metabolic
process of the plant which may decrease the resistance of the seedlings
to physiological and infectious diseases.

Beach (6) in l9h6 in Pennsylvania reported his work on damping-off
‘with several vegetables. He worked with spinach, peas, tomato and beets.
fie found that during January and February, Pythium sp. was more preva-

.lent, Fusarium sp. was less prevalent, and Rhizoctonia sp. was least

 

IDrevalent. On the contrary during May, June and July, Fusarium became

Inome prevalent, Rhizoctonia less prevalent, and Pythium least prevalent.

 

I3e:ach concluded that these results were due to the influence of environ-I’

IRental factors.

7

Cox (1}) in 1951 in Delaware investigated a disease causing losses
in red pine and white pine seed beds. He found that the cause of the

disease was a complex of leindrocladium scoparium, Rhizoctonia solani,

 

I)

and gythium sp.. In Delaware leindrocladium scoparium was found to be

 

the primary causal agent of a serious complex of diseases, including
damping-off, late damping-off, root rot, and seedling blight.

Strong (7?) in 1952 published a work on the control of damping-off
in forest nurseries in Michigan. He found that pathogenic species of

Fusarium, Rhizoctonia and Eythium were present in the nursery soils and

 

in the soils used in the greenhouse.
Vaartaja (82, 83, 8b) and Vaartaja 33 El (85, 86) published in re-
cent years a number of articles on damping—off and its control in Can-

adian forest nurseries. They found that Rhizoctonia solani or Eythium

 

sp. can be inhibited or parasitized by Aspergillus niger, Botrytis

 

 

 

 

cinerea, Penicillium sp., Trichoderma sp. and Gliocladium sp.. In
periods of heavy rains Bythium becomes high in virulence. He and his
colleagues found also that differences in susceptibility to damping-off
existed among tree species and among progenies of different trees. This

suggested genetical difference for resistance to damping—off.

MATERIALS AND METHODS

Plantings and Soils

 

In the first period of this year for the occurrence and behavior
of the different pathogenic to damping-off fungi, a number of flats con-
taining naturally infected soil from the Bogue Nursery were used in
the Plant Science Greenhouse. These flats of soil were regularly sowed
with black pine seed throughout the year. During the summer, sowings
were made out in the Bogue Nursery.

Seed were surface sterilized with l:lOOO solution of mercuric
chloride for 3 minutes, then washed thoroughly with sterile distilled
water (62). At the same time unsterilized seed were used, but these
seed were well washed with tap water before sowing.

Seed of black pine were sowed in flats or in the nursery beds at
a depth of S to 15 mm., usually 7 to 8 mm. (55), and were covered with
the same soil. After covering, the soil surface was gently pressed
down with a flat board. The number of seed sowed per row was always
more than a 100 depending on the germinability of the seed. Seeds were
sowed in sterilized soil in order to determine how many would germin-
ate, then enough seed were sowed so that 100 seedlings per row would.be
€Xpected to emerge. Watering was adjusted carefully to avoid over or
‘under watering the seedbeds.

The type of soil used is given a little later and was characterized

llWith the symbol (F). The pH of the soil was 5.5.
The pH of the soil was determined with an electrode pH meter. Equal
“Naights of soil and distilled water were stirred together in.a small

txaaker and the pH readings made. In order to change the pH 6f the soil

9

0.1 N hydrochloric acid and 0.1 N sodium hydroxide solutions were used
at first. This proved unsatisfactorxjhowever, since the soils adjusted
to the more alkaline ranges of pH developed a hard surface layer of
soil after a few days. This was probably due to the formation of sodium
carbonate. To avoid this condition different amounts and proportions
of 0.1 M phosphoric acid and 0.1 M sodium hydroxide (S) were used to

. u
make the adjustments of the soil pH, and also as buffers for seed germ-
ination tests at different pH levels in the laboratory. When these

chemicals were used in the soil the pH was checked for change after 2

or 3 days.

Isolation and Culture of Fungi

Damped-off seedlings were collected once or twice a week for isola-
tion in the laboratory. The seedlings were washed thoroughly with tap
water and surface sterilized with 70 percent alcohol or l:lOOO dilution
of mercuric chloride for 2 to 3 minutes (62). Next they were thor-
oughly rinsed for l to 2 minutes in distilled water. Finally root and
stem pieces from the diseased areas were planted on a nutrient medium
in Petri plates. Potato dextrose agar (Difco) and malt agar were the
media used (62). It was found necessary to add 2 to 3 drops of a 25
gpercent lactic acid to each plate in order to bring the pH of the medium
ibelow 5.0 and thus prevent bacterial growth.

For the isolation of species of Pythium from the soil a small
(piantity of soil was placed in a Petri dish in which enough sterile
ciistilled water was added to cover the bottom of the plate. Steril—
iizzd.half pieces of flax or hemp seed, or small pieces of boiled carrot

rxoot were then placed into the muddy water in the plate (50). When

lO
Phycomycetes were present, hyphae would become apparent after a few days,
growing out from the surfaces of the seed or carrot root. Small tips
of hyphae were then transferred to the nutrient agar plate for purifica-
tion. 'In several cases it was necessary to transfer hyphal material
from these first plate cultures to a maltose-peptone agar. In this
medium Pythium grew faster and could be separated from mixtures of dif-
ferent fungi, especially in mixture with Fusarium.

Another technique used in purification process was to examine
for isolated hyphal tips under low power of the microscope (lOOX) and
cut them out, together with an agar plug, with a small capillary tube.
These plugs were then transferred to a new plate (9, 62). In other
cases, using similar technique, small bits of mycelial mats were trans-
ferred aseptically to a drop of water on a microscope slide. This ma-
terial was teased apart until a single sporangium could be transferred
onto one needle. This was then streaked over the surface of the agar
in a new plate where isolated colonies started to grow after 2 or 3 days
along the streaks. From these a hyphal tips were cut and transferred
to new plates, and thus pure cultures of Pythium Species were obtained.
The same techniques were used to obtain pure cultures of Pythium species
directly from soil and from damped—off seedlings.

Several media were used for the growth and study of Pythium; po—
tato dextrose agar, corn meal agar, oatmeal agar, lima bean agar, water
agar, sterile distilled water only, and boiled halvesof hemp seed and
carrot.

Eythium isolates were grown in all of these media, but for each
species the growth, aerial mycelia, and the time and number of sporangia

and oospores formed differed considerably. Water cultures with hemp

ll
seed and carrot were used more extensively than the other media in these
studies. The cultures in the carrot and water medium induted earlier
and more abundant oospore formation than was obtained in the hemp seed
water medium.

Single spore isolation was used for Fusarium isolations (62). For

Rhizoctonia solani purification was obtained by using hyphal tip cut-.

 

tings.

The fungi used to infest soil were grown on a mixture of vermicu-
lite and cornmeal medium in Petri plates. This medium was about one
half the depth of the plate and it was pressed down a little with a
spatula to prevent the scattering of the particles when autoclaving.

In this way contaminants could be avoided more successfully during
growth of the pathogen. After suitable growth had developed in these
plates measured amounts were used as inoculum to infest the soil.

The soil samples were autoclaved after placement in the pots or in
the flats for one to one and one half hours at 15 pounds pressure.
After removal from the autoclave they were allowed to stand for about
one week in order to avoid possible undesirable influences resulting
from the soil sterilization which might affect seed germination and
growth, or affect the fungus inoculum (h8, 70). The inoculum was then
mixed with the soil in the pots or flats, and the seeds were sown.

The pots and flats were watered regularly to prevent undue drying.
For a period immediately after sowing a plastic sheeting cover on a
'wooden frame standing one and a half feet above the soil was used.

Adjustment of the humus content of the soil was made by the method
described by Piper (58) while the method for the mechanical analysis of

the soil was that described by Bouyoucos (7).

12

The percentage content of each soil type is given below:

Humus percent Sand percent Silt percent Clay percent
Sand (SA) O 100 O 0
Soil (D) 2.0b 89.0 2.98 5.98
Soil (E) 2.61 89.b3 1.99 5.97
Soil (F) 3.3b 86.00 b.00 6.66
Muck (M) b6.60 -- _- __
Clay 1.25 70.75 9.00 19.00

No mechanical anaLysis of sand was made since it was considered to

consist solely of Sand without humus, silt or clay material. Only the

humus content was measured in the muck soil.

SA.

SP.

The soils used were collected from the following places:
Sand collected from North, Michigan.

Sand like the above. In this series pots were watered with a
synthetic nutrient solution for fungi.

Sand like above. In this series sand was mixed with 10 percent
peat used in Plant Science Greenhouse.

Soil collected from Woodlot Chittenden Nursery, Wellston, Michigan
Soil collected from seedbeds Chittenden Nursery, Wellston, Michigan'

Bogue, University Forestry Nursery, Michigan State University,
East Lansing, Michigan

1
Muck soil, used in Plant Science Greenhouse, Michigan State Uni-
versity, East Lansing, Michigan.

Clay soil, used in Plant Science Greenhouse, Michigan State Uni-
versity, East Lansing, Michigan.

Rematode Isolation

 

For the study of nematodes which were present in diseased damping-

<3ff seedlings and in soil the funnel technique was used (23). Nematodes

1}
isolated from sections of stems and roots of diseased seedlings placed
in Petri plates were studied. These cultures having nematodes were put
in a blender and using the funnel technique nematodes were isolated.
Study of nematodes was also accomplished by the modified contact slide
technique which was described by the author, and the older one of

Cholodny (11) and Kzinchkova (9b).

A Modification of Modified Cholodny Soil Slide Technique
For the study of soil microorganisms (microflora and microfauna)
several techniques were used. One of them was the dilution plate tech-
nique. The other was the Cholodny (11) contact slide technique. It
was found that this work was slow and exact identifications were not
easy to do with soil fungi, since many of them grew on the slide as
mycelium. Krinchkova (9b) and Wright (9b) used a modification of the
Cholodny technique. Sterilized glass micro-slides were placed in
sterile petri dishes and coated with malt agar; after hardening of the
agar aseptically the slides were dried in an oven at LEO to 500C..
These slides with the dried agar on them were put in the soil of flats
in the greenhouse or in nursery beds where the damping-off study was
(done. A spatula was used to open a hole and make a flattened smooth
:surface; on that smooth surface the clean surface of the glass slide
unis leaned and the soil was pushed slowly onto the agar surface. The
Eilides were left in the soil from 2 to h days. After that the slides
I'~T~13.‘l“e:rem'oved carefully dried, stained and examined. This technique
Enas; an improvement of the older one of Cholodny (11). Because the
Slides with agar were placed in the soil for only 2 to h days and then

‘VGBIIZ dried, soil fungi etc. did not have time to establish themselves

lb

and produce the characteristic fruiting bodies necessary for identifi-
cation. The slides were covered with septate and non—septate hyphae.
Longer periods in the soil merely allowed the agar to dissolve in the
soil moisture.

The author's improvement of the above method was as follows:
After the removal of agar slides from soil, they were placed in steril-
ized Petri plates. Small pieces (2 X 2 mm) of the overgrown agar were
transferred to S X 5 mm blocks of malt or potato dextrose agar on
sterilized slides and this covered with a sterilized cover glass. The
mount was pressed a little and put on a small piece of mesh wire in a
sterilized Petri dish moist—chamber. After a few days abundant mycelia
and fruiting bodies were formed and identification became easier and

more accurate.

ISOLATION AND IDENTIFICATION OF SPECIES,
THEIR IMPORTANCE AND PATHOGENICITY
In the course of this study several fungi and other micro-
organisms were isolated, identified and studied. Isolation of a number
of some of the fungi were isolated from damped-off seedlings in Bogue

Nursery, and in flats of soil from the Bogue and Chittenden Nurseries.

The Genus Pythium

With the techniques which have already been described, the follow-
ing Pythium species were isolated, purified, identified and studied,
mainly from damped-off seedlings of black pine, and from soil. The
identifications were based on the following characteristics given by

several authorities.

Pythium irregulare Buisman: This species is a serious cause of

 

damping-off of coniferous seedlings (65, 66, 67). It was isolated
many times in the course of this study from damped-off seedlings and
from soil. Pure cultures, which were isolated from affected seedlings
sand from soil, proved highly pathogenic to black pine seedlings when
'they were grown in artificially infested soil.

The physical characteristics of P. irregulare are (50, 51):

Ptyphae: diameter 2-7 mu, branching freely.

Ehborangia: 10-30 mu in diameter, spherical to pyriform terminal or
intercalary. From sporangia 2005pores formed in a vesicle,
or they germinate as conidia.

ChDggonia: varying in shape, smooth or irregularly echinulate. The
echinulate oogOnia were less than 1 percent of the total.
Diameter of oogonia 2h-26 mu without projections, terminal

0

or intercalary, sessile or stalked.

l6
Oospores: Smooth aplerotic 10-20 mu in diameter.
Antheridia: Mainly monoclinous, sometimes diclinous, usually 1 to 2

per oogonium. Wall thickness about 1.5 mu.

Pythium debaryanum Hesse: This fungus is one of the most wide-

 

Spread of the Pythium species causing damping-off in coniferous seed-
lings (32, 6h). The fungus was isolated from damped-off seedlings and
from soil. Isolates from both origins proved highly pathogenic to black
pine seedlings.

Characteristics of the fungus (16, 32, 50, 51).

Hyphae: varying 3 to 7 mu in diameter.

Sporangia: 15-25 mu in diameter, spherical to oval, terminal or inter-
calary. Germination by zoospores, or as conidia with germ
tubes.

Oogonia: Oogonia were very difficult to find. This is also reported
by other workers (32). From over one hundred plates with
different media and age only a few oogonia were seen in
water agar cultures up to one and one half months old. In
water cultures with hemp seed and carrot in which the other
Pythium species studied formed abundant oogonia and oospores,
this Species produced no oogonia at all.

Oogonia 15-25 mu in diameter, Smooth spherical terminal or
intercalary.

C3C>Spores: 12-20 mu in diamter, aplerotic, smooth, wall thinner, about
l mu. Germination by germ tube.

Aditflderidia: Monoclinous and diclinous. One to as many as 6 per oogonium,

when monoclinous arising some distance below the oogonium.

17

Pythium ultimum Trow: This species was isolated from damped-off

 

seedlings of black pine and from soil.. It is the most common of the
Pythium species causing damping-off in the United States and is wide-
spread all over the world; Middleton (51) gives a long list of plants
and places throughout the world in which this fungus has occurred.
Pythium debaryanum and Pythium ultimum are quite similar in their char-
acteristics. Drechsler (16) considered and distinguished them as sep-
arate species.

Characteristics of the fungus (16, 50, 51).

Hyphae: 1.7-6.5 mu in diameter, long and branched.
Sporangia: 12-28 mu in diameter, spherical and terminal; or in some
cases intercalary barrel-shaped lb-l? mu, to 22.9 by 27.8 mu.
Sporangia act as conidia, germination only by a germ tube.
Oogonia: 19.6-22.9 mu in diameter, smooth usually terminal spherical.
OOspores: lh.7-18.3 mu in diameter, aplerotic, single spherical. Wall
thickness 1.5 mu. Germination directly by germ tube.
.Antheridia: usually 1 per oogonium, monoclinous, arising just below
the oogonium.

E. irregulare, P. debaryanum, and P. ultimum were isolated from
130th damped-off seedlings and from soil isolations in nursery beds and
ggreenhouse flats throughout the year. Pythium irregulare appeared to be
tide most common and probably the most highly pathogenic. Pythium ulti-

EEEEE was found second most prevalent, and Pythium debaryanum was third
ifIprevalence. Pathogenicity of these species was proved by many iso-
léitLions from black pine and scotch pine seedlings grown in sterilized

Soil which had been infested with these fungi.

 

 

 

 

 

 

 

 

 

 

Ir“
..._. -
.
n-r’;
v
....v

n
J';
.1._
.—
a
u.
.,.N
‘ 1
-~..
,..
.,_,
u
o
5‘

.1

18
In addition to the above, another Pythium species was isolated dur-
ing this study. However, it was proved to be non-pathogenic to black

pine seedlings.

Pythium perniciosum Serbinow: This species was first described in

 

Russia by Serbinow in 1912 as a parasite of tobacco seedlings and sever-
al other plants (50, 51). Drechsler (l7) doubted the existence of this
species, and thought that Serbinow had not purified his culture. Gil-
man (22) did not refer to this species, but Middleton (51) described

its isolation from rotted Euphorbia pulcherima plant.

 

 

The author isolated P. perniciosum from soil, and studied it in
pure culture. It grows well in water cultures on hemp seed and carrot,
where it produces abundant mycelium, sporangia, oogonia, and, in less
numbers, catenulate spherical conidia, 3-5 in a chain. It grows and

reproduced well in a number of the standard media, used for growing

cultures of Pythium.

Hyphae: 5-9.5 mu in diameter.

ESporangia: filamentous-like hyphae or arising from sac-like structure
of the hyphae, zoospores produced in a vesicle. Conidia-
like catenulate bodies 3-5 in number in a series germinat-
ing directly by germ tubes.

Cflbgonia: 18.9-50.0 mu in diameter, smooth, spherical, terminal on

A short mycelia branches.
OOspores: l8.1-J2.5 mu in diameter, aplerotic single, Serbinow (50,
51), and Middleton (51) state that germination is unknown.
In water cultures in hemp seed after 15 months in room en-
vironment the author observed germination. This oospore

germination was typically by a germ tube.

l9

Antheridia: l to 2, usually one, per oogonium monoclinous or diclinous.

The Genus Fusarium.

The genus Fusarium is probably one of the most important in caus-

ing damping-off and root rot in coniferous seedlings and other crops as

well, all over the world (12, 32, 6h, 79, 80, 81, 85, 9}). A large

number of Fusarium cultures were isolated from damped-off and root rot-

ted seedlings of black pine. No soil isolations were used, because of

the large number of species and strains, the parasitic and saprophytic
nature of this genus, and the difficulties of species identification.
Through single spore isolation (62) pure cultures of many were obtained.

The following species were identified from the two nurseries, Bogue

and Chittenden.
Fusarium solani (Mart.) Snyder and Hansen. /
Fusarium oxysporum (Schl.) Snyder and Hansen.

Fusarium moniliforme (Sheld.) Snyder and Hansen.

The Genus Rhizoctonia.

Rhizoctonia solani Kuhn is also a fungus which causes severe losses

 

to coniferous seedlings and other crops, causing both damping-off and
rwoot rot (57, 6b, 65, 66, 85). Many isolations of Rhizoctonia solani

unere made. After careful observation and study four distinct strains

(31‘ the fungus were established on their pathogenicity, cultural appear-
ance, growth and behavior (15, 116, 119).

Iglegpctonia solani strain N=l, isolated from damped-off seedlings.
.Ezligggctonia solani strain N=2, isolated from damped-off seedlings.

-5:LEJESEE2212.§91321 Strain N=b, isolated from damped-off seedlings.

Egligzggtonia solani strain N=87, isolated from root-rot seedlings.

20

Other Genera

Botrytis cinerea Persoon, was isolated from damped-off seedlings

 

and from soil. It is of much less importance in causing damping-off

of pine than the above three genera.

A number of other fungi and microorganisms were also isolated
throughout the course of this study. These isolations were obtained

from soil, through the modified contact soil slide technique or by

dilution plates, and from plates in which diseased tissues of damped-

off seedlings were planted.
Saprophytes contaminants or secondary parasites isolated from dis-

eased seedlings and identified as to species or to genera were as follows:

Mucor sp., from seedlings and soil.
Mucor hiemalis, from seedlings and soil.
Rhizopus nigricans, from seedlings and soil.

Alternaria sp., from seedlings and soil.

 

Aspergillus sp., from seedlings and soil.

.Penicillium sp., from seedlings and soil.

 

jgygorhynchus sp., from soil.

Sieotrichum sp., from soil.

Bremia sp., from soil.

Eligrospora sp., from soil.

_§flptryotrichum sp., from soil.

Tkartua.sp., from seedlings and soil.

fisftinomyces sp., from soil.
§i§fi§§ptomyces sp., from soil.
33Eicrteria, different species, from seedlings and soil.
Ekanuatodes, different species, from seedlings and soil.

11512112222, different species, from soil.

21

These fungi which were isolated from both soil and from diseased
plant tissues are generally considered to be saprophytes. In several
cases various workers (82, 85, 98) have considered them as possible
true parasites, however, although a number of organisms other than the
accepted damping-off parasites were found growing on the tissue plant-
ing plates it is probable that they were secondary. In some cases the
pathogens may have been killed, when decayed tissues absorbed too much
surface sterilizing chemicals, and thus allowed the appearance of the
saprophytic fungi or bacteria more resistant to the surface steriliza-
tion. In the favorable environment of the Petri plates bacteria or
saprophytes multiplied abundantly and often covered the surviving path-
ogens.

The author tested one of the so-called saprophytes, Rhizopus
nigricans, by infesting sterile soil in three pots. The pots kept well
watered and covered with cellophane bags. Surface sterilized black
pine seed were sowed in these pots. In a few days the pots were covered
‘with dense mycelium of this fungus. Complete killing of the seedlings
(accurred, mainly as postemergence damping-off. In natural environments,
luowever, these fungi must be considered saprophytes and only in condi-

tLions very favorable for their development as weak parasites.

’A

D
«\s

 

 

OBSERVATIONS ON CULTURAL APPEARANCE, GROWTH

The fungi isolated during the course of this work were studied as
to their growth at different pH levels and temperatures. The behavior
of these fungi in different artifical environmental conditions is im-
portant because it explains, in part, their differences in frequency and

severity in natural environments.

Bythium.

It can be seen from inspection of the data in Tables I and II that
the optimum temperatures for growth of the genus Pythium at a pH of 5.5
in potato dextrose agar are between 20 and 30° C. Roth and Riker (66)

found that the optimum for Pythium irregulare, a Wisconsin strain, was

 

280 c. Middleton (51) found that in corn meal agar at pH 6 the best

average growth in length over a 2b-hour period for P. debaryanum was at

 

28° C., for P. perniciosum at 31° C., and for P. ultimum at 28° C.. The

 

different Pythium species generally produced their best growth between
20° and 30° C.. After further studies of their growth habits at lower

temperature of 12° C. a great difference was noted; P. debaryanum

 

started to grow by the second day, 2. irregulare by the third day but

 

.E. perniciosum made no growth at all.

 

According to several investigators (36, 6b, 66) working with pH
relationships in the genus Pythium the minimum growth occurred between
ij 3.1 and h.6 and pH 7.2 to 9.6 while maximum optimum growth took
‘pflace between pH 3.5 to 8.3. In.the present work species of Pythium
.from Michigan were isolated and tested on potato dextrose agar at var-
‘ious pH levels adjusted with phosphate buffers. The cultures were grown

Eat an optimum constant temperature of 2b° C.

23

Table 1. Growth of various fungi at different pH levels, in an incubator
in the dark at 2b° C.

 

Average growth of colonies in radius in mm., on

 

 

 

 

 

 

 

 

 

Species pH , potato dextrose agar.
1 day 2 days 3 days b days 5 days 6 days 7 days
Pythium 8.5 17 26 39
debaryanum 5.5 20 h5 A5
6.5 12 26 b2
8.0 O O O
Pythium b.5 19 kt
irregulare 5.5 17 b5
6.5 15 39
8.0 11 26 lb
Pythium 8.5 23 AS
ultimum 5.5 18 h5
6.5 13 26‘ 80
8.0 2 8 12 18
Pythium 8.5 20 h5
perniciosum 5.5 23 h5
6.5 18 37
8.0 11 32 AS
Rhizoctonia h.5 15 35 b5+
solani No. l 5.5 13 3h b5+
"“"‘ 6.5 15 33 us
8.0 8 l8 hO
Rhizoctonia 8.5 8 18 25 31 36 h3
solani No. A 5.5 11 3O bb
6.5 l 5 18 25 3b hS
8.0 O l 8 15 21 30
Rhizoctonia 8.5 23 b5
solani No. 87 5.5 lb h5
""“' . 6.5 0 12 26 83
8.0 O O O O
Fusarium h.5 6 10 15 22 28 3S bl
solani No. 3 5.5 5 10 16 22 28 36 h2
6-5 6 10 17 23 30 37 83
8.0 h 10 16 21 27 33 39
Fusarium b.5 8 lb 20 28 3b bb
solani No. 25 5.5 6 13 20 28 36 DD
6.5 8 lb 23 30 b0 AS
8.0 6 12 2O 27 3h 82
Fusarium b.5 7 1h 20 27 3b b2
moniliformeNo.2b 5.5 b 10 18 26 3h b2 b5
6.5 8 15 23 31 bl AS
8.0 h 12 18 26 33 bl
Fusarium 8.5 5 10 17 26 32 hi
0 s orum No. 15 5.5 6 13 2O 29 36 bb
6.5 8 lb 22 30 39 h5+
8.0 6 12 18 25 32 to L3

 

 

 

 

 

 

 

 

m m s o m a a m n m m a 0 pm
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ma mm as cm

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as d s m a m m a o o o o o 6 NH masopoonasm
. m: an ma 5m
+mn an ma 0n
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ms ma as cm

m4 an an mm ma NH 0 ea H .62 Hamfiom

ma «a ma 6H s o m n N a a. o o 0 ma sacchuoumem
o o o o o c an
m4 0m 0m
m: «N am

ms Na a ma MM Ma oN a

N @H 0.... Edmo o CLO

o o o o o o o o S 523%
o o o o o 0 an
m: cm ma cm
ms NH am
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m4 mm @H m 0H Sacmxumbmo.

m: ma am am ma m m 0 NH aswnoxm
.06

ha ma NH as ea d w s e m 4 n m a seems
mama uDoCH momoodm

 

rm,cbws nmmm omOLSxmo cameo. CH as aw n soumlmsfipmm

\

m.m ma om ummm omOhbxop oomuoq co xsmo cm mopsbmuoaeob Scopommfio ca Hmcsm msomhm> mo cpsopo .HH m4m<H

 

oN.

2

mm

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~\:}i3g}c>c>~4~NUNUNCDCDr4—jxormCDCDcv—d—SEBCDCDr443()U\C>CDHUUNHqH3CD

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cm
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Edomob
EDHHmmam

 

mH .oz EstoMmNmo

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am.oz oELowHHHcoE
EdHhmmzm

 

 

mm .02 HcmHom
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fl .oz HcmHom
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Nw .oz HcmHOm
mHCOHuouHcm

26
Optimum growth of the Pythium species took place between pH b.5 and

6.5 with the best growth at pH 5.5. At pH 8.0 Pythium debaryanum failed

 

to grow, but the other three species grew satisfactorily (Table I).

Rhizoctonia.

 

Rhizoctonia solani Kfihn is a species found all over the world,

 

which can cause damping-off to coniferous seedlings (1b, 20, 32, 39,
6b, 65), and is pathogenic on many other hosts (15, b5, 57). Strains
of this fungus are known to have a wide range of pathogenicity on the
many different hosts (l3, 15, 32, b5, 57, 65). There are many strains,
and these strains have different cultural appearances and growth rates
in different media and environmental conditions (15, b6, 57).

Growth studies at different pH and temperatures were made of 5.
321321 cultures No. 1, 2, b, and 87 (Tables I, II). Potato dextrose
agar was used as culture medium. Small pieces, one-half to one centi-
meter in diameter, of inoculum were placed in the middle of each Petri
dish. Growth measurements recorded were the average of the largest
and smallest diameter of each colony.

The general appearance of the strains isolated remained constant
on the same medium at different pH levels and at room temperature; on
same medium, same pH and at different temperatures; and at same pH, same
temperature but different media.

The characteristics in appearance of the strains isolated are shown
in Table III.

From an examination of Table II it is seen that the four different
strains of Rhizoctonia solani differ in growth rates when grown on the

same medium at the same pH of 5.5 but at different temperatures. These

27

TABLE III. Cultural characteristics of strains of Rhizoctonia solani

 

 

 

 

One dayiold culture Seven day old culture
Strains Mycelium Concentric Mycelium Concentric
‘ appearance growth Sclerotia appearance growth Sclerotia
~ in agar rings in agar rings
plate plate
No. 1 white none none white none present
thin thin
No. 2 white none none' white present none
thick heavy
thick
No. b white to none none white to present none
brown brown
thick thick
No. 87 white none none white none none
quite " quite '
thin. thin

 

results corroborate the findings of other owrkers (15, b6, 57). Rhizoc-

tonia solani strain 87 grew rapidly at 16° C while strains 1 and b grew

 

less rapidly than the Pythium species. At 12° C Rhizoctonia strain 87

 

grew rapidly but strains 1 and b produced quite small colonies and the
total growth was less than the two pathogenic gythium species, B. debary-

anum and P. irregulare. (This shows that the growth of strains 1 and b

 

of Rhizoctonia is negligible at temperatures lower than 12° C as compared

 

'with the pathogenic species of Pythium.)

Rhizoctonia solani, a species which has a large number of races,

 

is expected to have a wide response to its environmental conditions,

It has been shown that the lower limits of growth are between pH 2.5 to
3.8, Optimum growth between pH b.5 to 7.0, and its upper limits of
growth from pH 6.7 to 9.1 (b6, 6b, 66). In our studies of the strains

<Df Rhizoctonia solani (Table I) strain No. l grew well from pH b.5 to

 

Ffli 8.0. The strain No. b showed a wider variation, best growth at pH

28
5.5, less at pH b.5 and 6.5, and still less at pH 8.0. The strain N0.
87 grew fastest at pH b.5 and 5.5, less at pH 6.5, with no growth at all
at pH 8.0.

The pathogenic ability of the different Rhizoctonia solani strains

 

is shown in Table XIV.

Fusarium.

Fusarium sp. are reported frommany countries as causing damping-
off and root-rot of Coniferous seedlings (1b, 32, 6b, 79, 80, 81, 82,
85), and other hosts (12, 7b, 93).

For the growth rate study of Fusaria, different species and strains,
were grown on potato dextrose agar at different pH levels at 2b° C and
in the dark (Table I). The differences found between the species and
strains of this genus were small and not significant under the condi-
tions of the experiment. This indicates that these species of Fusarium
studied, at least, should be considered stable culturally as to pH
effects. -

Temperature, on the contrary, influences the growth rate of Fusaria
considerably (Table II). The same medium, potato dextrose agar at pH
5.5 and same technique of measuring and recording was used as with

Eythium and Rhizoctonia. The Optimum growth temperatures for the

 

Fusaria studied were between 20° to 30° C., with smaller differences
among the species and strains. At 12° C. Fusarium solani strain No. 3

showed a small total growth, about equivalent to Rhizoctonia solani

 

strains No. l, and No. b. The difference being, however, that the
Fusarium had a small and constant rate of growth from the first day of

jplanting of the inoculum on the medium while Rhizoctonia did not begin

29
to grow for several days. The growth of the other Fusaria at 12° C. was

faster than that of Fusarium solani strain No. 3, but much'less than

 

that of the Bythiums at the same temperature. Fusarium roseuml, did

 

not grow at 37° C. and other species of Fusarium grew very slowly at
this high temperature although there was generally some growth at 37° C.
with the Fusaria studied it was about the same as or less than that of

Rhizoctonia solani strains No. l and No._b. This was in contrast to

 

Pythium which did not grow at all.

Numerous observations on cultural characteristics, of a number of
Fusaria isolations were made when grown on different media at pH levels
and under other environmental conditions. Differences in virulence are
shown in Table XIV.' All of the species were isolated from both damped-
off and root-rotted specimens of black pine seedlings. It was found
that all the species isolated from diseased seedlings can cause both.

damping—off and root-rot on black pine. Fusarium oxysporum was the most

 

common of a large number of isolations made during this study. The

other two species, F. solani, and E. moniliforme, were much less abund-

 

ant and of about equal frequenqy.
All of the fungus cultures used in this eXperiment all originated

from single spore isolations.

Pigmentation of Fusaria.

 

A large number of cultures were studied in replicate. They were
grown on potato dextrose agar and malt agar at pH 5. 5. The plates,
after planting, were held in three different environments, namely: at

room temperature about 21° C. in the dark (covered with Kraft paper); at
room temperature under diurnal light conditions (Not covered.with Kraft

 

1F. roseum was not isolated from diseased seedlings in any study, but
it was given to me by Mr. E. H. Pepper, and it is really F. roseum f.
cerealis (Cke) Snyder and Hansen (73).

30
paper); and in an incubator at 25° C. in the dark. Pigmentation was
generally more abundant in the absence of light or in reduced light
than when the cultures were grown under diurnal light conditions. This
is in agreement with the findings of other investigators (72, 96). Pig-
mentation varied with the medium used as has been reported by Brown and

Horne (10).

Zonation of Fusaria.

 

Previous workers (72, 96) have reported that light, especially
fluctuations in light, as well as temperature influence variations in
growth to produce zonation in the Fusaria. In the course of this study
several species and strains of Fusarium were grown on potato dextrose
agar at different pH levels and in incubatorsset at different tempera-
tures and without light. The results are shown in Tables IV and V.

TABLE IV. Zonation of Fusarium cultures in potato dextrose agar at
‘ different pH, in incubator, in dark, at 25° C.

W

 

 

 

 

pH F. solani 1:“. M _F_. moniliforme F. oxysporium
No. 3 No. 19
11.5 +3 + -b _
5.5 + - . . _ _
6,5 + + ’_ -
8.0 + + _ _

 

a+ zonation formed, b- no zonation.

F. solani (strains 3 and 19) formed zonation in potato dextrose

agar in the dark at 25° C. at all pH levels tested. The F. moniliforme

 

and F. oxysporium, under these conditions did not form zones. Thus

 

diurnal light or fluctuating temperature (72, 96) are not the only

31
factors which cause zonation in Fusarium. From the results it seems
that several species and strains possess the ability to form zones when
grown without light at certain temperatures as well as when grown in
diurnal light or at fluctuating temperatures. Different levels of pH
had no effect on zonation of F. £21321.

TABLE V. Zonation of Fusarium cultures in potato dextrose agar pH 5.5,
at different temperatures in dark.

 

 

 

 

 

Temp. F. solani F. solani F. F. F. roseum
°C. -'No. 3 ‘_ NET—25_ monfliforme oxysporium '_ '____—-
12 +a + + + —b
16 + _ _ - _

20 + _ - _ _
21, + - _ _ _
30 + - _ _ _
37 + . - - - -

 

a . b
+ means zonation formed, - means no zonation.

.E' solani strain No. 3 was the only one (Table V) forming zones on
potato dextrose agar at pH 5.5 in the dark at all temperatures tested

(12°-37° C.). This strain obviously represents a group possessing the

ability to form zonation under widely differing temperatures. From the' ‘

data in Tables IV and V it seems that temperature is a more important
factor in zonation than.pH, since with different temperatures we have
more variations than with different pH levels within the same species.

If. moniliforme anle. oxysporium formed zonation at 12° C. constant

 

 

Uzmperature, but no zonation at any other temperatures. ‘5. roseum

@IVe negative results in all temperatures tested.

32
Sporodochia.

Sporodochium formation was also studied. According to the litera-
ture (30, 72, 96) sporodochia are generally formed more abundantly in
light than in dark. At room temperature in potato dextrose agar pH
5.5 Fusarium species produced sporodochia more abundantly under the
influence of diurnal light, than those placed in the dark at room temp-
erature. At the same temperature on malt agar at pH 5.5 sporodochia
were produced in larger numbers under the influence of diurnal light
than in darkness. Some were also formed in the dark on malt agar.
These results, thus, generally confirm the reports of others on the
conditions influencing sporodochialproduction (10).

Many other studies on factors affecting variations in Fusarium
have been reported (10, 30, 52, 53, 5h, 71, 72, 96). These variations
were not included in this study but a few other observations were made.
F. oxysporum strain No. 15, for instance, in several cases under differ-

ent environmental conditions showed a turning of hyphal tips at about

b5 degrees from left to right.

'

Nematodes.

Nematodes were isolated from damped—off and root-rotted seedlings
in the Petri plate plantings of diseased stem and root tissues several
times in the course of this study. Nematodes have often been referred
tc>as causing diseases in plants. In coniferous seedlings they have
been.reported to cause disease, reduction in yield, poor quality plants,
and.that they are in a complex association with damping-off and root-rot
disease in several nurseries (19, 28, 37, 38, b1, 92).

During a year’s study of damping-off of black pine seedlings, and

from.several thousand isolations the presence of nematodes in the

33
damped-off seedlings did not appear to be significant. Table VI shows
the number of seedlings from which nematodes were isolated, and the
period of the year when it occurred. Nematodes isolated from pieces of
diseased tissues in plates, from soil, and by the contact slide technique,
revealed that the most abundant nematodes were species of the Family

Rhabditidae which are saprozooans. Another nematode which is parasitic

 

was isolated from diseased seedlings but in less numbers. The presence
of the characteristic stylet was observed in this nematode indicating
that it was a parasitic. However, it did not possess a vascular medium
esophageal bulb. No identification was made of any of these nematodes.
In Table VI the time of year and the number of nematodes recovered
is shown. Isolations from diseased seedlings were made from May, to
October. The number of nematodes recovered from diseased seedlings was
quite small. Thus in this case at Bogue Nursery nematodes seemed not
to play any important role in the appearance and severity of damping-

off.

SEASONAL DAMPING-OFF OF BLACK PINE

At the Bogue Nursery at Michigan State University, East Lansing,
Michigan, damping-off disease has been common since its establishment.

Strong (77) in 1952 published a report on control studies in this nursery.

Isolation studies.
Isolation from damped-off seedlings of black pine were made peri-
odically by the technique described previously. Table VI shows the

results of the isolations throughout the period of one year. In this

Table the main pathogenic fungi isolated are listed individually. Mis-
cellaneous fungi include many different fungi which normally are con-
sidered to be saprophytes or molds: Mucor, Aspergillus, Penicillium,
Alternaria, Geotrichum, Rhizopus and others.

At the same time studies of the soil microflora and to some extent
soil nlicrofauna were made. The dilution plate method was used. The
contact slide technique of Cholodny (11) as modified by Krinchova (9b)

sand the author was also used extensively. This study was repeated

:several times throughout the year. At different periods the following

1Fungi and microorganisms were isolated: different species of bacteria,
ill colonies or as single bacteria; Streptomyces sp., Pythium sp.,

E3c3tgztis sp., Rhizoctonia solani, Bremia sp., Fusarium sp., Nigrospora

 

31)., M3223 sp., Penicillium sp., Rhizopus nigricans, Actinomyces sp.,
m1; 119115323, W sp., Aspergillus sp., Geotrichum sp., m-
tIfiichum sp., many non-sporulating colonies both with septate and non-
seFVtate, hyaline and colored hyphae which could not be identified.
Nematodes, adults, larvae and eggs and different protozoa were also

found on the agar surface on the glass slides.

35

 

 

 

- 6H NH 1 N NH NH NH NH NH no eaHa NemHN emsoeeeeso teeasoeo
1 NH HH N mm mm mH m NA mm ocHQ HomHm mmsoccmoto topso>oz
N H m u Hm mm HH mm mm ocHQ HomHm omsoncmmbo Honopoo
- m NH - HN NN - HH NN NN esHa NemHN emaoecssao amassedem
N - - N «a Nm - NN mm mm seHd sumHN eHeHN .Ns<-NHsN
H u u 1 mm 00 m m mm 00 och HomHm mmsoncooto .m:<INHsh
NH - H N N Hm - NH oN NN meHa NumHN sHsHN mess
HH m N u m ON 1 m an Hm mcHQ HomHm mmzoncoobo ocsfi
NH N - - HN ON N N NN NN ecHd NudHN smsoeeeeto Nd:
- - N - NH NN N N NN cm meHa HumHN smaoeeeeao HHsda
- N N - NH NH 06 NH He NN seHd seesaw emsoecseao Hose:
1 N o u m HH mo NH Ho Hm mcHQ HomHm omsoHCNNHO poem:
- NH NN - NH NH NH NH HN Ha moseam .toz essenceeao .Hea-.ems
- N N - HH oN Nm mH oN mm eeHa Hoeoom essenceseo .Hea-.emn
- N NH H HH NH NH HH NN Nm seHd NemHN smashesseo ”Hem-.ems
N H
NMMMMMH Human N mmmmflmm. HMMWMM N R N R N
woOHmsmz HomHz mHHopomm mmmummmm.uoouHHm EdHHmmsm schuzm Nfiwam: .mbwwowmom .twmowmm mmwwwmm NNSWMMWMHO wzpcoz

 

mmcHHooom commomHo anm oonHomH machmmno

 

 

 

 

.\

Ill
lll

.mcpoHpcoo oHoHH ocm omsoccoebm boos: .
amok m Ho poHHoQ w udonmsoecb mammocuma Ho successooo paw mcHQ nouoom pcm HomHm mo HmonmcHdsmo .H> MHmHN

36

Table VI shows the frequency of appearance of the principal patho-
genic genera causing damping-off of black pine throughout the year.

Pythium was more prevalent as the cause of damping-off, and domin-
ated the other pathogenic species during warm and moist conditions of
the heated greenhouse during the winter months. During this period the
greenhousejizated and close, thus moisture was high, no evaporation of
water from flats and pots where seedlings were grown took place. Thus
this period must be considered the period when in flats and pots more
water was present, and at the same time enough temperature was present
too.

Fusarium was more prevalent and dominated all other species under
a wider range of environmental conditions. Thus under cooler environ-
ment of spring and autumn, and under warmer and drier conditions of sum-

mer Fusaria were the more prevalent species causing damping-off, with

second the Rhizoctonia solani, and third Pythium. This was probably due

 

to the fact that Fusaria as we mentioned previously are "strong" species
which means that they can live and grow satisfactorily under a wider
range of conditions of environment as compared with Pythium and Bhiégg-
‘tgnia, which are influenced more by their environment.

These results agree in a general way with those of Beach (6 ) in
Pennsylvania with several vegetable crops.

In Table VI is shown that the Fusarium, and especially 5. splani

(Schl.) Sny. and Han., E. oxysporum (Schl.) Sny. and Han., and E, moni-

 

liforme (Sheld.) Sny. and Han., are the most important year-round cause
of damping-off of black pine in Michigan, and possibly other conifers

as well. Rhizoctonia solani (Kuhn), Pythium irregulare Buisman, Pythium

 

ultimum Trow, and Pythium debaryanum Hesse, are only secondary in

37
importance. This is in contrast to the reports of Roth and Riker (65,

66, 67) who found that in Wisconsin nurseries Pythium irregulare and

 

Rhizoctonia solani are the main cause of damping-off on red pine.

 

With Fusarium, the most common year-round species was F. oxysporum,

followed by F. solani, and the least common F. moniliforme. With

 

Eythium generally throughout the year 5. irregulare was the most common,

 

_F_. ultimum less common, and P. debaryanum least common.

 

A number of questions have arisen during the process of this study.
One of these was; do the isolated fungi studied show any ability and
preference to attack seedlings from the time of emergence until they
become resistant to damping-off some 3 to b weeks later? The above
question is concerned with the postemergence damping-off of black pine.
From several thousands of isolations throughout the year, no species of
the three main pathogenic genera showed any Special ability to attack
earlier than any other species.‘ The ratio of isolated species from the
early time of damping-off to late time of damping-off was the same as
the ratio of the most common species to less common and least common,
which occurred at each period.~

Another question was; do pathogenic fungi which cause damping-off
work together and cause damping-off in the same individual seedling, or
do they avoid each other by competition or by some antibiotic means?
From the discussion of isolated organisms from damped-off seedlings
throughout the year and from Table VI we found that in many cases bac-

teria and various other fungi (Aspergillus sp., Penicillium sp., Mucor

 

 

sp., etc.) were present. However, bacteria, Aspergillus sp., Penicil-

 

lium sp., Mucor sp., etc. are saprophytes or weak parasites. Thus the

presence of all of them in so many cases must be considered as an

38
excellent example of suppression and antibiosis of the main pathogenic
fungi. It is probably easy for someone to say that the main pathogen
was already dead or was killed by the sterilizing agent, why then were
not the saprophytes or bacteria also dead or killed. From the work of
others it has been found that Pythium can be supressed by bacteria (83);
or that with the presence of eleven different saprophytes in sterilized
soil Pythium caused less damping—off than Pythium alone (32).

During this study it was found that bacteria and saprophytes in
plate cultures could suppress the main parasites. There is a consider-
able amount of literature on the subject of suppression, antibiosis,
and parasitism of several pathogenic fungi by saprophytic ones (25, 26,
b7, 59, 68, 69, 87, 88, 89, 90, 9b)-

Our original question was whether the main parasitic fungi among
Species of the same genus or among species of different genera were
working together in an individual diseased seedling or avoiding each
other by any means of competition or antibiosis etc., In order to give
an answer to the above question many combinations of the main pathogenic
fungi were planted in potato dextrose agar at pH 5.5 at room temperature
in the dark. Three plates were used in each case. The results found

are as follows:

 

 

Fusarium solani strain 3 and F. gxysporum +
Fusarium solani strain 3 and F. moniliforme strain 2b +
Fusarium solani strain 3 and F. roseum +

 

Fusarium roseum and F. moniliforme . , -

 

 

Fusarium moniliforme and F. oxysporum -

 

Fusarium roseum and P. debaryanum -

 

 

Fusarium solani strain 3 and P. irregulare -

 

39

Fusarium solani strain 3 and P. ultimum -

 

Fusarium solani strain 3 and R. solani strain 1 +

 

Fusarium moniliforme and R. solani strain 1 -

 

Fusarium moniliforme strain 2b and R. solani strain 87 +

 

Fusarium moniliforme and P. debaryanum -

 

 

Fusarium moniliforme and P. irregulare -

 

 

Fusarium oxysporum strain 1 and R. solani strain 1 -

 

 

Fusarium oxysporum strain 15 and P. ultimum j
Rhizoctonia solani strain 1 and P. ultimum +

 

Rhizoctonia solani strain 1 and P. debaryanum -

 

 

Rhizoctonia solani strain 1 and P. irregulare '3

 

 

Pythium irregulare and P. ultimum -

 

Pythium irregulare and P. debaryanum -

 

 

PfiMmuummam3.@mem -

 

 

In the above test - means no antibiosis apparent (fungi mixed), +
means antibiosis apparent (a clean zone was formed between the fungi
tested) and 1 means a slight antibiosis probably present, (a situation
among the first two.) In this third case the hyphae tips of the two
Opposite different fungi were approached by each other and probably
mixed a little between them, but a lighter zone was apparent.

From the above it was apparent that certain antibiosis was present.
For instance F. solani strain 3 showed antibiosis with the three

Fusaria tested, E. oxysEorum, E. roseum and F. moniliforme. The same

 

fungus showed also an antibiosis with Rhizoctonia solani strain 1, but

 

it does not Show any antibiosis with Pythium. (Gregory gt al (25) found

also that a Streptomyces isolate produced an antibiotic which was active

 

against some fungi but had low activity towards other fungi and bacteria).

b0
The same was also true of several other fungi and combinations used in
that test (Fig. 1). From the above it is clear that in pure cultures
several of the main pathogenic fungi avoid each other, several do not.
But the author, from the above results and his isolations from damped-
off seedlings believes that in nature as a general rule only one species
attacks and invades each individual damping-off seedling. In exception-
ally few cases the author isolated from the same seedling more than one
species of different genera; the same was found also by Brandt (8) who
in soil infested with pure culture mixtures, only rarely reisolated

Rhizoctonia and Fusarium together in the same diseased stem.

 

Another question was; when did the peak loss of seedlings take
place after emergence, and when was resistance attained? Roth and Riker
(67) found in Wisconsin on red pine and with Pythium irregulare and

Rhizoctonia solani that the highest percentage of damping-off occurred

 

in seedlings 11 to 13 days old, and relative resistance occurred after
about 1 month. Tint (80) by planting red pine seedlings of different
ages found that seedlings from 1 to 7 days old had the highest percent-
age of postemergence damping-off from Fusarium oxysporum and the re-
duction in damping-off corresponded with the increasing age of the host.

The data below (Table VII) illustrate a similar effect.

Seedling emergence started at eight days during the warmer summer
months, and under greenhouse heated cOnditions during winter months.
During spring and autumn cool conditions emergence started after 10 days.
It appears from this that, since the necessary moisture is available,
temperature is the next most important factor for seed germination. At
medium high temperatures germination is faster than at lower temperatures.

In nature seed germination is influenced by fluctuating temperature, but

Fig. 1. Upper half Rhizoctonia
solani strain 87, lower Fusarium
moniliforme, the two Species did
not mix, a clear line is apparent
between them.

 

b1

b2
under experimental conditions, medium high temperatures are more favor-
able for seed germination than lower ones (2, 27, 66, 81).
TABLE VII. Time of emergence and highest damping-off percentage

on black pine seedlings, as compared with the age of
seedlings.

 

 

Days to Highest percentage Seedlings developed

 

Time period emergence postemergence resiStance
damping-off
'Winter months 8 days 9 days after ' 25 days after
Greenhouse heated emergence emergence
‘Spring months 10 days 11 days after 27 days after
Greenhouse cool emergence emergence
Summer months 8 days 8 days-after 25 days after
Greenhouse-Field emergence emergence
Autumn months 10 days 11—12 days after 27 days after
Greenhouse cool emergence emergence

 

Black pine seedlings become resistant to damping-off nearly one
month after emergence (Table VII), the same as red pine in Wisconsin
(67). In Wisconsin the highest percentage of post-emergence damping-
off occurred at 11 to 13 days during summer months (67) but in Mich-
igan with black pine seedlings the highest damping-off during summer
was found to be.8 days after seedling emergence. This variance prob-
ably was due to the different host, different pathogenic fungus, vary-
ing soil type and pH, and finally the climatic conditions, since all
of them influence not only the good growth of the seedlings, but the
pathogenic fungi also.

With lower temperatures in Spring and Autumn black pine showed
the highest postemergence damping-off with seeds starting to emerge at
11 to 12 days respectively. From the data it is apparent that at

higher temperatures in summer or in a heated greenhouse, seedlings

b3
emerged earlier, more damping-off occurred earlier, and finally the
resistance of seedlings was attained earlier, than at the lower temper-
atures of Spring and Autumn. This is due probably to the better con-
ditions of plant growth which allowed the seedlings to pass all the
susceptible stages in a shorter time.

The total damping-off (preemergence plus postemergence) was found
to be quite high all year around under greenhouse conditions. this be-
ing in agreement with others (77, 79). From Table VI one can see that
preemergence damping-off is higher at the higher temperatures. Thus,
in the heated greenhouse, and during Summer months in the nursery, the
preemergence damping-off was higher than the postemergence damping-off,
and preemergence damping-Off was also higher at warm periods of winter
and summer than in cooler Spring and autumn}

The preemergence and postemergence damping-off is the same disease
expressed on the time and size Of seedling attacked. We can consider
that preemergence damping-off is a more severe type than postemergence
damping-off, since the same microorganisms cause both of them, but in
the first case it happens very early just as the seed starts to germin-
ate; in the postemergence damping-off it happens later, and after hypo-
cotyles and roots of seedlings are exposed to the fungi attack for a
longer period of time.

From Table VI we found that the higher the temperature the higher
the preemergence damping-off throughout the year, and this should natur-
ally happen with postemergence damping-off. Tint (81) found also that
the higher the temperature within limits the higher'the preemergence
damping-off.

Emergence of black pine seedlings was higher at warmer periods

than in cooler ones (Table VII) and the pathogenic fungi isolated grew

- 1*...- J. ‘
.A .1

bb
better at temperatures between 200 and 30° C. than at lower temperatures
they could not escape because the pathogens grow faster also.

Leach (bb) has pointed out that the severity of damping-off in all
combinations of host and pathogens was determined to a considerable de-
gree and was most severe at temperatures that were relatively less fav-
orable to the host than the pathogens as measured by the ratio of their

growth rates.

MODIFIED CHOLODNY SOIL SLIDE TECHNIQUE

This method was discussed under the Materials and Methods section.
The fungi found were reported in the chapter "Isolation and Identifica-
tion of Species, their Importance and Pathogenicity."

These main pathogenic fungi were always present in the soil. ‘Byt_-
13m Sp. were found to be abundant in the soil, by using the above
technique, even in periods in which they were not isolated from damped-
off seedlings. The other soil fungi and microorganisms were referred
to in an earlier chapter.

Data obtained in this study of soil microflora and microfauna by
the slide technique were poorly correlated with the damping-off of
black pine. The action of the main pathogenic fungi in this Study under
greenhouse conditions, seems to be correlated with the health and growth
of host and pathogens as they are influenced by the inorganic environ-
ment rather than the organic environment. In the greenhouse the patho-
genicity of damping-off fungi became greater than the competitive ability
of secondary microorganisms of the soil flora and fauna. It has been
pointed out in the literature, nevertheless, that the organic environ-
ment under certain or special conditions can reduce or control damping-

Off (25, 26, 32, b7, 87, 89).

BLACK PINE SEED GERMINATION

The damping-off disease occurs only from the time seed starts to
germinate until about 5 weeks after sowing. Since seedlings become re-
sistant in about 3 to b weeks, after emergence and since timing is a
very important factor for damping-off, it is easy to understand that
seed with a high germinative capacity and germination speed should have
more possibility of escaping the disease. The different germination
ability of seed depends upon its inherited capacity, the maturity of
seed, and the treatment and care of seed from the time of collection 3
until the time of sowing.

Many species of seeds are treated before they are sown in various
ways in order to produce better germination (1). Even although quick
seed germination is desirable, very little knowledge exists on the ef-
fect of such presowing treatments on the health of germinated seed in
relation to damping-off disease. One of these seed treatments, the pre-
soaking of seeds after Roth and Riker (66), had no apparent ill effects
on the seedlings. This is in contrast to one report (97) in the lit-
erature where dormant seeds of conifers had lower losses than pre-germ-
inated ones in Fusarium infested soil. The same was reported by Hansen
.EE.§£ (29), that the soaking of the seed of white pine and Norway pine
did not increase the rate of germination but increased injury from
damping-Off. 7
Black Pine Seed Germination and Moisture.

The first and most important factor in the germination of seed is
probably free moisture. Since the presence of free moisture is neces-

sary for seed germination, the next consideration must be the amount

b?

of water. The amount of water is important because it is inversely re—
lated to seed aeration and that is also an important factor in seed germ-
ination. (An experiment was designed to study these relationships. A
series of Petri plates were used. One filter paper was put on the bot-
tom of each plate, and one for seed covering. A series of plates con-
tained various quantities of water ranging from minimum to excess of the
required for normal germination. The results for the different moisture
levels are given in Table VIII.

All plates were held at room temperature (about 22° C.) and covered

with Kraft paper in order to have dark environment. Part of the plates

_g- .. _
fl ‘

were sealed with wide rubber bands in order to prevent evaporation, and L_.
retain constant moisture. The treatments were replicated four times
and-the experiment performed twice. Germination was considered com-
plete when the radicle was at least 2 mm in length.

Table VIII shows that seed required enough water quickly at the
beginning of germination. Thus there was better germination up to the
8th day in series B, C, D, E, in which seed could absorb more water
than in series A. With more water (seed 3/b or completely covered)
there was less germination, since aeration was the critical factor
(series F and G).

Although enough free moisture is necessary to initiate germination,
aeration seems to be the critical factor after 8 days, in this experi-
mentH Series A, with the best aeration, had the best germination of all.
Table VIII shows that with less water and better aeration the germina-
tion was better while with more water and poor aeration the germination

was reduced.

The sudden gain in germination of seeds in Series D after 11 days

TABLE VIII.

Black pine seed germination in
of water at room conditions in

b8

different quantities
darkness.

 

Water per plate

_—

:-

Percent germination in days

Remarks

 

7 8 11 13 15 21
A. 5 cc. a b Seed and paper enough
not sealed 16 28 76 96 100 100 moisture. In 13 days
2 cc. H20 was added.
B. 5 cc. Seed and paper enough
sea1ed 20 b0 72 8b 92 96 moistened, more than
in A.
C. 10 cc. Seed covered at least
not sealed 20 b0 52 8b 92 96 1/2 with water.
D. 10 cc. Same as C. but in 11
not sealed 20 b0 52C 92 96 96 day excess water was
poured off.
E. 10 cc. Seed covered at least
sea1ed 20 36 b0 61 8b 92‘ 1/2 with water.
F. 15 cc. Seed covered at least
sealed 16 32 36 60 8b 92 3/b with water.
G. 25 cc. Seed completely covered
sealed 8 20 22 b0 60 92 with water.

 

aSealedwith wide rubber band seal for dishes.

bFilter paper started to dry, 2 cc. more water was added per plate.

COn that day after counting the excess water was poured off.

b9
can be attributed to the improved aeration from draining the excess water.
Series C, where the water was not drained, showed less increase in germ-
ination. This experiment indicates that free moisture is required for
seed germination, but the necessary moisture is really quite low. Al-
though we had a better germination at the beginning with medium moisture,
after a few days it was evident that germination increased at the lower
moisture content (better aeration) more than at medium moisture content, L
and lowest germination occurred in the series with most water (less
aeration).

The above results apply to germination of black pine until the
thirteenth and fifteenth day. At the end of the twenty-first day, it
was found that coniferous seed like black pine could'give satisfactory
germination (92 percent) even if the seed were completely covered with
water as in series G. This germination would be considered satisfactory
except for the fact that it required approximately half again as long
as with less water (series A). This is an unfavorable condition since
these poorly aerated germinating seeds would be exposed for a longer
time to fungus attack. '

Germination of the same seed lot in greenhouse tests varied from

78 to 8b percent but in Petri dishes (Table VIII, A) it was 100 percent.

Black Pine Seed Germination and Temperature.

An experiment was designed to find the relationship of temperature
and germination of black pine seed. Four plates each containing 50
seeds were used per treatment. Watering was optimum and the plates
were held in incubators in dark at the designated temperatures. Part
of the treatments were constant temperatures (Table IX, NOS. 1-8) and

the remainder (No's. 9-17) were lb hours of a higher temperature and

TABLE IX. Black pine seed germination at different temperatures in the

50

 

 

 

dark.

No. Temperature Days
7 9 11 lb 16 19
1. 6°C (Constant) 0 b 12 16 28 32
2. 12°C (Constant) 0 b 12 16 28 32
3. 16°C (Constant) 0 12 2b b0 72 82
b. 20°C (Constant) b 20 32 56 80 8b
5. 2b°C (Constant) 16 36 56 80 92 92
6. 28°C (Constant) 12 28 bb 6O 72 8b
7. 31°C (Constant) 12 36 56 72 88 92
8. 37°C (Constant) 0 16 20 20 b0 bb
9. 2b°C(1b hrs), 16oc(10 hrs) 12 28 52 72 9b 96
10. 2b°C(1b hrs), 120c(10 hrs) 16 32 52 72 92 92
11. 2boc(1b hrs), 6°C(lO hrs) 12 20 36 60 8b 92
12. 370c(1b hrs), 16°C(1O hrs) b0 52 68 92 92 92
13. 37°C(lb hrs), 60c(10 hrS) 3b b6 60 80 8b 8b
lb. 31°C(lb hrs), 16°C(10 hrs) 1b 20 b0 80 96 96
15. 310c(1b hrs), 60c(10 hrs) 16 2b b8 80 88 92
16. 280c(1b hrs), 160c(10 hrs) 8 16 2b 60 8b 88
17. 28°C(1b hrs), 6°C(10 hrs) 10 18 36 6b 8b 88

 

. 119?”... Q, a- . --

51
10 hours of a lower temperature. The results (Table IX) Show that, in
incubators with constant temperatures, there was better germination be-
tween 16° and 31° C.. At the two lOwest temperatures (6° C. and 12° C.),
the germination was identical and only reached 32 percent throughout
the period. Only when the temperature reached 16° C. was the germination
significantly increased to 82 percent. Two maxima, 2b° C. and 31° C.,
were obtained for constant temperature conditions. Other workers (b,
66) have reported optimum germination around 2b-26° C. for red pine and
Haasis (27) found two optima at 2b° and 36° C. for red pine and for
lodgepole pine two optima at 2b° and b0° C. as well. At 37° C. constant
temperature there was another drop although still above that at 6° C.
and 120 C..

At fluctuating temperatures we obtained the following results:

The germination was more or less the same for 2b°-16° C., 2b°-12° C.,
2b°-6° C. as at a constant 2b° C.. This indicates that if one tempera-
ture was favorable for enough hours per day, the alternate temperature
did not change the amount of germination. When a higher temperature of
37° C. (known to be unfavorable when constant) was combined.with alter-
nating lower temperatures of 6° and 16° C. a favOrable germination con-
dition was produced. 0f the two, 16° C. was superior to 6° C. germina-
tion being 92 and 8b percent respectively (Table IX, No. 12 and 13).
At constant temperature of 37° C., 16° C. and 6° C. the germination was
bb, 82 and 32 percent respectively. At 31° C. higher temperature much
the same results were obtained with 2b° C. (Table IX, NO'S. lb, 15, and
9, 10, 11). At 28° C., as higher alternating temperature, there also

was better germination than at the constant temperatures 28° C., 16° C.,

or 60 C..

52

Summarizing the influence of alternative temperatures on black pine
seed germination, if one temperature of the alternative combination is
favorable or optimum in the case of constant temperatures, the change
of temperature had little influence and gave more less the same germ-
ination number as the favorable constant temperature. On the contrary,

if alternative temperatures are neither one favorable, the alternative

 

temperature stimulated germinatinn above that at any constant tempera- ‘ .1
ture used.
The Effect ofng on Black Pine Seed Germination. 1

Among other factors which may influence the germination of black
pine seed is pH. A series of experiments were conducted in order to
find any relationship of pH solutions and the germination of black pine
seed.

Seeds of black pine were put in Petri dishes with two layers of
filter paper at the bottom in order to retain more uniform moisture;
then seeds were covered with one filter paper. Four plates were used
in each treatment and the experiment repeated twice. Buffers were made
by using different combinations of 0.1 M phosphoric acid and 0.1 M
sodium hydroxide (5). Watering was normal to give best aeration and
germination. The plates were stored at room temperature. and covered
with Kraft paper in order to provide a dark environment. The results
of these germination tests are Shown in Table X.

A soil test was also used to determine the influence of pH on the
germination of black pine seeds. This experiment was conducted in the
Plant Science Greenhouse. The soil used contained 3.3b percent humus,
sand 86.00 percent, silt b.OO percent, and clay 6.66 percent (Type F,

Table XVII). The soils were also buffered with 0.1 M phosphoric acid

S3
and 0.1 M soidum hydroxide used in different proportions to give the
desired pH. Soil was sterilized etc. as described in Materials and
Methods. Four replicates were used for each pH value; 50 seeds were
sown in each pot. The watering was normal. The germination of black
pine seed in pot soil at different pH levels is shown in Table XI.

During the course of the study germination of black pine seed was
observed and also the influence of pH on the length of root and stem
was measured. The results of these observations are shown in Table XII.

By examination of Tables X and XI in which the germination of black
pine seed is shown in Petri plates and pot soil at different pH; it can
be seen that the pH of the solution strongly influenced total germina-
tive capacity and the germination curve of black pine seed. The in-
fluence of pH on earliness of germination is shown well in Table XI.
This test in soil at different pH levels showed that seed started to
germinate two days later at pH 7.0 than at pH b.5, and at pH 8.5 seed
started to germinate two days later than at pH 7.0.

The total germinative capacity of the same seed in Petri plates as
compared with that in soil differs for each pH value. This is probably
due to poorer aeration in pot soil. The germination capacity at each
pH value was also generally different. Thus the germination capacity
at pH b.5 in both Petri plates and soil was twice as large as that at
Ifli 8.5; the germination at pH 7.0 was found to be intermediate. These
findings are the opposite of those of Tint (80) who found a reduction

in emergence of Pinus nigra var. austriaca in acid media.

 

By comparing the two Tables X and XI it is found that the rate of
germination differs considerably. In Petri plates seed was considered

to have germinated when the radicle appeared. In pot soil, germination

5b

TABLE X. Black pine seed germination at different pH values in Petri
plates in dark at room temperature.

 

Total number of seed germinated. percent by days

 

 

6 ' 7 9 11 12 13 16
b. 5 60 y 68 86 88 88 88 88
7.0 38 b2 5b 59 59 59 59
8.5 26 31. bh b8 7 b8 b8 b8

 

TABLE XI. Black pine seed germination at different pH values in pot
soil in greenhouse.

 

 

Total number Of seed germinated, percent in days

 

pH 3 a

:12 1b l6 19 21' 23 26 28 31 33 35 37
b.5 ll 15 31 50 56 68 72 7b 7b 78 78 78
7.0 -- 5 17 32 b2 ‘ b8 so -56 56 56 56 56
8.5 -- -- b 8 23 29 32 32 32 32 32 32

 

 

aTime seedling root and stem measurements were taken (data in Table XII).

TABLE XII. Root and stem length of black pine seedlings at different
pH values in pot soil in greenhouse.

 

 

 

 

19 days after sown 33 days after sown
pH Root cm. Stem cm. Total Root cm. Stem cm. Total
length cm. length gm.
b.5 S.b5 3.25 8.70 6.50 3.b5 9.95'
7.0 3.90 2.60 6.50 V 5.80 3.20 9.00

8.5 3.30 2.50 5.80 5.00 3.20 8.20

 

55
whole germination process after seed started to germinate lasted only
five days. In soil after germination of seed started. it continued for
more than 15 days.

The results of root and stem measurements of seedlings grown at the
different pH levels is given in Table XII. Ten seedlings were collected
at random 19 and 33 days after sowing from each of four pots. The nin—
teenth day was the time of highest damping-off, and the thirtyieth was
the time when the seedlings had passed their susceptible stage.

In 19 days the length of roots at varying pH levels were different
and were longest at pH b.5, shOrter at pH 7.0, and shortest at pH 8.5.
Thus, at the time of highest damping-off and fungus activity the differu
ences in root length were most striking for the various pH levels. The
same trend was apparent at 33 days but there were smaller differences.l
Stem lengths differed also but in less degree, at different pH levels
on the 19th day. They were the same at 33 days at pH 7.0 as at 8.5
and only slightly longer at pH b.5. Jackson (b0) in liquid cultures
at different pH found total length of Douglas fir was greater at pH
3.5 and 5.5, and in quartz sand and with liquids the total length of
Douglas fir and Ponderosa pine were greater at pH b.5. Roth and Riker
(66) in pot soil with different pH found that the optimum range for
root growth of red pine extended from about pH b.7 to 6.0. From the
above results one can see that the influence of acidity on root and
total seedling growth is better at the acid side than at the alkaline
side of the pH scale. Different species Show a varying ability to grow

faster at the different levels on the acid side of the pH scale (b0, 66).

DAMPING‘OFF OF BLACK PINE SEEDLINGS

Relative Pathogenicity of Different Strains and Species.

In an earlier section we have discussed the fungus species isolated
from black pine damped-off seedlings. In order to determine if these
isolated fungi were the real cause of the disease, experiments were
conducted with pure cultures in soil.

The soil used in this experiment was at pH 5.5, and contained
humus 3.3b percent. Four or more pots were used for each treatment
and the experiment repeated one or more times. The soil was sterilized
and treated as described earlier. Cornmeal-vermiculite medium in Petri
plates was used for growing inoculum. One fourth of a plate of inoculum
was mixed into each b inch pot of soil in the first trial. Since this
amount of inoculum was found to be too heavy, one eighth of a plate
jper pot was used thereafter. Appropriate controls were uSed in each
set of experimnts in order to compare the percentage of healtby germ-
ination, and from this the percentage losses by damping-off. Watering
of the pots was as usual.

In order to have an accurate record of the temperature and relative
lunnidity a hygrothermograph was placed among the pots. Table XIII pre-
serflx; the record of temperature and relative humidity during this test.

It can be seen that the minimum temperature was more or less con-
staffl; throughout the period at about 22° C.; the higher temperatures were
(infaxnarable for seed germination, but the alternation of temperature has
beeni shown (Table IX) to reduce the unfavorable effects of high temper-
atures.

The relative humidity plays a considerable role to the maintenance

of'snail.moisture, and has less influence on damping-Off. When a 100

57

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58
percent relative humidity was obtained, however, in watered pots covered
with cellophane bags, Rhizoctonia solani grew abundant aerial mycelial
and could attack the above ground part of seedlings. This confirms.
the observations by Roth and-Riker (67). In this type of covered pots
the author got also complete postemergence damping-Off of black pine
seedlings using the common black mold Rhizopus nigricans.

Table XIV represents the relative pathogenicity of different Spec-
ies and strains of pathogenic fungi isolated from black pine seedlings.
Of the Pythium species the isolate of P. irregulare was the most path-
ogenic. This Species was the commonest isolated from diseased seedlings
and from soil from nursery and flats in the greenhouse. P. perniciosum,
as was expected, did not cause any damping-off of black pine seedlings.

The different strains of Rhizoctonia sOlani whose cultural and
growth characteristics were described in Tables I, II, and III showed
that they possess different pathogenic ability also. Even though all
of them showed a high total damping-off, there were noticeable differ-
ences in the preemergence and postemergence damping-off. Others have
also'reported that strains of B. gelani have different pathogenic ability
(13, 15, 32, 57, 65)-

In the Fusaria the different species and strains showed also cer-
tain differences in their total damping-off. Preemergence damping-off
inas generally more severe than postemergence but strain differences in
this respect could be seen as in 5. M strain 3 and 19 or 25. In
tilis study only in Fusarium solani were distinct strains found. For
time two other species 5. oxy5porum and F. moniliforme the many differ-

erfi; isolations studied proved to be culturally and pathogenically ident-

ical.

59

TABLE XIV. Relative pathogenicity in the greenhouse of strains and

species of isolated fungi on black pine seedlings.

 

.9

 

 

 

 

 

 

 

 

 

 

Number Damping-off percent Healthy

Species P815 Preem. Postem. Total I183;
Pythium irregulare b 83 11 9b 6
Pythium ultimum b 59 26 85 15
Pythium debaryanum b b9 33 82 18
Pythium perniciosum 'b O O O 100
Rhizoctonia solani strain 1 b 55 b0 95 5
Rhizoctonia solani strain b b 85 13 98 2
Rhizoctonia 221321 strain 87 b 70 18 88 12
Fusarium £91221 strain 3 b b9 39 88 12
Fusarium £21321 strain 25 b 85 10 95 5
Fusarium 521321 strain 19 b 68 6 7b 26
Fusarium oxysporum strain l5 16 7b 11 85 15
Fusarium moniliforme strain 2b 12 76 16 92 8

 

6O
Damping-off of Black Pine Seedlings and_pH.

It has been known for many years that acid soils reduced damping-
off losses, and in alkaline soil damping-off losses were heavy (32, b0,
6b, 66, 78, 80). This seems inconsistent with the present work Since it
was found that the pathogenic damping-off fungi really thrive in acid
environment, and in alkaline environment they grow less vigorously.

It is further assumed that in natural acid soil the antagonistic bac-
teria should compete with fungi better than in alkaline soil. As was
discussed earlier, when media with pH 5.5 were used for isolation of
pathogeniC‘fungi from damped-off seedlings, bacteria almost always
covered the entire tissue sections. It was necessary to put 2-3 drops
per plate of a 25 percent lactic acid solution in order to have a med-
ium with pH b to b.5, and in that medium only were the undesirable bac-
teria avoided.

' Although the pathogenic fungi thrive in acid media, and the com-
petitive bacteria in alkaline media, coniferous seedlings avoid damping-
Off better in acid media (soils). The reason for this is easy to find,
in that the seedlings themselves are favored by acid soils and grow in
a healthier condition. In the slightly acid soils minerals are easier
available to plants, and thus plants live in a healthier condition,
grow faster, and mature earlier (Table XV).

Black pine seed were sowed again in the same soil which was used
earlier. The pH of the soil was adjusted with buffers, and the soil
‘was sterilized as usual, and seeds were sterilized with l:lOOO mercuric
chloride. Fifty seeds were sowed per each b inch pot. Two fUngi,

Fusarium moniliforme and Rhizoctonia solani, were grown in Petri plates

_i

 

 

in cornmeal and vermiculite. One eighth of plate inoculum was used per

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Fig. 2. Black pine damping-off
at different pH levels of the
soil. Left pots with pH b.5 less
damping-off occurred, than at pH
7.0 center, or pH 8.5 right.

63

 

 

6b
b inch pot. Four pots were used, repeated twice, for each pH value;
watering was as usual. Temperatures and humidity during the course of
this study were measured and are shown in Table xva. Table XV gives
the results of this experiment.— At pH b.5 the preemergence damping-off
and especially the total damping-off was smaller than at pH 7.0 and pH
8.5. This is the case especially for Fusarium moniliforme (Fig. 2).

In Rhizoctonia solani strain No. b we had a total number of damping-off

 

of 80, alive 20, at pH b.5 and 100 percent damping-off at pH 7.0 and
pH 8.5. Fusarium moniliforme was less pathogenic in general than B.
£21221 strain No. b at the two lower pH levels but equally severe at
pH 8.5.

In general with both fungi we had less damping-off in acid soil
than in alkaline soil, this being in agreement with others (32, b0, 6b,
66, 78, 80).

Measurements of length of roots and stems at different pH of soil
were Shown in Table XII. Thus it was found that the root lengths gen-
erally were greater at pH b.5, less at pH 7.0, and least at pH 8.5;
this being in agreement with the work of others (b0, 66). Stern and
total length were less clearly affected.

Table X shows the influence of pH on seed germination. Thus at pH
14d; seeds started to germinate two days earlier than at pH 7.0, or b
diYE? earlier than at pH 8.5. But in acid soils not only germination
StaI‘lted earlier but it was approximately twice that in alkaline soils
(Table x1). Thus it is clear from all the above that in acid soils
1953‘ damping-off occurs not only because seedlings are grown better,
but sneeds also germinate earlier, faster, and in a total germination

twice: that of alkaline soils. Thus seedlings in this condition have

65
more possibilities to escape damping-off, and reach the maturity stage

earlier.

Black Pine Dampingeoff and Soil Quality.

It was referred earlier that temperature, moisture, aeration, pH
are some of the environmental factors which influence the germination,
the emergence, and the good and fast growth of seedlings, but at the
same time the growth condition and health of the pathogens. It was also
accepted that the main factor generally which determines the quantity
of damping-off is the growth condition and vigor of the seedlings, rather
than that of the pathogens.

Under good growth conditions vigor of the seedlings is influenced
not only by the quantitative and qualitative expression of environmental
factors such as temperature, moisture, aeration, pH, but also by the
quality (fertility etc.) of the medium (soil) in which seedlings are
grown. In nature the medium used is the soil and its type influences
the health and balanced growth of the seedlings, the pathogens, or both
of them, to affect the amount and kind of damping-off.

In order to find what relationships exist between soil quality and
damping-off, soil of different types was used. The origin and composi—
tion of soils was discussed under Materials and Methods. Composition
of the soils is given further in Table XVII.

All soils, before sterilization, were equalized at pH 6.0, by using
different proportions of phosphoric acid and'sodium hydroxide buffers.
After sterilization the pH was checked and corrected if necessany. In-
oculum was supplied from Petri dish cultures of vermiculite and corn-
meal. Two types of inoculations were made: "heavy" (Table XVI) in which

one third of a Petri dish culture was used per b inch pot, and "light"

66

 

 

 

 

 

 

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68

 

 

 

 

 

 

 

 

 

 

 

 

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69
inoculum (Table XVII) in which one eighth of a Petri dish culture was
used per b inch pot. Eight pots were used for each soil type and path-
ogen. Water was as usual. Temperature and relative humidity noted
during the progress of this test is given in Table XVIIa.

On comparing the damping-off for the various pathogens and soils
under two rates of inoculum it can be seen that the heavy type of in—
oculation (Table.XVI) was more severe than the light inoculation used
later (Table XVII).

In pure sand (series SA) there was less damping-off than in SS or
SP sands containing a fungus nutrient solution or peat (Table XVI).

In soils D, B, F with various percentages of humus, sand, silt, and
clay (c.f. Table XVII) we had less damping-off. It was lowest in ser-
ies F containing humus (3.3b percent) which probably represented a bet-
ter balanced fertilizer condition. In muck soil which contained b6.6O
percent humus, we had most damping-off.

Rhizoctonia solani strain b caused heavy losses in all types of

 

soils (Table‘XVI). Eythium irregulare gave heavier losses with muck soil

 

and in sand with fungus nutrient solution or with peat, and less with D,
E, F kind of soils. Even in the latter soils, however, the total losses
must be considered heavy. Eythium sp. are predominant in soils with

high humus content, and Rhizoctonia solani in soils with low humus con-

 

tent (32, 65). In the case of Bythium the present work gave more or‘

less the same results. With Rhizoctonia solani, however, in pure culture

 

inoculations there were heavy losses in all kinds of soils. These losses
were probably due in part to heavier amounts of inoculum and the favor-
able greenhouse environment. With Fusaria, again, there were heavy
losses on muck soil and sand plus nutrient fungus solution, sand plus

peat, and less damping-off with soil type F.

 

£31.31 was

70

In Table XVII it can be seen that a light inoculum with Fusarium
isglani produced most losses in clay soil, next most in the muck soil
and sand, less with soil D and least in soil F.

From the above it is clear that the type of soil is an important
factor in the occurrence of damping—off. The quality of soil influences,
along with the environmental conditions, both fungus growth, and the
balanced growth and vigor of the host. Thus it is apparent that the
physical, chemical and fertility properties of the soil are important
factors in damping-off. The experimental work confirms, in part the
results of Tint (80).

In Table XVII one can see that the root length and total length of
black pine seedlings 16 days old were greater in sand (no humus at all)
than in muck soil (humus b6.6O percent). Stems were shorter than roots
in all the soils tested, but in the case of muck soil the root-stem ratio
was smallest. The longer length of roots in sand is probably due to
better aeration which also influences seed germination. The increased
root lengths found in lower moisture, better aerated soils confirms a
report by Roth and Riker (61) on red pine.

The length and weight of green and dry seedlings are also shown in
Table XVII. Thirty three seedlings 16 days old were collected at random
from pots of each soil type. The average dry weight per centimeter of
seedling length was calculated also, since the length of seedlings
varied with the soil type. There was no correlation between dry weight
per centimeter and severity of damping-off.

The less damping-off, or the greater number_of healthy seedlings
occurred in soil of series F and D. This probably was due to a balanced

condition of soil fertility in which seedlings were grown neither so

71
fast, nor so slow. In case of sand seedlings grew too fast, and tissues
were too tender and attacked easier by pathogens. In muck soil seed-
lings grew too slow, thus they were exposed to fungus attack for a pro-
longed period. Probably seedlings in soil of series F and D possessed
a medium condition of growth between sand and muck, and thus less damp-

ing-off occurred.

Relationship of Dampingeoff and Root-rot of Black Pine Seedlings.

In many cases seedlings in flats in natural infected soil, or in
pots of pure cultures, or in the nursery, escaped damping—off and were
allowed to remain for more than a year. In a number of these cases,
especially in the favorable environment of the greenhouse, root-rot
sooner or later developed. Isolations were made from representative
root-rotted seedlings taken from natural infected soil in greenhouse
and nursery. In all cases Fusaria were found to be the primary cause

of root-rot. In a few cases Rhizoctonia solani was isolated also, but

 

it was doubtful that it was the root-rot agent since FUsarium was also
isolated from the same root section.

In several cases the phenomenon of weak parasitism, as pointed out
by Tint (79), was also observed. Black pine seedlings that escaped
damping-off in natural infected soil, started to show dryness of their
lower needles after several months. Root isolations proved that Fusaria
were present. If these seedlings were retained for one half to two
years they continued to be alive, even though their lower needles were
dried and even though Fusaria were living in their roots.

.5. solani, F. oxysporum and F. moniliforme strains from the Chit—

 

tenden nursery, and strains of the same species from the Bogue nursery

caused both damping-off and root-rot on black pine seedlings in pure

72
cultures in pot soil. The same Chittenden strains can cause a root-rot
complex with nematodes on white pine at the same nursery (61).

No Pythium or Rhizoctonia alone were isolated from root-rot seed-
lings of black pine in the greenhouse or in the nursery. These results

were similar to those of Riffle (61) with white pine.

DISCUSSION

Black pine damping-off was examined in natural infections in Mich-

igan nurseries and research was done mainLy in the greenhouse. During

the summer field experiments were also made in the nursery.
Under greenhouse conditions damping-off was heavier than in the

field, this being in accordance with work of others (77, 79, 82). It

was found also that if the same infested soil, either natural or inoc-

ulated, was used repeatedly the damping-off became more severe. This

means that under greenhouse conditions the main pathogens predominate
against their competitive microflora and microfauna.
In Michigan soil the following damping-off fungi pathogenic to

black pine were isolated: Pythium irregulare Buismann, E.u1timum

Trow, P. debaryanum Hesse, Fusarium solani (Mart-.) Snyder and Hansen,

 

E} oxysporum (Schl.) Sny. andHan., E. moniliforme (Sheld.) Sny. and
Iflar1., Rhizoctonia £21221 Kflhn, and Botrytis cinerea Persoon. Of the
atmyve several Fusarium sp. were the most important pathogens causing
damping-off of black pine under a wide range of conditions, and second-
aryr in importance were Rhizoctonia and Pythium. In the genus Fusarium
the most common species isolated under a wide range of conditions was
5* gxy5porum, and in fitbium it was _P. irregulare.

Pythium perniciosum was isolated from soil, and was of interest be-
Cause this is the second time in which this Pythium was reported in the
thitxed States (SO, 51). In this fungus oogonium germination was un-
lu‘Cfivri (SO, 51), but the author observed germination in two cases in
”atual‘ cultures. These occurred on hemp seed held in this culture for

I; InOnths in room environment. Germination was by a germ tube which had

7b
a branch about bO mu from its base. This species showed no pathogen-
icity to black pine seedlings.

The principal pathogenic species and strains isolated showed dif-
ferences in culture appearance, growth, and pathogenicity. Growth
studies of these fungi were done in different environments. Pythium
grew satisfactoriLy from pH b.5 to 8.0, with pH 5.5 being the most
favorable, this agrees with the work of others (36, b0, 6b, 66). Byt_—
ium also grew better in potato dextrose agar at pH 5.5 between 20° C.
and 30° C., this also being in a general agreement with the work of
others (51, 66).

From Rhizoctonia solani four different strains were isolated; these

 

were found to have stable cultural appearance in different environments,
different growth, and pathogenicity. Rhizoctonia solani grew faster be-
tween 200 c. and 300 c. generally for all strains. At 12° c. the dif-
ferent strains grew in different ways, and two strains of it slower than
Bythium at the same temperature. The growth of Rhizoctonia in different
pH varied. Best growth generally occurred in the slight acid media,
and its growth in alkaline media was less, this being in agreement with
others (b6, b9, 6b, 66).

In the case of the Fusaria growth rates were smaller than Pythium

and Rhizoctonia but as a whole the different species and strains tested

 

showed less differences under the varying environments used. This means
inlat Fusaria are less influenced by the changes of their environments
shad that they are really "strong" fungi. The above probably explains
‘why Fusaria predominate under a variety of conditions throughout the
year. Others have found that Fusaria are widely adapted, and generally

can.cause severe losses in higher temperatures (6b, 81, 93). Pigmentation

75

in Fusaria was found to be more abundant under dark, and it is influenced
also by the media used, this being in agreement with others (10, 72, 96).
The results of this study showed that pH of the medium is net an import-
ant factor for zonation. And further it was found that all the damp-‘
ing-off Fusaria formed zonation in the lowest constant temperature of
12° C.. This is not in agreement with the work of others (72, 96);
that zonation in Fusaria is influenced onLy by fluctuating temperature
or light. It was also found that several strains of different species
can form zonation under a wide range of environmental conditions.
Sporodochium formation was proved to occur more abundantty in light
than in dark, this being in agreement with others (30, 72, 96). Media
influenced also sporodochia formation.

Isolations from damped-off seedlings of different fungi throughout
the year proved that species of the main pathogenic genera Fusarium,

Pythium, Rhizoctonia, and their importance in different seasons of the

 

year are the same in Michigan with black pine as in Pennsylvania with
several vegetable plants (6).

Pythium was more prevalent under the warm and moderately humid con-
ditions of closed heated greenhouses during winter months. Pythium is
responsive to moisture and caused more severe damping-off of seedlings
and predominated more over the other main pathogens the more the soil
was saturated. Pythium causes negligible losses in dry soils (6, 61).
During the warmer and drier summer months, Pythium losses were neglig-
ible in open, well aerated greenhouses or in the field. Other patho-
genic genera predominated over Pythium at that time. The above means
that, in a natural infested soil at medium high temperatures in which

flzthium lives together with other pathogenic damping-off fungi and with

I

70

a great number of other competitive microorganisms, Pythium predominar
tion depends mostly on moisture. The higher the soil moisture content
for a prolonged period of time the higher the losses from damping-off
of Pythium on black pine seedlings. In drier conditions the other path-
ogenic fungi were predominant.

Fusaria were predominant under a variety of conditions throughout
the year. 'As was mentioned earlier, Fusaria are less influenced by

environment than Pythium or Rhizoctonia which are influenced by more

 

limited conditions. Fusaria are "strong" species while Rhizoctonia

 

become secondary in importance in many periods of the year. The pres-
ent work also confirms that of Roth and Riker (66) who pointed out that
Rhizoctonia was influenced less by environment than Pythium.

From several thousands of isolations throughout the year in natural
infected soil the main damping-off causing fungi did not show any spec-
ial ability to attack black pine seedlings earlier than did other spec-
:ies. The rate of isolation of different species of fungi was accord-
ing to the final percentage ratio per each pathogenic genus for each
set of seedlings.

When the main pathogenic fungi were allowed to interfere with each
other in culture media it was found that several of them possessed a
Strong antibiotic property. Gregory gt. _a_l_ (25) found an antibiotic
FHVDduced by a Streptomyces which was active against some fungi, but had
lfinw activity towards other fungi and bacteria. The earlier results of
true author's work and from his isolation studies, showed that in natural
filfected soil in which damping—off occurs, individual damping-off seed-
111195 can be attacked and invaded by one only and in very few cases by

more than one pathogenic fungus.

77

Black pine seeds germinated two days earlier in warm than in cool-
er periods, and black pine seedlings developed resistance to damping-
off after 25 and 27 days after emergence respectivery. During warmer
periods both the preemergence damping-off and postemergence damping-
off of black pine was higher than during cooler periods. This confirms
the results of others (32, b3, bb, 66), who found that conifer damping»
off was generally more severe in the warmer soils and that seedlings
were better able to escape the disease under cool conditions.

The author's modification of the older contact-slide technique for
the study of soil microflora (11, 9b) was used. The advantages of
the new modified technique was that it was faster, and more accurate
for the identification of soil fungi.

Seed germination is an important factor in damping-off disease.
since it is evident that in case of slow germination, pathogenic fungi
have more opportunities to attack and cause severe types of preemergence
damping-off.~ Black pine seed germinated under different free moisture
conditions showed that a medium amount of moisture is necessary at the
beginning for a good start, but within a few days about the time the
new radicle started to appear, aeration became the most important fac-
tor for black pine seed germination. In other words germination was
favored by medium moisture initially and a reduction in moisture after
the process had begun. The above is of importance in nurseries practice,

because it is clear that a few days moderate watering is necessary after
sowing and then watering must be much lighter thereafter. In the lower
moisture content test and in good aeration 100 percent germination re-
sulted with the same seed lot which in the practice gave 78 to 8b per-

cent germination. The reason for this difference must be probably due

78
to the poorer aeration of seed in soil, as compared with the better
aeration in low moisture content in Petri plates in the above experiment.

At constant temperature with normal moisture black pine germination
showed a double peak curve with its optima at 2b° C. and 31° C.. The
same was found for other species by Haasis (27). Alternating medium
temperatures showed good germination as well (Table IX). Alternating
a low temperature with an unfavorably high temperature produced favor-
able germinatibn conditions.-

At different pH levels of b.5, 7.0 and 8.5, in Petri plates and
soil there was better germination at the lower pH b.5 and successively .
poorer at the more alkaline. Lower pH also induced earlier germination.
Here again'there was better germination in Petri plates than in soil at
each pH level, probably due also to the poorer aeration in soil. Aera-
tion also is probably the reason that germination was completed in S
to 6 days, in plates after it had started, while in soil the same pro-
cess required about 20 days. - *

From all the above it is apparent that germination is a very im-
portant factor in connection with damping-off disease, since under
special environments germination takes place quickly, and a high total
germination number occurs, thus seedlings more readily escape the
disease. Under other environments on the contrary, germination starts
later, continues for a longer time, and gives finally a lower germin-
ation number. All these conditions are favorable for the development
of severe damping-off.

Black pine damping-off was lighter in soil with pH b.5 than in soils
with pH 7.0 or pH 8.5; this being in agreement with the work of others

(32, b0, 6b, 66, 78, 80). Black pine was found to grow better at pH

79
b.5 than at pH 7.0 and pH 8.5; this was also in agreement with the
work of others (b0, 66) on red pine. This is probably one explanation
for the lower damping-off in medium acid soil; plants grew faster and
were in a nutritional equilibrium of minerals made possible by the soil
acidity. But this is not the only explanation-of the less occurrence
of damping-off in acid soils, because it was found by the author that
in acid soils germination of black pine seeds started earlier, the
speed was greater, and the total germination number twice as much as
that in alkaline soils, and hence the seedlings reach the maturity
stage earlier, when they become resistant to damping—off.

Soil type and fertility influence plant growth, the pathogens, and
the soil microorganisms. Less damping-off of black pine occurred in
soils of medium fertility and in conditions in which black pine grew
well on the average than in sand, muck, or in more clayey soil. Black
pine grew larger seedlings in sand soils, and plants with heavier unit
of dry weight perilength in muck soil. It is postulated that, under a
medium type of soil condition and fertility, seedlings did not grow so
fast as to become excessively tender and susceptible to pathogenic fungi
as they did on sandy soil. Neither did they grow so slowly as to be
exposed to fungus attack for long periods as was the case on muck soil.

In several reports (75, 78, 79), less damping-off occurred with
coniferous seedlings in coarse sand cultures or in cases in which coarse
sand.was used as seed cover. In our case a fine sand less than 1 mm. in
diameter particle was used in the greenhouse. In Table XVII it was
found that neither the longer seedling length in sand soil nor the

heavier seedlings in muck soil were correlated with a lower incidence

of damping-off.

80

Seedlings of black pine escaping damping-off in greenhouse flats
with natural infected soil; in pots in pure cultures; or in nursery beds
were allowed to continue growth. The root-rot was heavier in greenhouse
(77, 79, 82) probably due to more favorable environment for the patho-
genic organisms and the tenderness of seedlings than in the nursery.
In seedlings of one or more years, weak parasitism was found to be as-
sociated with FUSarium Sp.. These seedlings grew satisfactorily and
only their lower needles started to dry, these seedlings in the opinion
of the author were good stock for planting. From the results of this
study weak parasitism seems to depend on the age in which the host was
attacked and also to the environmental conditions. Tint (79) wrote
that weak parasitism and virulence of Fusaria was dependent to the
species and the nature of their nutrition prior to inoculation and the

conditions under which the experiments took place.

'SUMMARY

The causal agents of damping-off of black pine in Michigan forest

nursery soils proved to be Pythium irregulare, P. ultimum, P. debaryanum,

 

 

Rhizoctohia solani, Fusarium solani, F. oxysporum, E. moniliforme, and

 

 

 

Botrytis cinerea. Culture and soil isolations proved that prevalence

of,gythium'was; Pythium irregulare first, 2. ultimum second, 2. debary-

 

anum third. Among Fusaria, F. oxysEorum isolations were more common,

.5' solani was second, and F. moniliforme was third.

 

From soil isolations Pythium perniciosum was identified, this re-

 

port being the second in this country, but when tested proved to be
non-pathogenic to black pine seedlings.

In culture studies Pythium generally grew better at pH 5.5, and in
a temperature between 20° and 30° C. Four definite strains of £31322-
£2313 solani were isolated with characteristic cultural appearances and
different pathogenicity. The strains showed also differences in growth

in different temperatures and pH of medium. Rhizoctonia in cultures

 

generally grew faster between‘2Oo and 30° C., and at the lowest temper

ature tested 120 C. was slower than Pythium. Rhizoctonia generally

 

grew well at pH b.5, 5.5 and 6.5, and its growth at Ph 8.0 was satis—
factory.
Fusaria grew slower than Pythium and Rhizoctonia, but the differences

among species and strains used in different
environments were less than those of Bythium.and Rhizoctonia. Pig-

 

 

mentation in Fusaria was more abundant under dark, and was influenced
by the medium used” Zonation in Fusaria was not influenced by the pH
of the medium; it was influenced by diurnal light; and at the lowest

temperature tested 12° C. all pathogenic Fusaria formed zonation. It

82
was also found that several strains of Fusaria possess the ability to
form zonation despite their enviornmental conditions.

Antibiotic properties were found when the main pathogenic fungi
were grown together to interfere with each other. Thus Fusarium solani
strain 3 proved to possess considerable antibiotic ability when it was
allowed to grow with several other of the pathogenic fungi; several of
the other fungi proved also to possess antibiotic ability in less degree

than F. solani, these fungi are Rhizoctonia solani strain 1, FUSarium

 

moniliforme, anle. oxysporum.

 

From isolations of damping-off seedlings and the above antibiosis
of several fungi, it was concluded that in natural infected soil, gen-
erally only one of the pathogenic genera attack and invade each seed-
ling; more than one genera were isolated from the same seedling only in
a few cases.

Nematodes saprophytic and parasitic species were isolated from
black pine damped-off seedlings, but due to the small number of cases,
they cannot be considered important factors in the occurrence of damp-
ing-off.

Isolations from greenhouse and field around a year showed that
Pythium was more prevalent during winter months in a closed heated green-

house, with Fusarium second in importance and Rhizoctonia third. In

 

spring in a greenhouse which was open and cooler Fusarium became the

more prevalent species with Rhizoctonia second, and Pythium third in

 

importance. During summer months in greenhouse and field Fusarium was

the more prevalent, Rhizoctonia was second in importance, and gythium

 

was third. In several cases during summer Pythium did not appear at

all in isolations. In fall again in greenhouse which was cooler,

83
Fusarium was most prevalent, Rhizoctonia was less prevalent and Pythium
was least prevalent. From the above it is clear that Fusarium is the
main cause of damping—off in Michigan soils, and that Fusarium is really
a "strong" species since in a natural infected soil and under a wide
variety of conditions it dominated in many cases the other pathogenic
fungi.

During warmer periods the preemergence damping-off was higher than
during cooler periods. The same was true for postemergence damping-off
and consequently for total damping off.

Studies on black pine seed germination showed that at the begin-
ning a medium quantity of moisture, and not too much moisture, is neces-
sary for initiation of seed germination. After a few days aeration be-
came the critical factor, and total germination proved best at the
lower moisture content tested. At constant temperature black pine seed
germination showed two optima at 2bO C. and 31° C.. Alternating temper-
atures resulted in the same or better germination than constant temper-
atures. As to the relation of black pine and germination and pH of
the medium, it was proved that at pH b.5 total germination number was
approximately twice that at pH 8.5, and it occurred in much less time.
From the above it is clear that pH, moisture, and temperature, are very
important factors for the rapid and better germination of black pine
seeds, and this of course is corellated to the occurrence of damping-
off disease since rapidly germinating seed and fast growing seedlings
reach their resistant stage sonner. Black pine seedlings reached matur-
ity 25 to 27 days after emergence, and seeds germinated under favorable
environments reach this stage earlier, and can escape damping-off.

The soil type and fertility influence also the growth of plants,

8';
of pathogens, and the soil microorganisms generally. Thus less damping-
off occurred in black pine seedlings in a soil of medium fertility
where seedlings did not grow so fast as in the case of sand, or so
slow as in the case of muck soil. It was found that the larger seed-
lings grew in sand but were probably tender, while the seedlings in
muck soil were heavier in dry organic matter, but they grew too slowly
and were proved to be attacked more severely by damping-off fungi.

For the study of soil microflora and microfauna a modification
of the older Cholodny and Krinchkova soil slide technique was used.
This modification proved useful since less time and more accurate ident-
ification of soil fungi were obtained.

The practical applications of this study are that in forest nurser-
ies in order to avoid as much as possible damping-off, the soil must
be light, slightly acid, and of medium fertility. After sowing the
watering, when no progenminated seeds are used, must be medium for sev-
eral days and quite light after. And since the temperature is also an
important factor, time of sowirlg must be tested in each locality and

for each plant species used.

10.

ll.

12.

13.

lb.

15.

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