INTERACTION OF SEED-COAT MICROFLORA AND SOIL
MICROORGANISMS AND ITS EFFECTS ON PRE- AND
POST-EMERGENCE OF SOME CONIFER SEEDLINGS'
M. I. TIMONIN
Forest Pathology Laboratory, Saskatoon, Saskatchewan
Received May 24, 1963
Abstract
Analyses of seed samples of Pinus banksiana Lamb., P. contorta Dougl. var.
latifolia Engelm., and Picea glauca (Moench) Voss demonstrated the presence
of 12 genera of fungi. Pythium, Phytophthora, and Rhizoctonia were not isolated
but typical symptoms of damping-off disease were observed among the seedlings
grown in sterilized soil. Surface sterilization of seeds resulted in a significant
reduction in emergence of P. banksiana and P. glauca seedlings but not on
P. contorta var. latifolia. The cause of this difference is discussed. The postemergence mortality of P. banksiana seedlings resulting from surface-sterilized
seeds grown in sterilized and unsterilized soil was significantly higher than that
among the seedlings from untreated seeds. Mortality of P. contorta var. latifolia
seedlings was significantly higher among the seedlings from untreated seeds, and
that of P. glauca seedlings was higher among the seedlings from untreated seeds
and only in unsterilized soil.
Introduction
The microflora of the seed-coat of coniferous and deciduous trees has been
investigated by many workers and its effects on the deterioration of viability
of the stored seed have been recently reviewed (3, 4, 8, 9, 12, 16). However,
the study of the effect of seed-coat microflora on the preemergence and postemergence survival of the seedlings has been neglected. On this subject only a
few papers have been published.
Thus, Gibson (2) in East Africa, working with seeds of Pinus patula
reported that the saprophytic fungi, A sper gillus , Afucor, , Rhizopus, Trichoderma,
and Trichothecium of the seed-coat microflora could, under favorable conditions,
invade tissues of the germinating seed and kill the seedling. The fungi were
able to invade the seed, according to him, through the damaged seed-coat.
The field experiments of Lawrence and Rediske (6) with isotope-tagged seeds
demonstrated that the seed-coat microflora was directly responsible for decay
of the seed and, indirectly by the weakening of seed vigor, predisposed it
to the attack of soil-borne pathogenic fungi. Shea (13) reported that under
laboratory conditions 18 species of fungi commonly associated with seedcoat microflora were capable of destroying Douglas-fir seedlings, under conditions favorable to fungus growth.
In Canada, as far as the author is aware, the seed-coat microflora and its
effects on the preemergence and postemergence survival of the seedlings have
not been investigated. The only references related to the seed-coat microflora on record are those of Salisbur y (10, 11). According to him there was no
definite correlation between high fungus spore load and low viability of
Douglas-fir seeds.
'Contribution No. 904, Forest Entomology and Pathology Branch, Department of Forestry,
Ottawa, Canada.
Canadian Journal of Microbiology. Volume 10 (1964)
18
CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 10, 1964
The present investigation was planned to study the seed-coat microflora
as well as its effect, if any, on the preemergence and postemergence survival
of the seedlings.
Materials and Methods
Species of seeds used in this experiment and locality of their origin were
as follows: jack pine (Pinus banksiana Lamb.) lot 1958, Montreal Lake;
lodgepole pine (Pinus contorta Dougl. var. latifolia Engelm.) lot 1957 Cypress
Hills; white spruce (Picea glauca (Moench) Voss) lot 1960, Emma Lake. The
samples of seeds were obtained from the forest nursery at Prince Albert,
Sask.
The germination was determined by the (routine) standard method in
washed sand in growth chambers at 70 to 72° F with continuous illumination
of 75 ft-c intensity. The final count of germinated seeds was made 30 days
from planting (18).
The percentages of impurities were determined by weight on 10-g samples.
The impurities were separated from the seeds by hand under the dissecting
microscope.
To estimate the percentages of moldy seeds in a sample, seeds of uniform
size were surface-sterilized in 3% aqueous solution of calcium hypochlorite for
5 minutes, washed in three changes of sterilized distilled water, and placed,
20 per petri dish, under aseptic conditions, on malt extract agar (Difco)
containing 7.5% NaC1 (1) and peptone dextrose rose bengal — streptomycin
(100 µg/ml) agar (5). One hundred seeds of each sample were used for this
purpose. Seeds which developed fungus growth were counted as moldy, and
all colonies developed were subcultured on potato dextrose agar (Difco) for
identification.
The numbers of seed-coat fungi and bacteria were estimated by the dilution
plate method, using peptone dextrose rose bengal — streptomycin (100 pg/m1)
agar (5), potato dextrose agar (Difco) acidified to pH 4.2 — 4.5 for estimation
of fungi, and soil extract agar and nutrient agar (Difco) for bacteria. For
preparation of the dilution the seeds (5 g) were suspended in sterile distilled
water (100 ml) and were shaken by hand for 5 minutes.
To evaluate the effect of seed-coat microflora and the soil microorganisms
on survival of the seedlings, local soil was used. This soil was classified as
Bradwell Association of sandy loam texture with neutral hydrogen ion concentration and contained 4% of organic matter (by ignition)*. Forty-eight
quarter-gallon glazed crocks were filled with this soil to about 1 in. from the
top, and divided into two lots, one of which was autoclaved for 21- hours at
15 lb pressure.
Seeds of each species, uniform in size and without impurities, surfacesterilized and unsterilized, were planted, 20 seeds per crock, in four replicates
of each treatment.
Moisture content of the soil was then adjusted to 55% of the total moistureholding capacity and maintained at this level by the constant weight method.
The pots were kept in the greenhouse under continuous illumination (250 ft-c)
*Dr. W. L. Hutcheon, Chairman, Soil Science Department, University of Saskatchewan,
personal communication.
TIMONIN: EMERGENCE OF CONIFER SEEDLINGS
19
at 60-75° F. The emerged seedlings were counted daily and those that died
were marked with toothpicks.
Results
The data summarized in Table I demonstrate the content of impurities in
various seed species. The impurities consisted of broken needles, seed-wings,
damaged seeds, and resin. Thus, the lodgepole pine sample contained a higher
percentage of impurities than the jack pine or white spruce. However, the
impurities of lodgepole pine carried a lower fungus spore and bacteria load
than the impurities of jack pine and white spruce. The data also indicate the
difference in the fungus spore load carried by different seed species. Thus,
jack pine seeds carried four times as many fungus spores as seeds of lodgepole
pine and nearly six times as many as seeds of white spruce. The numbers of
bacteria also varied considerably in different sources of impurities as well as
in various species of seed. It is of interest to note that impurities carried a
markedly higher fungus spore and bacteria load than the seeds of the same
origin. Thus, impurities contained 62, 11, and 28 times as many fungus spores
and 5, 9, and 4 times as many bacteria as the seeds of jack pine, lodgepole
pine, and white spruce respectively. These dense microbial populations will
be an important source of inoculum when introduced into soil.
The data also indicated that surface sterilization had no appreciable effect
on the germination of jack pine and white spruce seeds. However, there was a
slight increase in germination of lodgepole pine seeds.
Furthermore, the data also showed that all species contained a high percentage of moldy seeds. The white spruce sample contained a higher percentage of moldy seeds than the samples of lodgepole pine or of jack pine.
By visual examination under the dissecting microscope, Cytosporella sp.
was observed on the broken needles in the jack pine sample. The growth of
mycelium or fruiting bodies was not observed on the seeds in any samples
investigated. However, it was noticed that a high percentage of white spruce
seeds had minute wounds on the seed-coat.
The microbiological analysis of seed-coat microflora revealed the presence
of species of Alternaria, Aspergillus, Cephalosporium, Chaetomium, Cladosporium, Cylindrocarpon , Fusarium, Gliocladium, Penicillium, Pullularia,
TABLE I
Numbers of fungi and bacteria per gram of coniferous seed, impurities, and percentage
of moldiness
Germination
Seed species
Jack pine
Impurities (6.2%)
Lodgepole pine
Impurities (8.2%)
White spruce
Impurities (6.2%)
Surfacesterilized,
%
67
—
78
—
72
—
Not surfacesterilized,
Fungi
Bacteria
Moldiness,*
(thousands) (thousands)
%
%
68
87.5
112.0
11.3
—
5,494.5
666.0
—
21.5
70
64.0
15.1
—
247.5
618.8
—
71
3,080.0
13.8
19.5
—
387.1
12,554.0
—
*Moldiness = % of surface-sterilized seeds that developed fungus growth.
20
CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 10, 1964
Trichoderma, and others as yet not identified. It was also found that seeds of
white spruce and jack pine carried a heavy load of Aspergillus spores. Several
isolates of Aspergillus from moldy seeds morphologically closely resemble
A. restrictus, the organism responsible for damage to grain in storage (1).
This fungus was able to invade the tissues of radicle and cotyledon of jack
pine (Fig. 1).
The effect of seed and soil treatments on the emergence and postemergence
mortality of the seedlings determined 120 days after planting is shown in
Table II.
Emergence
The data presented indicate that the emergence of jack pine and white
spruce seedlings from untreated seeds was significantly higher than the
emergence from surface-sterilized seeds, in sterilized as well as in unsterilized
soil. On the other hand, the emergence of lodgepole pine seedlings from sterilized seed was significantly higher than that from untreated seeds and occurred
only in sterilized soil.
Sterilization of soil appreciably increased the emergence of white spruce
seedlings from surface-sterilized seeds and that of jack pine seedlings from
untreated seeds.
Soil treatment did not affect the emergence of lodgepole pine seedlings.
Postemergence Mortality
The data (Table II) indicate that mortality of jack pine seedlings from
surface-sterilized seeds grown in sterilized as well as in unsterilized soil was
significantly higher than the mortality among the seedlings from untreated
seeds. The mortality of lodgepole pine seedlings on the other hand was
significantly higher among the seedlings from untreated seeds. The loss of the
white spruce seedlings was significantly higher among those from unsterilized
seeds and only in unsterilized soil.
The sterilization of soil significantly reduced the mortality of the lodgepole
pine seedlings from surface-sterilized as well as from unsterilized seeds, whereas
the decrease in mortality of jack pine and white spruce seedlings due to sterilization of soil occurred only among the seedlings from unsterilized seeds.
TABLE II
Effect of sterilization of seed and soil on the percentages of emergence and postemergence loss
of three coniferous species
Seed surface-sterilized
Seed unsterilized
Soil treatment
A*
Unsterilized
Sterilized
55.0
65.0
Unsterilized
Sterilized
11.4
5.8
B
C
Emergence
70.0
72.5
73.8
65.0
Postemergence loss
21.4
15.3
11.5
6.9
L.S.D. between emergence mean (P =.05) =7.0.
L.S.D. between postemergence loss means (P =.05) =4.8.
*A =Jack pine; B =lodgepole pine; C =white spruce.
A
B
C
45.0
51.3
75.0
78.8
37.5
65.0
22.2
14.6
11.7
3.2
16.7
11.5
PLATE I
FIG. 1.
Seed coat (left) and radicle (right) of jack pine seed attacked by A sper gillus sp.
Titnonin—Can. J. Microbiol.
TIMONIN: EMERGENCE OF CONIFER SEEDLINGS
21
Discussion
In considering the results presented in Table II it should be remembered
that the pathogenicity of the isolated seed-coat and soil microorganisms was
not determined. The chief criterion of the harmful or beneficial effects of seedcoat microflora and soil microorganisms was the percentage of emergence and
postemergence survival of the seedlings.
The data presented in Table II demonstrate a significant reduction in
emergence of jack pine and white spruce seedlings from surface-sterilized
seeds as compared with percentages of germination of surface-sterilized seeds
in sand cultures (Table I). In both cases the seeds used were from the same
surface-sterilized lot. The environmental conditions in sand cultures, however,
were quite different from those of the greenhouse soil cultures. These differences according to Spaulding (17) and Tint (19) could be due to better aeration
in the sand cultures, which probably stimulates emergence and allows the
fungi less opportunity to act upon radicles of the seedlings. In this experiment,
therefore, the fungi causing the moldiness of the seeds after surface-sterilization
(Table I) could become an important factor in the emergence of seedlings. In
this respect it is of interest to note the reports by Simmonds (15) and Ledingham et al. (7). According to them, when wheat seeds were surface-sterilized
prior to inoculation with Helminthosporium sativum, the treatment resulted
in significantly more lesions on the seedlings compared with those developing
from seeds not surface-sterilized. Furthermore, according to them the seed
obtained from greenhouse-grown plants without surface sterilization, on
inoculation also developed a high disease rating. They demonstrated that
seeds from greenhouse-grown plants had a microflora different from that of
seeds from field-grown plants.
The different results obtained with lodgepole pine seeds could possibly be
due to the different seed-coat microflora from that of jack pine and white
spruce seeds. In this respect Aspergillus sp., which was frequently isolated from
jack pine and white spruce seeds (Fig. 1), was not isolated from lodgepole
pine seeds.
The "classical organisms" that cause postemergence damping-off disease
of seedlings, such as Pythium, Phytophthora, and Rhizoctonia, were not isolated
from the seed samples investigated in this experiment. However, the dampingoff symptoms occurred in sterilized soil planted with surface-sterilized seeds.
This would tend to indicate that "saprophytes" of the seed-coat microflora
could, under favorable conditions, attack seedlings and produce symptoms of
damping-off. In this respect the results are in agreement with the findings of
Shea (14), who isolated only species of Aspergillus, Penicillium, and Trichoderma from Douglas-fir seedlings affected by damping-off.
Acknowledgments
The statistical analyses were made by Dr. R. K. Misra and Dr. F. Sosulsky,
University of Saskatchewan, for which the author expresses his sincere
appreciation. The technical assistance of Mrs. A. Leonard is also gratefully
acknowledged.
22
CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 10, 1964
References
CHRISTENSEN, C.
M. Deterioration of stored grains by fungi. Botan. Rev. 23, 108-134
(1957).
I. A. S. Saprophytic fungi as destroyers of germinating pine seed. E. African
Agr. J. 22, 203-206 (1957).
HOLMES, G. D. and BuszEwIcz, G. The storage of seed of temperate forest tree species.
Part I. Forestry Abstr. 19, 313-322 (1958).
HOLMES, G. D. and BUSZEWICZ, G. The storage of seed of temperate forest species. Part II.
Forestry Abstr. 19, 455-476 (1958).
JOHNSON, L. F., CURL, E. A., BOND, J. H., and FRIBOURG, H. A. Methods for studying
soil microflora-plant disease relationships. Burgess Publ. Co., Minneapolis, Minn.
1960.
LAWRENCE, W. H. and REDISKE, J. H. Fate of broadcast seed. Weyerhaeuser Forestry
Research Center. 1961.
LEDINGHAM, R. J., SALLANS, B. J., and SIMMONDS, P. M. The significance of the bacterial
flora on wheat seed in inoculation studies with Helminthosporium sativum. Sci. Agr.
29, 253-262 (1948).
NOBLE, M., DE TEMPE, J., and NECRGAARD, P. An annotated list of seed-borne diseases.
Commonwealth Mycol. Inst. Kew, Surrey, England. 1958.
ORLOVA, A. A. Necotorye dannye o mikoflore semian drevesnykh i kustarnikovykh
porod. Tr. Inst. Lesa Akad. Nauk SSSR, 16, 281-296 (1954).
SALISBURY, P. J. Some aspects of conifer seed microflora. Can. Dept. Agr. Forest Biol.
Div. Bi-Monthly Progr. Rept. 9(6), 3-4 (1953).
SALISBURY, P. J. Moulds of stored Douglas-fir seed in British Columbia. Can. Dept. Agr.
Forest Biol. Div. Interim Rept. 1955.
SHEA, K. R. Problem analysis: Molds of forest tree seed. Weyerhaeuser Timber Co.,
Forestry Res. Center. 1957.
SHEA, K. R. Mold fungi on forest tree seed. Weyerhaeuser Co. Forestry Res. Note 31
(1960).
SHEA, K. R. 1961. Field survival of Thiram-treated Douglas-fir seed. Weyerhaeuser Co.
Forestry Res. Note 38 (1961).
SIMMONDS, P. M. The influence of antibiosis in the pathogenicity of Helminthosporium
sativum. Sci. Agr. 27, 625-632 (1947).
SoHoLov, D. V. I togi fitopatologischeskikh ekspertizy drevesnykh semian na Leningradskoi
seinennoi kontrolnoi stancii. Lesnoe Khoziaistvo. 1940(4). Quoted in Tr. Inst. Lesa
Akad. Nauk SSSR, 16, 281-296 (1954).
SPAULDING, P. The damping-off of coniferous seedlings. Phytopathology, 4, 73-88 (1914).
SWOFFORD, T. F. Proposed standards for tree seed testing for species in the Eastern and
Southern United States. Region 8 Tree Seed Testing Laboratory, Macon, Georgia.
1959.
19. TINT, H. Studies in the Fusarium damping-off of conifers. I. The comparative virulence
of certain Fusaria. Phytopathology, 35, 421-439 (1945).
GIBSON,