INTERACTION OF SEED-COAT MICROFLORA AND SOILMICROORGANISMS AND ITS EFFECTS ON PRE- ANDPOST-EMERGENCE OF SOME CONIFER SEEDLINGS'

M. I. TIMONINForest Pathology Laboratory, Saskatoon, Saskatchewan

Received May 24, 1963

AbstractAnalyses of seed samples of Pinus banksiana Lamb., P. contorta Dougl. var.

latifolia Engelm., and Picea glauca (Moench) Voss demonstrated the presenceof 12 genera of fungi. Pythium, Phytophthora, and Rhizoctonia were not isolatedbut typical symptoms of damping-off disease were observed among the seedlingsgrown in sterilized soil. Surface sterilization of seeds resulted in a significantreduction in emergence of P. banksiana and P. glauca seedlings but not onP. contorta var. latifolia. The cause of this difference is discussed. The post-emergence mortality of P. banksiana seedlings resulting from surface-sterilizedseeds grown in sterilized and unsterilized soil was significantly higher than thatamong the seedlings from untreated seeds. Mortality of P. contorta var. latifoliaseedlings was significantly higher among the seedlings from untreated seeds, andthat of P. glauca seedlings was higher among the seedlings from untreated seedsand only in unsterilized soil.

IntroductionThe microflora of the seed-coat of coniferous and deciduous trees has been

investigated by many workers and its effects on the deterioration of viabilityof 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 post-emergence survival of the seedlings has been neglected. On this subject only afew papers have been published.

Thus, Gibson (2) in East Africa, working with seeds of Pinus patulareported 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 wereable to invade the seed, according to him, through the damaged seed-coat.The field experiments of Lawrence and Rediske (6) with isotope-tagged seedsdemonstrated that the seed-coat microflora was directly responsible for decayof the seed and, indirectly by the weakening of seed vigor, predisposed itto the attack of soil-borne pathogenic fungi. Shea (13) reported that underlaboratory conditions 18 species of fungi commonly associated with seed-coat microflora were capable of destroying Douglas-fir seedlings, under con-ditions favorable to fungus growth.

In Canada, as far as the author is aware, the seed-coat microflora and itseffects on the preemergence and postemergence survival of the seedlings havenot been investigated. The only references related to the seed-coat micro-flora on record are those of Salisbur y (10, 11). According to him there was nodefinite correlation between high fungus spore load and low viability ofDouglas-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 microfloraas well as its effect, if any, on the preemergence and postemergence survivalof the seedlings.

Materials and MethodsSpecies 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 CypressHills; white spruce (Picea glauca (Moench) Voss) lot 1960, Emma Lake. Thesamples of seeds were obtained from the forest nursery at Prince Albert,Sask.

The germination was determined by the (routine) standard method inwashed sand in growth chambers at 70 to 72° F with continuous illuminationof 75 ft-c intensity. The final count of germinated seeds was made 30 daysfrom 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 dissectingmicroscope.

To estimate the percentages of moldy seeds in a sample, seeds of uniformsize were surface-sterilized in 3% aqueous solution of calcium hypochlorite for5 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 thispurpose. Seeds which developed fungus growth were counted as moldy, andall colonies developed were subcultured on potato dextrose agar (Difco) foridentification.

The numbers of seed-coat fungi and bacteria were estimated by the dilutionplate method, using peptone dextrose rose bengal — streptomycin (100 pg/m1)agar (5), potato dextrose agar (Difco) acidified to pH 4.2 — 4.5 for estimationof fungi, and soil extract agar and nutrient agar (Difco) for bacteria. Forpreparation of the dilution the seeds (5 g) were suspended in sterile distilledwater (100 ml) and were shaken by hand for 5 minutes.

To evaluate the effect of seed-coat microflora and the soil microorganismson survival of the seedlings, local soil was used. This soil was classified asBradwell Association of sandy loam texture with neutral hydrogen ion con-centration and contained 4% of organic matter (by ignition)*. Forty-eightquarter-gallon glazed crocks were filled with this soil to about 1 in. from thetop, and divided into two lots, one of which was autoclaved for 21- hours at15 lb pressure.

Seeds of each species, uniform in size and without impurities, surface-sterilized and unsterilized, were planted, 20 seeds per crock, in four replicatesof each treatment.

Moisture content of the soil was then adjusted to 55% of the total moisture-holding 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 diedwere marked with toothpicks.

ResultsThe 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 higherpercentage of impurities than the jack pine or white spruce. However, theimpurities of lodgepole pine carried a lower fungus spore and bacteria loadthan the impurities of jack pine and white spruce. The data also indicate thedifference in the fungus spore load carried by different seed species. Thus,jack pine seeds carried four times as many fungus spores as seeds of lodgepolepine and nearly six times as many as seeds of white spruce. The numbers ofbacteria also varied considerably in different sources of impurities as well asin various species of seed. It is of interest to note that impurities carried amarkedly higher fungus spore and bacteria load than the seeds of the sameorigin. Thus, impurities contained 62, 11, and 28 times as many fungus sporesand 5, 9, and 4 times as many bacteria as the seeds of jack pine, lodgepolepine, and white spruce respectively. These dense microbial populations willbe an important source of inoculum when introduced into soil.

The data also indicated that surface sterilization had no appreciable effecton the germination of jack pine and white spruce seeds. However, there was aslight increase in germination of lodgepole pine seeds.

Furthermore, the data also showed that all species contained a high per-centage of moldy seeds. The white spruce sample contained a higher percent-age 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 ofmycelium or fruiting bodies was not observed on the seeds in any samplesinvestigated. However, it was noticed that a high percentage of white spruceseeds had minute wounds on the seed-coat.

The microbiological analysis of seed-coat microflora revealed the presenceof species of Alternaria, Aspergillus, Cephalosporium, Chaetomium, Clado-sporium, Cylindrocarpon , Fusarium, Gliocladium, Penicillium, Pullularia,

TABLE INumbers of fungi and bacteria per gram of coniferous seed, impurities, and percentage

of moldiness

Seed species

Germination

Fungi(thousands)

Bacteria(thousands)

Moldiness,*%

Surface-sterilized,

%

Not surface-sterilized,

%Jack pine 67 68 87.5 112.0 11.3

Impurities (6.2%) — — 5,494.5 666.0 —Lodgepole pine 78 70 21.5 64.0 15.1

Impurities (8.2%) — — 247.5 618.8 —White spruce 72 71 13.8 3,080.0 19.5

Impurities (6.2%) — — 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 ofwhite spruce and jack pine carried a heavy load of Aspergillus spores. Severalisolates of Aspergillus from moldy seeds morphologically closely resembleA. restrictus, the organism responsible for damage to grain in storage (1).This fungus was able to invade the tissues of radicle and cotyledon of jackpine (Fig. 1).

The effect of seed and soil treatments on the emergence and postemergencemortality of the seedlings determined 120 days after planting is shown inTable II.

EmergenceThe data presented indicate that the emergence of jack pine and white

spruce seedlings from untreated seeds was significantly higher than theemergence from surface-sterilized seeds, in sterilized as well as in unsterilizedsoil. On the other hand, the emergence of lodgepole pine seedlings from steril-ized seed was significantly higher than that from untreated seeds and occurredonly in sterilized soil.

Sterilization of soil appreciably increased the emergence of white spruceseedlings from surface-sterilized seeds and that of jack pine seedlings fromuntreated seeds.

Soil treatment did not affect the emergence of lodgepole pine seedlings.

Postemergence MortalityThe data (Table II) indicate that mortality of jack pine seedlings from

surface-sterilized seeds grown in sterilized as well as in unsterilized soil wassignificantly higher than the mortality among the seedlings from untreatedseeds. The mortality of lodgepole pine seedlings on the other hand wassignificantly higher among the seedlings from untreated seeds. The loss of thewhite spruce seedlings was significantly higher among those from unsterilizedseeds and only in unsterilized soil.

The sterilization of soil significantly reduced the mortality of the lodgepolepine seedlings from surface-sterilized as well as from unsterilized seeds, whereasthe decrease in mortality of jack pine and white spruce seedlings due to steril-ization of soil occurred only among the seedlings from unsterilized seeds.

TABLE IIEffect of sterilization of seed and soil on the percentages of emergence and postemergence loss

of three coniferous species

Soil treatment

Seed unsterilized Seed surface-sterilized

A* B C A B C

EmergenceUnsterilized 55.0 70.0 72.5 45.0 75.0 37.5Sterilized 65.0 65.0 73.8 51.3 78.8 65.0

Postemergence lossUnsterilized 11.4 21.4 15.3 22.2 11.7 16.7Sterilized 5.8 11.5 6.9 14.6 3.2 11.5

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.

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

DiscussionIn considering the results presented in Table II it should be remembered

that the pathogenicity of the isolated seed-coat and soil microorganisms wasnot determined. The chief criterion of the harmful or beneficial effects of seed-coat microflora and soil microorganisms was the percentage of emergence andpostemergence survival of the seedlings.

The data presented in Table II demonstrate a significant reduction inemergence of jack pine and white spruce seedlings from surface-sterilizedseeds as compared with percentages of germination of surface-sterilized seedsin sand cultures (Table I). In both cases the seeds used were from the samesurface-sterilized lot. The environmental conditions in sand cultures, however,were quite different from those of the greenhouse soil cultures. These differ-ences according to Spaulding (17) and Tint (19) could be due to better aerationin the sand cultures, which probably stimulates emergence and allows thefungi 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. Inthis respect it is of interest to note the reports by Simmonds (15) and Leding-ham et al. (7). According to them, when wheat seeds were surface-sterilizedprior to inoculation with Helminthosporium sativum, the treatment resultedin significantly more lesions on the seedlings compared with those developingfrom seeds not surface-sterilized. Furthermore, according to them the seedobtained from greenhouse-grown plants without surface sterilization, oninoculation also developed a high disease rating. They demonstrated thatseeds from greenhouse-grown plants had a microflora different from that ofseeds from field-grown plants.

The different results obtained with lodgepole pine seeds could possibly bedue to the different seed-coat microflora from that of jack pine and whitespruce seeds. In this respect Aspergillus sp., which was frequently isolated fromjack pine and white spruce seeds (Fig. 1), was not isolated from lodgepolepine seeds.

The "classical organisms" that cause postemergence damping-off diseaseof seedlings, such as Pythium, Phytophthora, and Rhizoctonia, were not isolatedfrom the seed samples investigated in this experiment. However, the damping-off symptoms occurred in sterilized soil planted with surface-sterilized seeds.This would tend to indicate that "saprophytes" of the seed-coat microfloracould, under favorable conditions, attack seedlings and produce symptoms ofdamping-off. In this respect the results are in agreement with the findings ofShea (14), who isolated only species of Aspergillus, Penicillium, and Tricho-derma from Douglas-fir seedlings affected by damping-off.

AcknowledgmentsThe statistical analyses were made by Dr. R. K. Misra and Dr. F. Sosulsky,

University of Saskatchewan, for which the author expresses his sincereappreciation. The technical assistance of Mrs. A. Leonard is also gratefullyacknowledged.

22 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 10, 1964

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