THE SIGNIFICANCE OF SOIL MICROORGANISMS AS A LIMI- TING FACTOR IN INFECTION OF CLOVER BY SCLEROTINIA TRIFOLIORUM ERIKSS. AT DIFFERENT TIMES OF THE YEAR Anna-Marja Halkilahti Department of Plant Pathology, University of Helsinki Received May 27, 1964 Antibiotic microorganisms limiting the growth of fungi are found very fre- quently in the soil (4, 5, 27). Some of these microorganisms are also presumed to restrict the mycelial growth in the soil of the causal agent of clover rot ( Sclerotinia trifoliorum Erikss.) (9, 21). Microbes with such an antibiotical effect on artificial culture media have been isolated from various parts of Finland (21, 22, 23, 24). It was observed during these studies that environmental factors, such as temperature and the quality of the growth medium, have a marked effect on the antibiotical capac- ity of the microbes (10, 22). The present paper describes investigations on the antagonistic effectivity of the soil due to microorganisms (cf. Pohjakallio, 20) on the extent of clover rot infection in the soil. The principal object of these studies was the variation in the antagonistic effectivity of the soil at different times of the year. A second point investigated was the effect of the numbers of living microbes in the soil on clover rot infection. Experimental methods The trials were begun each year in April and continued until November or December. During this time, when the ground was not covered by snow, soil sam- ples were taken about once a week from cultivated land. The samples were put in sterilized petri dishes (0 9 cm); 5 dishes per sample were used in 1961 and 6 in the other years. In addition, soil from each sample was autoclaved and put into 3 petri dishes. The soil was moistened with sterilized water. Mycelia of S. trifoliorum were transferred to the surface of the soil in the centre of the dish. Observations were subsequently made on the spreading of the mycelia on the soil surface. Since the mycelia of the clover rot fungus are often invisible to the naked eye, clover seed was sown in each dish so that the antagonistic capacity of the soil could be determined on https://www.c-info.fi/en/info/?token=BjwyCeQNNycxrbbg.NfNg6R_jkuIZWVV6nDr18Q.bR6vYnbT1UJw4XvZXa6D0OBD_JefRkjm2SPtDnizJtFHigcJ5oJ3FplhcqNFrbWJ019gAP0ln8I0eI4T6HpZ00Itk7FKmAQcDhkpDjOntEq34nElclYKB-JPgKK59YbFwQ_oCXmwvMSBY9fD8qimKMFGHGTFH-QBz_1jNpw19g-39fQ 121 the extent of the infection of the clover seedlings. Just before seeding, the seed was dressed with a solution of Ceresan and carefully rinsed with sterilized water. In sowing the nearest seeds were located at least 1.2 cm from the transferred mycelium of S. trifoliorum (Fig. 1). In the years 1957—61 the petri dishes were kept inathermostat at a temperature of 7—lo°C and in 1961—63 they were kept outdoors in a shaded place. Accordinlyg, within one year (1961) similar trials were carried out both in the thermostat and outdoors. The extent of infection of the clover seedlings by S. trifoliorum was eval- uated after the two-week test period. If the fungus had infected the seedlings this was evidently caused by a reduction in the antagonistic effectivity of the soil. If the fungus did not produce infection, this was held to indicate an increased antagonistic capacity of the soil. In the tables showing the results of the trials, the percentages of the petri dishes in which the clover seedlings were infected by clover rot are in- dicated. Soil samples were taken from a depth of o—s cm from the following fields: a) fallow, b) limed fallow, c) winter rye, d) soil where clover has been sown (with spring wheat as nurse crop) in the year of the test, as well as soil where clover had been sown in the previous year, e) unlimed area, and f) limed area. The main soil type was sandy clay with a pH of 5,5—6,0. Ground limestone was applied to the Fig. 1. The growth ofS. trifoliorum mycelia in the soil samples was determined by sowing dower in the petri dishes; the smallest distance from the seeds to the transferred mycelia in the centre of the dish was 1.2 cm. 122 surface layer of the soil (0—10 cm) in the spring before the commencement of the trials. In 1962 an amount of 12 000 kg/ha was applied, with the result that the soil became alkaline. In the following year, 1963, only 6 000 kg/ha were used. In 1962 the pH of the fallow was 5.4—5.8, that of the limed fallow 7.3 —8.0 and that of the clover field 6.0—6.5. In 1963 the pH values were: fallow 5.1—5.5, limed fallow 6.2— 7.0, clover field 5.6—6.3 and limed clover field 6.3—7.5. The pH of the soil samples was not determined in the other years. Mycelia of S. trifoliorum for the trials were cultured at a temperature of 7 —lo°C on Henneberg agar (dist. water 100 ml, glucose 10 g, CaCl 0.01 g, MgS04 0.05 g, KN03 0.2 g, NH 4H2P04 0.2 g, peptone 1 g). After two weeks of growth the mycelia were transferred in the form of agar tablets I—21 —2 mm thick (0 0.5 cm) to the surface of the soil in the petri dishes. The clover rot fungus degenerated when continually cultured on artificial me- dium and had to be replaced twice with a more vigorous growth. In 1957—58 a single-spore strain isolated at Viik was used, in 1959—60 a saltant of this strain was used, and in the years 1961—63 the fungus employed had been isolated in the autumn of 1960 from mycelia growing in an infected red clover plant in the field. During several years a sporadic decline in growth and also in the infective capacity of the clover rot fungal mycelia was observed. No reason for this could be found. Counts of microorganisms in the soil samples Directly before the antagonistic effectivity of the soil was determined, microbe counts were made of the soil samples. These counts were made by means of the dish dilution method (14), which is based on the development of living cells into colonies when growing on artificial media. The substrate used was soil extract agar prepared in the following manner: The soil extract consisted of soil and water in a ratio of 1:1 which was autoclaved for 30 min; a small amount of CaC03 was added and mixed; the solution was clarified by centrifuging and water was added to the original volume. The final agar medium consisted of 1000 g soil extract, 0.2 g K 2HP04 , 15 g agar; pH adjusted to about 6.0 with NaOH. Since the substrate was soil extract, the colo- nies consisted principally of native (autoctonic) soil microorganisms (14). Before the counting was undertaken, the soil samples were diluted by adding 1 g soil to 99 ml 0.05 % peptone solution, which was further diluted (1:10). The most suitable dilution proved to be 1:100 000. An 1-ml portion of it was added to a petri dish of liquid soil extract agar (+45°C). The dishes (2 replicates per the soil sample) were held for 14 days at a temperature of about +2O°C, after which the number of colonies was visually counted. Among the various microorganisms in the soil samples, the bacteria proper comprised about 82—93 %, the actinomycetes about I—61 —6 % and the fungi about 5—16 %. Correlation calculations for the trial results were performed according to Mudra (15). In the outdoor trials determinations were made of the 123 Table 1. Infection of red clover seedlings by 5. trifoliorum in sterilized and unsterilized soil samples. % of petri dishes Total no. Year Soil sample ... . , . , , Trialv with infected of seedlings dishes " Sterilized Unsteri- Sterilized Unsteri- soil lized soil soil lized soil A. THERMOSTAT (7-10°C) 1957 Fallow 100 39 93 186 17.4.-13.12. 1958 Winter rye field 99 57 138 276 14.4.-10.12. 1959 Fallow 100 45 189 378 2.4.-17.12. Clover field (year of sowing) 99 38 189 378 2.4.-17.12. 1960 Fallow 100 22 135 270 21.4.-20.12. Clover field (Ist year) 98 23 135 270 21.4.-20.12. Clover field (year of sowing) 99 20 126 252 29.4.-20.12. 1961 Fallow 91 15 90 150 17.4.- 9.12. Clover field (Ist year) 88 15 90 150 17.4.- 9.12. Clover field (year of sowing) 86 6 90 150 17.4.- 9.12. Average 97 31 Total 1275 Total 2460 Av. temp, during 4-day period before start of trial: ■" ( 10.1 15.0'C 15.1-20.0°C 20.1/C < Source of soil sample . . c. . , ~ ~ o,- t . ;lr Autumn Spring Autumn Summer Summer Summer (taken in April - December) v. +T~ZZ „ Of Of f Of Of of % o■§ % 6-g % 6 * % 6 % % 6•£ % 6-S g. =5 6. =5 6, =5 6. 6, 'S 6. '-a 901 Fallow 80 (20) 16 (25) 53 (30) 5 (20) 0 (40) 0 (5) Clover field (Ist year) 55 (20) 36 (25) 47 (30) 15 (20) 3 (40) 0 (5) Clocer field (year of sowing) 65 (20) 24 (25) 57 (30) 5 (2(1) 0 (40) 0 (5) 962 Fallow 01 (30) 57 (30) 10 (42) 14 |72] IT (12) Limed fallow 25 (36) 13 (24) 0 (42) 0 (72) 0 (12) Clover field (Ist year) 47 (36) 60 (30) 5 (42) 8 (72) O (12) 963 Fallow 75 (18) 58 (18) 44 (18) 33 (42) 2 (48) Limed fallow 42 (18) 25 (18) 17 (18) 12 (42) 0 (48) Clover field (Ist year) 71 (18) 67 (18) 56 (18) 17 (42) 0 (48) Limed clover field (Ist year) 17 (18) 0 (18) 6 (18) 0 (42) 0 (48) Average 55 32 26 9 1 0 (Total) (240) (231) (288) (444) (348) (15) Fig. 3. The number of soil microorganisms as well as the percentage of infection of clover in petri dishes by S. trifoliorum in soil samples taken from limed and unlimed fields between April and November (total number of dishes about 190 in 1962, 144 in 1963; cf. Fig. 4). Table 4. Numbers of microorganisms in soil samples taken in April —December. Results grouped according to average temperatures during the 4-day periods before the counts were made. (The figures in brackets indicate the numbers of soil samples counted). No. of microorganisms (in millions per 1 g soil) Year Source of soil .. „ 10,-0-5.O°C 5.1-10.0°C 10.-15.0°C 15.1-20.0°C 20.1°C< 0-21 C sample Whole tria Spring Autumn Spring Autumn Summer Summer Summer period 1958 Winter rye field 2.3 (8)16.2 (9) 10.7 (4) 18.0 (8) 12.4(12) 7.7 (4) 36.0 (1) 12.3(46) 1959 Fallow 6.3 (7) 7.0 (20) 7.0 (5) 7.2 (6) 6.5 (10) 6.7 (12) 6.6 (3) 6.8 (63) Clover field (year of sowing) 6.5 (7) 12.6 (20) 6.8 (5) 15.0 (6) 8.8 (10) 11.3 (12) 8.7 (3) 10.6 (63) 1960 Fallow 8.8 (4) 19.1 (11) 6.9 (1) 15.7 (6) 15.9 (9) 15.4 (12) 16.2 (2) 15.7 (45) Clover field (Ist year) 8.3 (4) 14.3 (11) 7.1 (1) 14.3 (6) 13.4 (9) 12.3 (12) 9.9 (2) 12.7 (45) Clover field (year of sowing) 11.4 (2) 16.5 (11) 15.7 (6) 17.1 (9) 13.8 (12) 9.7 (2) 15.2 (42) 1961 Fallow 13.5 (2) 6.8 (5) 13.6 (5) 8.6 (6) 8.9 (3) 11.6 (8) 11.9 (1) 10.4 (30) Clover field (Ist year) 12.2 (2) 7.1 (5) 19.4 (5) 8.2 (6) 13.0 (3) 12.0 (8) 11.4 (1) 11.7 (30) Clover field (year of sowing) 10.2 (2) 6.8 (5) 14.0 (5) 14.9 (6) 8.5 (3) 9.1 (8) 7.1 (1) 11.3 (30) 1962 Fallow 5.3 (3) 7.8 (4) 6.0 (2) 9.4 (7) 7.6 (12) 4.8 (4) 7.4 (32) Limed fallow 7.7 (2)12.1 (4) 9.3 (2) 13.5 (7) 11.5 (12) 5.9 (4) 10.9 (31) Clover field (Ist year) 5.7 (3)10.6 (4) 10.8 (2) 15.1 (7) 10.7 (12) 8.8 (4) I 1.0 (32) 1963 Fallow 8.8 (3) 5.9 (3) 10.6 (3) 6.4 (7) 5.6 (8) 6.9 (24) Limed fallow 13.0 (3) 6.9 (3) 13.2 (3) 8.3 (7) 9.4 (8) 9.7 (24) Clover field (Ist year) 10.9 (3) 8.5 (3) 12.5 (3) 9.0 (7) 6.9 (8) 8.9 (24) Limed clover field (Ist year) 15.0 (3) 7.8 (3) 18.3 (3) 10.7 (7) 11.8 (8) 12.2 (24) Average 6.9 12.0 10.2 13.2 10.6 10.2 11.5 11.0 (Total) (46) (121) (49) (Nil) (132) (132) (Hi) (585) and at different times of the year (Fig. 4). In general, however, there were relatively small variations in infection between the soil samples taken at the same time from the various areas of the field cultivated in different ways. Nevertheless, in some autumns infection was less marked in the samples taken from clover fields than in those from fallow (Fig. 4). Furthermore, liming appreciably reduced the extent of infection (Table 1; Figs. 3 and 4). In the outdoor trials, clover was more severely infected in the spring and autumn than in the summer; often it did not become infected at all in the summer (Fig. 4; Table 3). The maximum infection occurred late in the autumn, at a time when the temperature was lowest (0—5°C) (Table 3). In the thermostat trials (7 —10°C) the clover rot fungus generally caused considerable infection also in the soil samples taken in the summer (Fig. 4; Table 2). A statistically significant negative correla- tion occurred between the infection of clover by S. trifoliorum and the temperature during the 2-week trial period (Table 5, B). 126 127 Table 5. The effect of the number of soil microorganisms and the temperature on the infection of red clover by S. trifoliorum, as determined by correlation calculations. (The infection was judged on a scale of 0 6 in which 6 = plants in all the 6 replicate dishes were infected; in 1961 the scale was 0 —5). Regression coefficients b, b 2Year Soil sample taken in No. of .. . ,r No. of microbes Av. temp, during April December trials , , . . „ , , . .r start of trial 2-week trial (mill, per 1 g soil) (X) A. THERMOSTAT (7-10X) 1957 Fallow 31 - 1958 Winter rye field 46 -0.05 1959 Fallow 63 -0.04 Clover field (year of sowing) 63 —o.23*** 196(1 Fallow 45 -0.06 Clover field (Ist year) 45 -0.02 » » (year of sowing) 42 —O.OB 1961 Fallow 30 -0.10 Clover field (Ist year) 30 -0.05 » » (year of sowing) 30 —0.04 B. OUTDOORS 1961 Fallow 28 -0.12 -0.17** Clover field (Ist year) 28 +O.Ol -o.l9*** » » (year of sowing) 28 +0.03 -o.2l*** 1962 Fallow 32 -0.14 -0.22** Limed fallow 31 -0.04 -o.ll** Clover field (Ist year) 32 -0.09 -0.20** 1963 Fallow 24 +0.28 -0.25** Limed fallow 24 -0.05 -o.lB*** Clover field (Ist year) 24 -o.oB** -o.4B*** Limed clover field (Ist year) 24 +0.03 -0.05* In the thermostat trials (7 —10°C) (Table 2), as well as in the outdoor trials when the temperature was s—lo°C (Table 3), 5. trifoliorum generally caused more severe infection of clover in the spring than in the autumn. In 1961, however, in- fection was much more severe in the autumn than in the spring (Tables 2 and 3). As a rule, the maximum numbers of microorganisms in the soil were found in the autumn and the minimum numbers in the spring (Table 4). In 1961, however, they were more numerous in the spring. Larger amounts of soil microbes were often encountered in the samples taken from the clover field than in those from the fallow- ed area; this was especially evident in the autumn. Liming resulted in an increase in the numbers of microorganisms (Fig. 3). In the thermostat trials (7—10°C) there was always a negative correlation between the infection of the clover plants and the numbers of microorganisms in the soil at the onset of the trial. However, only in one case was this correlation statistically significant (Table 5, A). In the outdoor trials the correlation between these factors varied from year to year (Table 5, B). Taking into account, however, 128 only those results obtained at the temperature range s—lo°C,5—10°C, it can be seen that when there were small numbers of microorganisms in the soil, S. trifoliorum gen- erally grew better than when the numbers were large (Fig. 6). Discussion In general, clover plants become infected with the clover rot fungus (Scleroti- nia trifoliorum )in the autumn. The damage may continue during the winter and spring, but in the middle of the summer the disease generally does not occur. This Fig. 4. The infection of red clover seedlings by S. trifoliorumin soil samples in different months. The amount of infection is denoted by the columns whose height indicates the percentage of pelri dishes in which the seedlings were infected. The number of trials in each month is shown by the numerals; in each trial there were 6 petri dishes (■"> in 1961). WvVVV\A W = the mycelia of S. trifoliorum used for inoculating the soil samples grew poorly (cf. p. 122) has been attributed mainly to the fact that the dry conditions usually accompanying the high temperatures of the summer check the growth of the clover rot fungus (2, 3,7, 13, 17, 18, 19, 25, 29). However, in the present trials, the infection was generally lightest in the middle of the summer also when the clover was grown in petri dishes where the soil was constantly moist. This is most clearly seen in the outdoor trials (Fig. 4, B) performed during the warmest period of the summer (Fig. 5) and indicates that the temperature had a preventive effect on clover rot infection especially during the actual time of the trial (Table 3). In checking infection the Fig. 5. Long-term mean temperatures (I °C) a«rf precipitation (I mm) a? Helsinki in the months April December 1921 1950 as well as their deviations at Viik 1957—1963 129 130 temperature scarcely played a direct part, since it is known that the mycelia pf Sclerotinia trifoliorum can grow throughout a temperature range of o—33°C0—33°C (16), the optimum being 13—20°C (7, 11, 16, 18). Autoclaving of the soil had a marked effect in increasing the amount of infec- tion of the clover seedlings growing in it (Table 1). This may show that the micro- organisms normally occurring in soil limited the extent of clover rot infection in unsterilized soil. The formation of mycelia from sclerotia on the surface of auto- claved soil is also very abundant (6). Furthermore, it is known that autoclaving changes the soil nutrients into a form readily utilizible by fungal organisms (30), but at present it is not known whether this could lead to an increase in clover rot infection. The number of soil microorganisms was not found, however, to be greater during the warm part of the summer whenclover rot infection was slight (Table 3; Fig. 4, B) than in the spring and autumn (Table 4; cf. 28). On the other hand, at relatively low temperatures (5—10°C) the extent of infection decreased as the number of soil microbes increased (Table 5, A; Fig. 6). As a rule, there were less microbes in the soil in the spring than in the autumn (Table 4). Correspondingly, clover rot infection was usually (at s—lo°C5—10°C temperatures) more severe in the spring than in the autumn (Tables 2,3). In this case, accordingly, the number of microorganisms determined the antagonistic power of the soil. At other temperatures such a corre- lation was not observed (Table 5, B), which suggests that the effect of soil micro- organisms in hindering clover rot infection (Table 5, B) is mainly due to their antag- onistic influence. As is generally known, a rise in temperature increases the rate of metabolism of microbes (26) as well as the formationof many antibiotic substances (cf. 12). On certain artificial media, the infection-checking effect of the soil micro- organisms was also found to be enhanced by an increase in the temperature (Fig. 7). Fig. 6. The numbers of soil microorganisms and the amount of infection of red clover by S. trifolio- rum in soil samples in trialsperformed outdoors at a temperature of 5 10°C. 131 It is known (10, 16) that S. trifoliorum can grow in a wide range of pH concentra- tions, between 2.1 and 8.0; its optimum is pH 5.5—6.5 (16). In the current trials, treating fields with lime which raised the soil pH to the alkaline side appreciably increased the number of microorganisms and at the same time reduced the amount of clover rot infection (Fig. 3). In earlier studies (1, 16, 18, 19) the soil reaction was not known to have influenced the occurrence of clover rot on the field. This was possibly due to the fact that the fungus spread in the form of spores directly on the leaves of the clover plants (cf. 9, 13, 25). In the present trials, on the other hand, the clover became infected from the mycelia growing in the soil; such mycelial infection may also take place under normal field conditions (7, 8, 31). The clover plants were less severely infected by the fungus in limed than in unlimed soil samples also in the cases when the soil was sterilized (Table 1). Like- wise, the infection was somewhat weakened at high temperatures even in the steriliz- ed samples (Table 1). This can be taken as being due to the fact that the autoclaving of the soil although it killed the bacteria did not completely destroy all the antibiotic substances in the soil. However, since the effect of soil sterilization in enhancing clover rot was even more distinct in the thermostat (7 —10°C) trials than in those performed outdoors under varying temperature conditions, it appears that also other factors did to some extent modify the results. In this case attention can Fig. 7. The effect of a soil inoculum transferred to nutrient agar medium on the growth of S. trifoliorum at different temperatures. S. t r ifol i o r urn was inoculated to the lower edge of the dish, the soil to the upper edge. The growth is 2 weeks old. The soil contained microorganisms such as Muco r, Trichoderma and bacteria. 132 be drawn to the fact that sometimes due to unknown reasons the mycelial growth of the fungus declined, a phenomen on which often occurred during the warm part of the summer (Fig. 4). Conclusions In the petri dish trials the mycelia of Sclerotinia trifoliorum in sterilized soil samples generally infected clover quite readily. In unsterilized samples the infection was less severe and showed marked variations in different years and at different times of the year. In the outdoor trials clover became less infected in the middle of the summer than in the spring and autumn. On the other hand, in the thermostat trials, where the temperature was constantly 7—lo°C, the temperature at the time of taking the soil samples did not have a pronounced effect on the extent of clover infection. Liming of the soil caused a marked decrease in the severity of clover rot infection. In some autumns clover plants growing in soil samples taken from fallow were more seriously infected than those growing in soil from a clover field. As a rule, however, there were only slight variations in the extent of infection in the soil samples taken at the same time from the various areas of the field cultivated in different ways. In general, the highest numbers of microorganisms in the soil were found in the autumn and the lowest in the spring. In clover fields there were often more micro- organisms particularly in the autumn than in fallow. Liming caused an in- crease in the numbers of soil microbes. S. trifoliorum infected clover very severely at temperatures of o—s°C;0—5°C; in the range s—2l°C the infection grew generally milder the more the temperature was found to be rising. At temperatures of s—lo°C5—10°C an increase in the numbers of soil microbes resulted in a decrease in the infection of the clover. 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A. 1945. Microbial antagonisms and antibiotic substances. 350 p. New York. (28) Waksman, S. A. & Starkey, R. L. 1950. The soil and the mikrobe. 260 p. New York. (29) Valle, O. 1930. Apilamädästä ja sen tuhoista. Suomen laiduntalous 2: 33 46. (30) Wright, J. M. 1955. The production of antibiotics in soil. 11. Production of griseofulvin by Peni- cillium nigrigans. Ann. Appi. Biol. 43,2: 288 296. (31) Ylimäki, A. 1956. Additional Experiments on the Chemical Control of Clover Rot. Valt. maatal. koet, julk. 148: 31 49. 134 SELOSTUS: MAAN PIENELIÖSTÖN MERKITYKSESTÄAPILAN SCLEROTINIA TRIFOLIORUM ERIKSS.- INFEKTIOTA RAJOITTAVANA TEKIJÄNÄ VUODEN ERI AIKOINA ANNA-MaRJA H ALKU.AHTI Helsingin yliopiston kasvipatologian laitos Petrinmaljakokeissa Sclerotinia trifoliorumin rihmasto saastutti steriloiduissa maanäyt- teissä apilaa yleensä verraten hyvin. Steriloimattomissa maanäytteissä sen saastutuskyky oli pienempi ja eri vuosina sekä eri vuodenaikoina varsin vaihteleva. Ulkona suoritetuissa kokeissa apila saastui lievemmin keskikesällä kuin keväällä ja syksyllä. Sen sijaan termostaattikokeissa, joissa lämpö- tila oli jatkuvasti 7 10°C, ei maanäytteen ottamisajankohdanlämpötilalla ollut selvää vaikutusta apilan saastumiseen. Kalkitus heikensi apilamätäsienen saastutuskykyä huomattavasti. Eräinä syksyinä apilamätä- sieni saastutti ankarammin kesannosta kuin apilapellosta otetussa mullassa kasvanutta apilaa. Yleensä ei apilamätäsienen saastutuskyky kuitenkaan kovin paljon vaihdellut saman peltolohkon eri tavoin viljellyistä osista keskenään samaan aikaan otetuissa maanäytteissä. Mikrobeja oli maassa yleensä eniten syksyllä ja vähiten keväällä. Apilapellossa oli mikrobeja usein, etenkin syksyisin, enemmän kuin kesantomaassa. Kalkitus lisäsi mikrobien määrää. S. trifoliorum saastutti apilaa hyvin ankarasti 0— 5 °C:n lämpötiloissa ja 5 —2l°C;n lämpötiloissa yleensä sitä lievemmin mitä korkeampi lämpötila oli. s—lo°C;n lämpötiloissa apilan saastuminen heik- keni maan mikrobiluvun noustessa. Korkeammat lämpötilat, jotka eivät suurentaneet mikrobien lukua maassa, lisäsivät kuitenkin maan antagonistista voimaa.