by Bacteria in Soil: Clay Minerals and ph

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1979, p /79/ /07$02.00/0 Vol. 38, No. 6 Influence of Environmental Factors on Antagonism of Fungi by Bacteria in Soil: Clay Minerals and ph WILLIAM D. ROSENZWEIGt AND G. STOTZKY* Laboratory of Microbial Ecology, Department of Biology, New York University, New York, New York Received for publication 19 September 1979 The soil replica plating technique was used to evaluate the influence of clay minerals and ph on antagonistic interactions between fungi and bacteria in soil. In general, the antagonistic activity of bacteria towards filamentous fungi was greater in soil than on agar. The spread of Aspergillus niger through soil was inhibited by Serratia marcescens when the organisms were inoculated into separate sites in soil, and this antagonistic effect was maintained when the soil was amended with 3, 6, 9, or 12% (vol/vol) montmorillonite, whereas the addition of kaolinite at a concentration of 3% reduced the antagonism and at 6, 9, or 12% totally eliminated it. Similar results were obtained with the inhibition of A. niger by Agrobacterium radiobacter and of Penicillium vermiculatum by either S. marcescens or Nocardia paraffinae. When A. niger and S. marcescens were inoculated into the same soil site, A. niger was inhibited in all soils, regardless of clay content, although the extent of inhibition was greater as the concentration of montmorillonite, but not of kaolinite, increased. A. niger was inhibited more when inoculated as spores than as mycelial fragments and when inoculated 96 h after S. marcescens, but a 1% glucose solution reduced the amount of inhibition when the fungus was inoculated 96 h after the bacterium. When the ph of the soil-clay mixtures was altered, the amount of antagonism usually increased as the ph increased. Antagonism appeared to be related to the cation-exchange capacity and the ph of the soil-clay mixtures. Bacillus cereus and another species of Bacillus showed no activity in soil towards A. niger under any of the environmental conditions tested, even though the Bacillus sp. significantly inhibited A. niger and seven other fungi on agar. Soil is a reservoir for many microbial pathogens, including ones for human beings (16, 27, 31) and plants (4, 10, 14, 30, 33). Although numerous attempts have been made to control or eliminate these pathogens in soil by the use of other microorganisms, biological control has only been partially successful (9-11, 36). The methods used in an attempt to control pathogens have been either direct, by introducing antagonists into soil (9, 10, 11), or indirect, by altering the physicochemical properties of soil and, thus, stimulating the naturally occurring antagonistic microbiota (1, 3, 9, 27, 30, 31, 33). The present study used nonpathogenic microorganisms as model systems to evaluate the influence of some physicochemical environmental factors on the control of microbes in soil. Antagonistic interactions between microbes were studied by a technique that allowed for repeated observations of numerous sites with a minimum of disturbance of the cells and soil t Present address: Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, NJ particles. Problems resulting from artificial growth surfaces were eliminated, and conditions that exist in soil in situ were simulated (24, 29). MATERIALS AND METHODS Microorganisms. Microorganisms were obtained from the stock culture collection of the Laboratory of Microbial Ecology at New York University, the American Type Culture Collection, and the Midwest Culture Service. Media. Nutrient-cycloheximide agar (nutrient agar [Difco], 23.0 g; cycloheximide [Actidione, Upjohn Co.], 100 mg; and water, 1 liter, ph 6.8) was used for replica plating of bacteria from soil; bacterial cultures were also maintained and grown on this medium without cycloheximide. Sabouraud-dextrose-rose bengal-streptomycin agar (Sabouraud-dextrose agar [Difco], 65.0 g; rose bengal [Allied Chem. Co.], 33.3 mg; streptomycin sulfate [Sigma Chem. Co.], 80 mg; and water, 1 liter, ph 5.6) was used for replica plating of fungi from soil; fungal cultures were also maintained and grown on this medium without rose bengal and streptomycin sulfate. Toxin assay agar (glucose, 30.0 g; NaNO3, 3.0 g; K2HPO4, 1.0 g; MgSO4. 7H20, 0.5 g; KCl, 0.5 g; peptone 1120

2 VOL. 38, 1979 [Difco], 10.0 g; agar [Difco], 15.0 g; and water, 1 liter, ph 6.5) was used in pure culture studies on the ability of bacteria to produce toxins against filamentous fungi. Soils. Soil obtained from the Kitchawan Reseach Laboratory of the Brooklyn Botanic Garden, Ossining, N.Y., was amended with either montmorillonite (Volclay, Panther Creek-Aberdeen, American Colloid Co.), with a cation-exchange capacity (CEC) of approximately 60 milliequivalents (meq) per 100 g (25), or kaolinite (Continental, R. T. Vanderbilt Co.), with a CEC of approximately 6.5 meq/100 g (25), to yield approximate concentrations of 3, 6, 9, and 12% clay (vol/vol). The clays and soil were thoroughly mixed in an electric cement mixer, and the soil-clay mixtures were stored in metal garbage cans (ca. 75 liters) lined with plastic. Some chemical and physical properties of the soil, unamended and amended with clay, have been presented elsewhere (2). X-ray diffraction analysis showed that this soil does not naturally contain montmorillonite-type clay minerals but does contain mica-illite, kaolinite, and vermiculite. Soil replicator. The soil replica plating apparatus has been described elsewhere (24, 29). Experimental design. (i) Soil studies. The soilclay mixtures were placed in plastic bags, and water was added to bring the mixtures to 2% above their 1/ 3-bar tension water content determined with a 5-bar pressure plate extractor (Soilmoisture Equipment Corp., Santa Barbara, Calif.). After equilibration for 24 h at 4 C, the mixtures were passed through a sieve with 2-mm openings, and 40 g of the sieved mixture was dispensed into a heavy walled petri dish and leveled by gentle tapping on a large rubber stopper. Desired inoculation sites were marked, with the aid of a template, by making small depressions in the soil surface, and the soil plates were autoclaved for 0.5 h at 121 C and 15 lb/in2, during which time the extra 2% water was lost and the sterilized soil-clay mixtures were at their 1/3-bar tension water content. Fungi were grown for 3 to 5 days on slants of Sabouraud-dextrose agar, and bacteria were grown for 2 to 3 days on slants of nutrient agar, both at 25 ± 2 C, and the slants were then flooded with 0.85% sterile saline and agitated on a Vortex-Genie. In some studies, 0.1 ml of fungal inoculum (either as spores or as mycelial fragments) was placed in the center of the soil plate, and 0.1 ml of a bacterial suspension was placed near the periphery, approximately 2.5 cm from the center of the soil dish. In other studies, 0.1 ml of the fungal and 0.1 ml of the bacterial suspensions were both placed into the same site in the center of the soil dish; either both were added concurrently, or the fungus was added 96 h after the bacterium. Control plates were centrally inoculated with the fungus only. After inoculation, all soil plates were placed in the humidifier-incubator, which was maintained at 25 ± 20C. The soil dishes were replicated 1, 7, 14, and 21 days after inoculation, although additional replications were made at intermediate times in some experiments, and the replicated agar dishes were incubated at C. Replication has been described elsewhere (24, 29). Radial growth of the bacteria and fungi through soil was determined and mapped after the various time intervals, and the growth rates of the fungi under EFFECT OF CLAYS AND ph ON ANTAGONISM OF FUNGI 1121 various environmental conditions, in the presence or absence (control) of bacteria, were calculated. The results of replications on day 1 were used as the initial diameter of the inoculum in the soil. Growth rates, in millimeters per day, were calculated by dividing the amount of radial spread of the fungus in soil after a certain period of time, minus the initial radius of the inoculum, by the number of days of growth (i.e., from day 1 to the time of measurement). In some experiments, the bulk ph of the soil-clay mixtures was altered by adding either HCI or NaOH to the water used to adjust the mixtures to the 1/3- bar water content. The ph of the mixtures was measured before each experiment to verify that the desired ph was attained. Three replicate soil plates were used for each variable, and experiments were performed at least twice. All data were analyzed statistically with a Tektronic model 31 calculator programmed by a cassette tape to yield the mean and standard error of the mean. Most data were also analyzed by the Student's t test (22). (ii) Pure culture studies. Bacteria, including actinomycetes, and one yeast were also tested for their ability to inhibit filamentous fungi on agar. A bacterium or yeast was inoculated 2.5 cm from the periphery of a dish containing toxin assay agar, and after 48 to 72 h, a filamentous fungus was inoculated onto the center of the dish. After sufficient time for the filamentous fungus to overgrow the agar dish (48 to 96 h), the plates were scored for inhibition (+, a clear zone around the bacterial or yeast colony) or lack of inhibition (-, the filamentous fungus grew up to or over the bacterial or yeast colony) of the fungus. RESULTS AND DISCUSSION Effect of bacteria and a yeast on growth of filamentous fungi in K6M soil or on agar. Ten bacteria, including three actinomycetes, and a yeast were tested for their antagonistic activities toward eight filamentous fungi in Kitchawan (K) soil amended with 6% montmorillonite (K6M) and on agar (Table 1). In soil, Serratia marcescens inhibited six fungi; Agrobacterium radiobacter, Nocardia paraffinae, Streptomyces nodosus, and Rhodotorula rubra inhibited four each; and Micromonospora chalcea inhibited two. Bacillus cereus, Bacillus sp., Erwinia herbicola, Micrococcus agilis, and Sarcina lutea were nonantagonistic. Penicillium was the most sensitive genus, followed by Aspergillus and Fusarium; Rhizopus stolonifer and Cunninghamella echinulata were not inhibited by any of the organisms. On agar, only a Bacillus sp. (isolated from laboratory air) and S. nodosus showed antagonistic activity towards any of the eight fungi. The Bacillus sp. inhibited all fungi, and S. nodosus inhibited all except C. echinulata and R. stolonifer (Table 1). Antagonism was more widespread in soil than on agar: six organisms were antagonistic to fungi

3 1122 ROSENZWEIG AND STOTZKY TABLE 1. Influence of various bacteria, including actinomycetes, and a yeast on the growth offungi on agar and in soil amended with 6%o montmorillonite F. oxy- A. niger A. terreus P. as- P. vermni- sporum f. F. solani f. C. echinu- R. stoloni- Organism perum culatum sp. tinans conglu- sp. pisi lata fer Agar Soil Agar Soil Agar Soil Agar Soil Agar Soil Agar Soil Agar Soil Agar Soil Gram-positive eubacteria Bacillus sp. +a -b + + _ _ + + _ Gram-negative eubacteria A. radio bacter S. mar cescens Actinomycetes M. chalcea N. paraffi nae S. nodosus Yeast R. rubra a Inhibition of growth. 'No inhibition of growth. in soil, whereas only two bacteria were antagonistic on agar. However, eight fungi were inhibited on agar and only six were inhibited in soil, and the Bacillus sp., which inhibited all fungi on agar, had no effect on any of the fungi in soil. Antagonism on agar was probably limited to amensalism, because competition for nutrients rarely, if ever, occurs on media with high nutrient levels. The strain of S. nodosus used was a known producer of the antifungal antibiotics amphotericin A and B (35), and it and the Bacillus sp. produced diffusible amensalistic substances, as shown by the clear zones around their colonies on agar plates seeded with the individual fungi. No zone of antifungal activity was found around any of the bacterial colonies in soil, suggesting that amensalistic substances were not produced by these bacteria in soil or, if produced, were inactive (19-21). The antagonistic activity observed in soil was probably the result of some form of competition. Influence of clay minerals on antagonistic interactions between bacteria and fungi in soil. When the fungus and bacterium were inoculated into different sites of the soil, A. niger was significantly inhibited by S. marcescens in the Kitchawan (K), K3K, K3M, K6M, K9M, and K12M soils, but there was no significant inhibition in the K6K, K9K, and K12K soils (Table 2). A. niger was inhibited by A. radiobacter only in the Kitchawan (unamended) and in the montmorillonite-amended soils, and neither B. cereus nor Bacillus sp. showed antagonism in any of the soils. P. vermiculatum was inhibited by S. marcescens in all soils except in APPL. ENVIRON. MICROBIOL. the K9K and K12K soils, and the fungus was inhibited by N. paraffinae only in the K6M, K9M, and K12M soils. When A. niger and S. marcescens were inoculated simultaneously into the same site in the soil, the growth of the fungus was inhibited in all soils, regardless of the type of clay mineral added, although the inhibition was significantly greater in soil amended with montmorillonite than in unamended soil or in soil amended with kaolinite. In general, as the concentration of montmorillonite increased, the inhibition increased, but there was no correlation between the concentration of kaolinite and the amount of inhibition (Table 3). When S. marcescens was inoculated 96 h before A. niger, the degree of inhibition was even greater than when both organisms were inoculated simultaneously and, in general, increased as the concentration of montmorillonite increased. There was no correlation between the concentration of kaolinite and the level of inhibition (Table 3). When A. niger was inoculated with 1 ml of a 1.0% glucose solution 96 h after S. marcescens, the amount of antagonism was less than when the fungus was inoculated after 96 h without glucose, suggesting that the antagonism was due to the utilization of soil nutrients by the bacterium during the 96 h before the inoculation of the fungus and that the inhibition resulted primarily from competition for nutrients, especially carbon. Mishra and Pandey (18) also reported that fungistasis increased as the interim between the incubation of nonsterile soil and the inoculation of the test fungus increased. When A. niger and S. marcescens were inoculated

4 VOL. 38, 1979 TABLE 2. Influence of adding various concentrations of kaolinite or montmorillonite to soil on the inhibition of the growth of A. niger by S. marcescensa aclayy ph b GrowthC % of controld pe None ± ± 3.23 <0.001 K3K ± ± 6.91 <0.050 K6K ± ± 4.38 >0.100 K9K ± ± 3.25 >0.200 K12K ± ± 3.25 >0.200 K3M ± ± 9.72 <0.025 K6M ± ± 4.83 <0.001 K9M ± ± 5.58 <0.010 K12M ± ± 0.0 <0.001 a Fungus was inoculated into the center of the soil plate, and the bacterium was inoculated near the periphery. bph of the soil-clay mixtures before inoculation. c Mean linear radial extension of A. niger after 12 days (in millimeters, x ± standard error of the mean). d Mean percentage of control ± standard error of the mean based on control plates containing only A. niger, extension in all control plates after 12 days was 35.0 ± 0.0 mm. eprobability, two-tailed t test comparing experimental growth to control. simultaneously, the nutrient levels in the soil were apparently capable of supporting growth of both the fungus and the bacterium. The degree of inhibition, regardless of whether the organisms were inoculated into the same or different sites, was related to both the type of clay mineral present and the ph of the soil-clay mixtures. Certain clay minerals, such as montmorillonite, have been shown to enhance the growth of bacteria both in soil (5, 12, 13, 15) and in pure culture (25, 26, 32) by maintaining the ph of the environment at a level suitable for sustained growth by the exchange of basic cations from their exchange complex for H+ produced during metabolism. This stimulation of bacterial growth and, therefore, the enhanced depletion of nutrients in the presence of montmorillonite was apparently partially responsible for the inhibition of A. niger by S. marcescens. Kaolinite, which had a CEC that was approximately 1/10 that of montmorillonite (6), does not maintain a ph suitable for sustained bacterial growth (25, 26, 32), and the inhibition of A. niger by S. marcescens was reduced in the presence of 3% kaolinite and totally eliminated at higher concentrations, even though the CEC of the K6K, K9K, or K12K soils was higher than that of the K3K soil. This apparent contradiction between the lack of fungal inhibition and the increased CEC of soils amended with various concentrations of kaolinite suggests that either EFFECT OF CLAYS AND ph ON ANTAGONISM OF FUNGI 1123 the CEC was not the only soil factor that affected the inhibition of A. niger by S. marcescens or that the CEC of the kaolinite-amended soils, which ranged only from 8.38 to 9.61 meq/ 100 g of oven-dry soil, was below a threshold level necessary for sufficient buffering of the soil solution to sustain bacterial growth (26). Influence of ph on antagonistic interactions between bacteria and fungi in soil. The initial ph of the soil-clay mixtures also appeared to affect the interaction between A. niger and S. marcescens. The bulk ph of the unamended soil was 5.1, whereas that of the soilmontmorillonite mixtures ranged from 5.4 to 5.7 and that of the soil-kaolinite mixtures ranged from 4.6 to 4.8. The minimal ph for growth and survival of S. marcescens is approximately 4.5 (7), and the bacterium was an ineffective inhibitor of A. niger in the soil-kaolinite mixtures because their low ph resulted in sufficient reductions in its growth and metabolism to preclude its ability to antagonize A. niger. The higher ph of both the unamended soil and the soil amended with various concentrations of montmorillonite allowed better growth of the bacterium and resulted in greater inhibition of A. niger. The ph of the various soil-clay mixtures reflected both the ph of the bulk soil solution and the surface ph of the numerous soil microhabitats. The ph at the surface of individual microbial cells and soil particulates, especially of clay minerals, is different from the bulk ph (8, 17, 28). Inasmuch as microbial activity in soil occurs, to a large extent, near the surface of clays and other particulates, the microbes may have been exposed to a ph different from that measured, and this ph may be more important in determining the extent of antagonism between bacteria and fungi than the bulk ph (28, 34). The measured ph of a 1:2 kaolinite-water suspension was 4.2 and, thus, lower than both the ph of any of the soil-kaolinite mixtures and the minimal ph for the growth of S. marcescens. Conversely, the measured ph of a 1:2 montmorillonite-water suspension was 7.6, which was considerably higher than the ph of any of the soil-montmorillonite mixtures and was suitable for good growth and metabolism of S. marcescens. When the fungus and bacterium were inoculated into separate sites of the soil, S. marcescens inhibited A. niger in the Kitchawan (ph 5.1) and K6M (ph 5.5) soils but not in the K6K (ph 4.8) soil (Table 4). When the ph of the Kitchawan soil was adjusted to 4.8 or 5.5, S. marcescens still inhibited the growth ofa. niger, and there were no significant differences in the amount of inhibition at the various ph values. However, lowering the ph of the K6M soil to

5 1124 ROSENZWEIG AND STOTZKY TABLE 3. Growth rates ofa. niger alone and in combination with S. marcescens in soil amended with various concentrations of kaolinite or montmorillonitea Growth rate" System Clay added Without bacte- P % of control' With bacterium rium A. niger and S. None 2.09 ± ± < ± 2.44 marcescens inoculated K3K 1.95 ± ± < ± 0.0 at the same time K6K 2.29 ± ± < ± 2.44 K9K 2.29 ± ± < ± 2.57 K12K 1.67 ± ± < ± 2.35 K3M 1.67 ± ± < ± 3.62 K6M 1.11 ± ± < ± 2.14 K9M 0.83 ± ± 0.80 < ± 4.44 K12M 0.60 ± ± < ± 3.28 A. niger inoculated 96 h None 1.57 ± ± < ± 2.67 after S. marcescens K3K 1.74 ± ± < ± 2.33 K6K 1.25 ± ± < ± 4.98 K9K 1.81 ± ± < ± 2.40 K12K 1.46 ± ± < ± 0.0 K3M 0.90 ± ± < ± 4.36 K6M 0.56 ± ± < ± 2.84 K9M 0.43 ± ± < ± 2.31 K12M 0.28 ± ± < ± 2.52 A. niger inoculated 96 h None 2.00 ± ± < ± 0.0 after S. marcescens in K6K 2.40 ± ± < ± 2.44 a 1.0% glucose solution K6M 1.08 ± ± < ± 2.04 a Fungus and bacterium inoculated into the same site in the center of the soil plate. 'Mean growth rate, in millimeters per day, ± standard error of the mean based on measurements of linear radial extension. Growth rates for A. niger were determined 7 days after inoculation of the fungus. 'Probability, two-tailed t test comparing growth rate with bacterium to without bacterium. d Mean experimental (with bacterium) percentage of control (without bacterium) ± standard error of the mean. TABLE 4. Influence ofph on the inhibition by S. marcescens of the growth ofa. niger in K6K or K6M soil' Natural soil ph-adjusted soil' Clay added Pf ph" Growth' % of controld ph" Growth' % of controld None ± ± ± ± 4.71 > ± ± 2.74 >0.200 K6K ± ± ± ± 0.0 >0.500 K6M ± ± ± ± 4.80 <0.050 afungus inoculated into the center of the soil plate and the bacterium near the periphery. b ph of the soil-clay mixtures before inoculation. 'Mean linear radial extension of A. niger after 12 days (in millimeters, x ± standard error of the mean). d Mean percent of control ± standard error of the mean, based on control plates containing only A. niger; extension in all control plates after 12 days was 35.0 ± 0.0 mm. e ph of the soil was adjusted by the addition of either 1 N HCl or 1 N NaOH. f Probability, two-tailed t test comparing growth in natural soil to ph-adjusted soil. 4.8 significantly decreased the inhibition of growth of A. niger, but raising the ph of the K6K soil to 5.5 did not result in any inhibition. B. cereus and Bacillus sp. were nonantagonistic, regardless of the ph or clay mineral content of the soil. When the fungus and bacterium were inoculated into the same site of the soil and the bulk ph was increased, the amount of inhibition generally increased: e.g., in the Kitchawan soil (ph APPL. ENVIRON. MICROBIOL. 5.1), the mean radial growth rate of A. niger was 2.09 ± mm/day (61.5 ± 2.44% of control); when the ph was adjusted to 5.5, the growth rate decreased to 1.46 ± mm/day (55.3 ± 4.65% of control); and when it was adjusted to 4.8, the growth rate increased to (76.6 ± 2.33% of control) (Table 5). When the ph of the K6K soil was increased from 4.8 to 5.5, the growth rate decreased from ( % of control) to

6 VOL. 38, mm/day (70.3 ± 0.0% of control), and when the ph of the K6M soil was decreased from 5.5 to 4.8, the growth rate increased from 1.11 ± (34.1 ± 2.14% of control) to 1.39 ± 0.0 mm/ day (48.8 ± 0.0% of control). Although soil ph was an important factor in determining the amount of inhibition ofa. niger by S. marcescens, the clay mineralogy was also involved. At equivalent ph values, S. marcescens was a better inhibitor of A. niger in the K6M soil than in the K6K or Kitchawan soils, and inhibition was greater in the Kitchawan than in the K6K soil at ph 5.5, although it was equal at ph 4.8. Sensitivity of spores versus mycelium of A. niger to antagonism by S. marcescen& When both A. niger and S. marcescens were inoculated into the same site, the fungus was EFFECT OF CLAYS AND ph ON ANTAGONISM OF FUNGI 1125 inoculated predominantly as spores, and the initial interaction was between the bacterium and spores, whereas the interaction was primarily between the bacterium and growing fungal mycelium when the fungus was inoculated into the center of the soil dish and the bacterium was inoculated near the periphery. When A. niger was inoculated with the bacterium as mycelial fragments, the fungus was still inhibited in all soils, but the amount of inhibition in the Kitchawan and K6M soils was significantly less than when the fungus was added as spores (Table 6). There was no significant difference in the level of inhibition between spores and mycelium in the K6K soil. In all soil-clay mixtures, the growth rate of the fungus, in either the absence or presence of S. marcescens, was significantly greater when A. niger was added as mycelial TABLE 5. Influence ofph on the growth rates ofa. niger alone and in combination with S. marcescens in K6K or K6M soil' Growth rate' Clay added phb Pd % of control' With bacterium Without bacterium None ± < ± 2.33 None 5.1f 2.09 ± ± < ± 2.44 None ± ± < ± 4.65 K6K 4.8f 2.29 ± ± < ± 2.44 K6K ± ± < ± 0.0 K6M ± ± 0.0 < ± 0.0 K6M 5.5f 1.11 ± ± < ± 2.14 a Fungus and bacterium inoculated into the same site in the center of the soil plate. b ph of the soil-clay mixtures before inoculation. 'Mean growth rate, in millimeters per day, ± standard error of the mean, based on measurements of linear radial extension. Growth rates for A. niger were determined 7 days after inoculation of the fungus. d Probability, two-tailed t test comparing growth rate with bacterium to without bacterium. 'Mean experimental (with bacterium) percent of control (without bacterium) ± standard error of the mean. f Natural ph of the soil-clay mixtures. TABLE 6. Growth rates ofa. niger, added as either spores or mycelial fragments, alone or in combination with S. marcescens, in K6K or K6M soir Growth rate' System ~ Clay P fcnrl System added With bacte- Without bac- P % of control" rium terium A. niger added as None 2.09 ± ± < ± 2.44 spores K6K 2.29 ± ± < ± 2.44 K6M 1.11 ± ± < ± 2.14 A. niger added as None 3.54 ± ± < ± 0.0 mycelial frag- K6K 3.26 ± ± < ± 0.0 ments K6M 3.47 ± ± < ± 0.0 a Fungus and bacterium inoculated, at the same time, into the same site in the center of the soil plate. 'Mean growth rate, in millimeters per day, ± standard error of the mean, based on measurements of linear radial extension. Growth rates for A. niger were determined 7 days after inoculation of the fungus. c Probability, two-tailed t test comparing growth rate with bacterium to without bacterium. d Mean experimental (with bacterium) percentage of control (without bacterium) ± standard error of the mean.

7 1126 ROSENZWEIG AND STOTZKY fragments than as spores. Steiner and Lockwood (23) found that the mycelial filaments of eight species of fungi, including three aspergilli, were less sensitive to soil fungistasis than were the corresponding spores (conidia), and they concluded that mycelia represented the least sensitive stage of a fungus to fungistasis. The increased sensitivity of A. niger to inhibition by S. marcescens when both were inoculated into the same site was apparently a result of the greater sensitivity of the spores than of the mycelial filaments. These data indicate that the inhibition of fungi by bacteria in soil is influenced by the clay mineralogy, ph, nutrient levels, type of fungal propagule present, and spatial relations of the organisms. Furthermore, the results of these studies with model systems suggest that altering the physicochemical properties of soil in situ (e.g., incorporation of montmorillonite) might stimnulate the naturally occurring antagonistic microbiota and, thereby, exert an indirect control of soil-borne pathogenic fungi. ACKNOWLEDGMENTS Gratitude is expressed to A. L. Leaf for the soil analyses and to American Colloid Co. and R. T. Vanderbilt Co. for providing the clay minerals. LITERATURE CITED 1. Alexander, M Microbial ecology. John Wiley and Sons, New York. 2. Babich, H., and G. Stotzky Effect of cadmium on fungi and on interactions between fungi and bacteria in soil: influence of clay minerals and ph. Appl. Environ. Microbiol. 33: Bashi, E., and N. J. Fokkema Environmental factors limiting growth of Sporobolomyces roseus, an antagonist of Cochliobolus sativus, on wheat leaves. Trans. Br. Mycol. Soc. 68: Broadbent, P., K. F. Baker, and Y. 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