Vol. 38, No. 6APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1979, p. 1120-11260099-2240/79/12-1120/07$02.00/0

Influence of Environmental Factors on Antagonism of Fungiby Bacteria in Soil: Clay Minerals and pH

WILLIAM D. ROSENZWEIGt AND G. STOTZKY*

Laboratory ofMicrobial Ecology, Department of Biology, New York University, New York, New York 10003

Received for publication 19 September 1979

The soil replica plating technique was used to evaluate the influence of clayminerals and pH on antagonistic interactions between fungi and bacteria in soil.In general, the antagonistic activity of bacteria towards filamentous fungi wasgreater in soil than on agar. The spread of Aspergillus niger through soil wasinhibited by Serratia marcescens when the organisms were inoculated intoseparate sites in soil, and this antagonistic effect was maintained when the soilwas amended with 3, 6, 9, or 12% (vol/vol) montmorillonite, whereas the additionof 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. nigerby Agrobacterium radiobacter and of Penicillium vermiculatum by either S.marcescens or Nocardia paraffinae. When A. niger and S. marcescens wereinoculated into the same soil site, A. niger was inhibited in all soils, regardless ofclay content, although the extent of inhibition was greater as the concentrationof montmorillonite, but not of kaolinite, increased. A. niger was inhibited more

when inoculated as spores than as mycelial fragments and when inoculated 96 hafter S. marcescens, but a 1% glucose solution reduced the amount of inhibitionwhen the fungus was inoculated 96 h after the bacterium. When the pH of thesoil-clay mixtures was altered, the amount of antagonism usually increased as thepH increased. Antagonism appeared to be related to the cation-exchange capacityand the pH of the soil-clay mixtures. Bacillus cereus and another species ofBacillus showed no activity in soil towards A. niger under any of the environ-mental conditions tested, even though the Bacillus sp. significantly inhibited A.niger and seven other fungi on agar.

Soil is a reservoir for many microbial patho-gens, including ones for human beings (16, 27,31) and plants (4, 10, 14, 30, 33). Although nu-merous attempts have been made to control oreliminate these pathogens in soil by the use ofother microorganisms, biological control hasonly been partially successful (9-11, 36). Themethods used in an attempt to control pathogenshave been either direct, by introducing antago-nists into soil (9, 10, 11), or indirect, by alteringthe physicochemical properties of soil and, thus,stimulating the naturally occurring antagonisticmicrobiota (1, 3, 9, 27, 30, 31, 33).The present study used nonpathogenic micro-

organisms as model systems to evaluate theinfluence of some physicochemical environmen-tal factors on the control of microbes in soil.Antagonistic interactions between microbeswere studied by a technique that allowed forrepeated observations of numerous sites with aminimum of disturbance of the cells and soil

t Present address: Department of Biochemistry and Micro-biology, Rutgers University, New Brunswick, NJ 08903.

particles. Problems resulting from artificialgrowth surfaces were eliminated, and conditionsthat exist in soil in situ were simulated (24, 29).

MATERIALS AND METHODSMicroorganisms. Microorganisms were obtained

from the stock culture collection of the Laboratory ofMicrobial Ecology at New York University, the Amer-ican Type Culture Collection, and the Midwest Cul-ture Service.

Media. Nutrient-cycloheximide agar (nutrient agar[Difco], 23.0 g; cycloheximide [Actidione, UpjohnCo.], 100 mg; and water, 1 liter, pH 6.8) was used forreplica plating of bacteria from soil; bacterial cultureswere also maintained and grown on this medium with-out 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; fungalcultures were also maintained and grown on this me-dium 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

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EFFECT OF CLAYS AND pH ON ANTAGONISM OF FUNGI 1121

[Difco], 10.0 g; agar [Difco], 15.0 g; and water, 1 liter,pH 6.5) was used in pure culture studies on the abilityof bacteria to produce toxins against filamentous fungi.

Soils. Soil obtained from the Kitchawan ReseachLaboratory of the Brooklyn Botanic Garden, Ossining,N.Y., was amended with either montmorillonite (Vol-clay, Panther Creek-Aberdeen, American Colloid Co.),with a cation-exchange capacity (CEC) of approxi-mately 60 milliequivalents (meq) per 100 g (25), orkaolinite (Continental, R. T. Vanderbilt Co.), with aCEC of approximately 6.5 meq/100 g (25), to yieldapproximate concentrations of 3, 6, 9, and 12% clay(vol/vol). The clays and soil were thoroughly mixed inan electric cement mixer, and the soil-clay mixtureswere stored in metal garbage cans (ca. 75 liters) linedwith plastic. Some chemical and physical properties ofthe soil, unamended and amended with clay, havebeen presented elsewhere (2). X-ray diffraction anal-ysis showed that this soil does not naturally containmontmorillonite-type clay minerals but does containmica-illite, kaolinite, and vermiculite.

Soil replicator. The soil replica plating apparatushas been described elsewhere (24, 29).Experimental design. (i) Soil studies. The soil-

clay mixtures were placed in plastic bags, and waterwas added to bring the mixtures to 2% above their 1/3-bar tension water content determined with a 5-barpressure plate extractor (Soilmoisture EquipmentCorp., Santa Barbara, Calif.). After equilibration for24 h at 4°C, the mixtures were passed through a sievewith 2-mm openings, and 40 g of the sieved mixturewas dispensed into a heavy walled petri dish andleveled by gentle tapping on a large rubber stopper.Desired inoculation sites were marked, with the aid ofa template, by making small depressions in the soilsurface, and the soil plates were autoclaved for 0.5 hat 121°C and 15 lb/in2, during which time the extra2% water was lost and the sterilized soil-clay mixtureswere at their 1/3-bar tension water content.

Fungi were grown for 3 to 5 days on slants ofSabouraud-dextrose agar, and bacteria were grown for2 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 somestudies, 0.1 ml of fungal inoculum (either as spores oras mycelial fragments) was placed in the center of thesoil plate, and 0.1 ml of a bacterial suspension wasplaced near the periphery, approximately 2.5 cm fromthe center of the soil dish. In other studies, 0.1 ml ofthe fungal and 0.1 ml of the bacterial suspensions wereboth placed into the same site in the center of the soildish; either both were added concurrently, or thefungus was added 96 h after the bacterium. Controlplates were centrally inoculated with the fungus only.After inoculation, all soil plates were placed in thehumidifier-incubator, which was maintained at 25 ±20C.The soil dishes were replicated 1, 7, 14, and 21 days

after inoculation, although additional replicationswere made at intermediate times in some experiments,and the replicated agar dishes were incubated at 25+ 2°C. Replication has been described elsewhere (24,29). Radial growth of the bacteria and fungi throughsoil was determined and mapped after the various timeintervals, and the growth rates of the fungi under

various environmental conditions, in the presence orabsence (control) of bacteria, were calculated. Theresults of replications on day 1 were used as the initialdiameter of the inoculum in the soil. Growth rates, inmillimeters per day, were calculated by dividing theamount of radial spread of the fungus in soil after acertain period of time, minus the initial radius of theinoculum, by the number of days of growth (i.e., fromday 1 to the time of measurement).

In some experiments, the bulk pH of the soil-claymixtures was altered by adding either HCI or NaOHto the water used to adjust the mixtures to the 1/3-bar water content. The pH of the mixtures was meas-ured before each experiment to verify that the desiredpH was attained.

Three replicate soil plates were used for each vari-able, and experiments were performed at least twice.All data were analyzed statistically with a Tektronicmodel 31 calculator programmed by a cassette tape toyield the mean and standard error of the mean. Mostdata were also analyzed by the Student's t test (22).

(ii) Pure culture studies. Bacteria, including ac-tinomycetes, and one yeast were also tested for theirability to inhibit filamentous fungi on agar. A bacte-rium or yeast was inoculated 2.5 cm from the peripheryof a dish containing toxin assay agar, and after 48 to72 h, a filamentous fungus was inoculated onto thecenter of the dish. After sufficient time for the fila-mentous fungus to overgrow the agar dish (48 to 96h), the plates were scored for inhibition (+, a clearzone around the bacterial or yeast colony) or lack ofinhibition (-, the filamentous fungus grew up to orover the bacterial or yeast colony) of the fungus.

RESULTS AND DISCUSSION

Effect of bacteria and a yeast on growthoffilamentous fungi in K6M soil or on agar.Ten bacteria, including three actinomycetes, anda yeast were tested for their antagonistic activi-ties toward eight filamentous fungi in Kitchawan(K) soil amended with 6% montmorillonite(K6M) and on agar (Table 1). In soil, Serratiamarcescens inhibited six fungi; Agrobacteriumradiobacter, Nocardia paraffinae, Strepto-myces nodosus, and Rhodotorula rubra in-hibited four each; and Micromonospora chalceainhibited two. Bacillus cereus, Bacillus sp., Er-winia herbicola, Micrococcus agilis, and Sar-cina lutea were nonantagonistic. Penicilliumwas the most sensitive genus, followed by As-pergillus and Fusarium; Rhizopus stoloniferand Cunninghamella echinulata were not in-hibited by any of the organisms.On agar, only a Bacillus sp. (isolated from

laboratory air) and S. nodosus showed antago-nistic activity towards any of the eight fungi.The Bacillus sp. inhibited all fungi, and S. no-dosus 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

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TABLE 1. Influence of various bacteria, including actinomycetes, and a yeast on the growth offungi onagar 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. conglu- sp. pisi lata fertinans

Agar Soil Agar Soil Agar Soil Agar Soil Agar Soil Agar Soil Agar Soil Agar Soil

Gram-positiveeubacteria

Bacillus sp. +a -b + + _ + - + _ + - + _ _ _Gram-negative

eubacteriaA. radio- - + + + - - - + - - - -

bacterS. mar- - + - + - + - + - + - + -

cescensActinomycetesM. chalcea - - - - - - + - +N. paraffi- - - - - - + - + - + - + -

naeS. nodosus + - + - + + + + + + + + -

YeastR. rubra - + - + - + - +

a Inhibition of growth.'No inhibition of growth.

in soil, whereas only two bacteria were antago-nistic on agar. However, eight fungi were in-hibited on agar and only six were inhibited insoil, and the Bacillus sp., which inhibited allfungi on agar, had no effect on any of the fungiin soil.

Antagonism on agar was probably limited toamensalism, because competition for nutrientsrarely, if ever, occurs on media with high nutri-ent levels. The strain of S. nodosus used was aknown producer of the antifungal antibioticsamphotericin A and B (35), and it and the Ba-cillus sp. produced diffusible amensalistic sub-stances, as shown by the clear zones aroundtheir colonies on agar plates seeded with theindividual fungi. No zone of antifungal activitywas found around any of the bacterial coloniesin soil, suggesting that amensalistic substanceswere not produced by these bacteria in soil or,if produced, were inactive (19-21). The antago-nistic activity observed in soil was probably theresult of some form of competition.Influence of clay minerals on antagonis-

tic interactions between bacteria and fungiin soil. When the fungus and bacterium wereinoculated into different sites of the soil, A. nigerwas significantly inhibited by S. marcescens inthe Kitchawan (K), K3K, K3M, K6M, K9M,and K12M soils, but there was no significantinhibition in the K6K, K9K, and K12K soils(Table 2). A. niger was inhibited by A. radio-bacter only in the Kitchawan (unamended) andin the montmorillonite-amended soils, and nei-ther B. cereus nor Bacillus sp. showed antago-nism in any of the soils. P. vermiculatum wasinhibited by S. marcescens in all soils except in

the K9K and K12K soils, and the fungus wasinhibited by N. paraffinae only in the K6M,K9M, and K12M soils.When A. niger and S. marcescens were inoc-

ulated simultaneously into the same site in thesoil, the growth of the fungus was inhibited inall soils, regardless of the type of clay mineraladded, although the inhibition was significantlygreater in soil amended with montmorillonitethan in unamended soil or in soil amended withkaolinite. In general, as the concentration ofmontmorillonite increased, the inhibition in-creased, but there was no correlation betweenthe concentration of kaolinite and the amountof inhibition (Table 3).When S. marcescens was inoculated 96 h be-

fore A. niger, the degree of inhibition was evengreater than when both organisms were inocu-lated simultaneously and, in general, increasedas the concentration of montmorillonite in-creased. There was no correlation between theconcentration of kaolinite and the level of inhi-bition (Table 3). When A. niger was inoculatedwith 1 ml of a 1.0% glucose solution 96 h after S.marcescens, the amount of antagonism was lessthan when the fungus was inoculated after 96 hwithout glucose, suggesting that the antagonismwas due to the utilization of soil nutrients by thebacterium during the 96 h before the inoculationof the fungus and that the inhibition resultedprimarily from competition for nutrients, espe-cially carbon. Mishra and Pandey (18) also re-ported that fungistasis increased as the interimbetween the incubation of nonsterile soil and theinoculation of the test fungus increased. WhenA. niger and S. marcescens were inoculated

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TABLE 2. Influence of adding variousconcentrations of kaolinite or montmorillonite to

soil on the inhibition of the growth ofA. niger by S.marcescensa

aClayY pH b GrowthC % of controld pe

None 5.1 23.9 ± 0.74 68.3 ± 3.23 <0.001

K3K 4.8 30.6 ± 1.55 87.3 ± 6.91 <0.050K6K 4.8 33.3 ± 0.83 95.2 ± 4.38 >0.100K9K 4.7 33.9 ± 1.11 96.8 ± 3.25 >0.200K12K 4.7 33.9 ± 0.74 96.8 ± 3.25 >0.200

K3M 5.4 25.0 ± 2.50 71.4 ± 9.72 <0.025K6M 5.5 23.3 ± 0.83 66.7 ± 4.83 <0.001K9M 5.6 21.7 ± 2.64 61.9 ± 5.58 <0.010K12M 5.7 20.0 ± 0.0 57.1 ± 0.0 <0.001a Fungus was inoculated into the center of the soil

plate, and the bacterium was inoculated near theperiphery.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 was35.0 ± 0.0 mm.

eProbability, two-tailed t test comparing experi-mental growth to control.

simultaneously, the nutrient levels in the soilwere apparently capable of supporting growth ofboth the fungus and the bacterium.The degree of inhibition, regardless of

whether the organisms were inoculated into thesame or different sites, was related to both thetype of clay mineral present and the pH of thesoil-clay mixtures. Certain clay minerals, suchas montmorillonite, have been shown to enhancethe growth of bacteria both in soil (5, 12, 13, 15)and in pure culture (25, 26, 32) by maintainingthe pH of the environment at a level suitable forsustained growth by the exchange of basic cat-ions from their exchange complex for H+ pro-duced during metabolism. This stimulation ofbacterial growth and, therefore, the enhanceddepletion of nutrients in the presence of mont-morillonite was apparently partially responsiblefor the inhibition of A. niger by S. marcescens.

Kaolinite, which had a CEC that was approx-imately 1/10 that of montmorillonite (6), doesnot maintain a pH suitable for sustained bacte-rial growth (25, 26, 32), and the inhibition of A.niger by S. marcescens was reduced in the pres-ence of 3% kaolinite and totally eliminated athigher concentrations, even though the CEC ofthe K6K, K9K, or K12K soils was higher thanthat of the K3K soil. This apparent contradic-tion between the lack of fungal inhibition andthe increased CEC of soils amended with variousconcentrations of kaolinite suggests that either

the CEC was not the only soil factor that af-fected the inhibition of A. niger by S. marces-cens or that the CEC of the kaolinite-amendedsoils, which ranged only from 8.38 to 9.61 meq/100 g of oven-dry soil, was below a thresholdlevel necessary for sufficient buffering of the soilsolution to sustain bacterial growth (26).Influence of pH on antagonistic interac-

tions between bacteria and fungi in soil.The initial pH of the soil-clay mixtures alsoappeared to affect the interaction between A.niger and S. marcescens. The bulk pH of theunamended soil was 5.1, whereas that of the soil-montmorillonite mixtures ranged from 5.4 to 5.7and that of the soil-kaolinite mixtures rangedfrom 4.6 to 4.8. The minimal pH for growth andsurvival of S. marcescens is approximately 4.5(7), and the bacterium was an ineffective inhib-itor of A. niger in the soil-kaolinite mixturesbecause their low pH resulted in sufficient re-ductions in its growth and metabolism to pre-clude its ability to antagonize A. niger. Thehigher pH of both the unamended soil and thesoil amended with various concentrations ofmontmorillonite allowed better growth of thebacterium and resulted in greater inhibition ofA. niger.The pH of the various soil-clay mixtures re-

flected both the pH of the bulk soil solution andthe surface pH of the numerous soil microhabi-tats. The pH at the surface of individual micro-bial cells and soil particulates, especially of clayminerals, 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 andother particulates, the microbes may have beenexposed to a pH different from that measured,and this pH may be more important in deter-mining the extent of antagonism between bac-teria and fungi than the bulk pH (28, 34). Themeasured pH of a 1:2 kaolinite-water suspensionwas 4.2 and, thus, lower than both the pH of anyof the soil-kaolinite mixtures and the minimalpH for the growth of S. marcescens. Conversely,the measured pH of a 1:2 montmorillonite-watersuspension was 7.6, which was considerablyhigher than the pH of any of the soil-montmo-rillonite mixtures and was suitable for goodgrowth and metabolism of S. marcescens.When the fungus and bacterium were inocu-

lated into separate sites of the soil, S. marces-cens inhibited A. niger in the Kitchawan (pH5.1) and K6M (pH 5.5) soils but not in the K6K(pH 4.8) soil (Table 4). When the pH of theKitchawan soil was adjusted to 4.8 or 5.5, S.marcescens still inhibited the growth ofA. niger,and there were no significant differences in theamount of inhibition at the various pH values.However, lowering the pH of the K6M soil to

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TABLE 3. Growth rates ofA. niger alone and in combination with S. marcescens in soil amended withvarious 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 ± 0.078 3.40 ± 0.113 <0.001 61.5 ± 2.44marcescens inoculated K3K 1.95 ± 0.070 2.71 ± 0.113 <0.005 72.0 ± 0.0at the same time K6K 2.29 ± 0.179 2.85 ± 0.070 <0.050 80.4 ± 2.44

K9K 2.29 ± 0.070 2.78 ± 0.070 <0.010 82.4 ± 2.57K12K 1.67 ± 0.208 3.06 ± 0.080 <0.005 54.6 ± 2.35K3M 1.67 ± 0.114 3.16 ± 0.160 <0.005 52.8 ± 3.62K6M 1.11 ± 0.070 3.26 ± 0.070 <0.001 34.1 ± 2.14K9M 0.83 ± 0.160 3.68 ± 0.80 <0.001 22.6 ± 4.44K12M 0.60 ± 0.113 3.54 ± 0.069 <0.001 17.0 ± 3.28

A. niger inoculated 96 h None 1.57 ± 0.160 2.80 ± 0.113 <0.005 56.1 ± 2.67after S. marcescens K3K 1.74 ± 0.160 3.06 ± 0.208 <0.010 56.9 ± 2.33

K6K 1.25 ± 0.113 2.85 ± 0.069 <0.001 43.9 ± 4.98K9K 1.81 ± 0.175 2.85 ± 0.179 <0.020 63.5 ± 2.40K12K 1.46 ± 0.133 2.99 ± 0.133 <0.005 48.8 ± 0.0K3M 0.90 ± 0.070 2.78 ± 0.208 <0.005 32.4 ± 4.36K6M 0.56 ± 0.113 2.50 ± 0.069 <0.001 22.4 ± 2.84K9M 0.43 ± 0.069 2.80 ± 0.179 <0.001 15.5 ± 2.31K12M 0.28 ± 0.113 2.79 ± 0.241 <0.001 10.0 ± 2.52

A. niger inoculated 96 h None 2.00 ± 0.0 3.06 ± 0.168 <0.005 65.4 ± 0.0after S. marcescens in K6K 2.40 ± 0.139 3.00 ± 0.069 <0.050 80.0 ± 2.44a 1.0% glucose solution K6M 1.08 ± 0.069 2.87 ± 0.139 <0.001 37.6 ± 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 5.1 23.9 ± 0.74 68.3 ± 3.23 4.8 24.4 ± 1.55 69.8 ± 4.71 >0.5005.5 21.7 ± 1.86 61.9 ± 2.74 >0.200

K6K 4.8 33.3 ± 0.83 95.2 ± 4.80 5.5 35.0 ± 0.0 100.0 ± 0.0 >0.500K6M 5.5 23.3 ± 0.83 66.7 ± 4.80 4.8 27.2 ± 0.83 77.7 ± 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 ofgrowth of A. niger, but raising the pH of theK6K 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 ofthe soil.When the fungus and bacterium were inocu-

lated into the same site of the soil and the bulkpH was increased, the amount of inhibition gen-erally increased: e.g., in the Kitchawan soil (pH

5.1), the mean radial growth rate of A. nigerwas 2.09 ± 0.078 mm/day (61.5 ± 2.44% of con-trol); when the pH was adjusted to 5.5, thegrowth rate decreased to 1.46 ± 0.121 mm/day(55.3 ± 4.65% of control); and when it was ad-justed to 4.8, the growth rate increased to 2.29+ 0.070 (76.6 ± 2.33% of control) (Table 5).When the pH of the K6K soil was increasedfrom 4.8 to 5.5, the growth rate decreased from2.29 + 0.179 (80.4 + 2.44% of control) to 2.15 +

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0.0 mm/day (70.3 ± 0.0% of control), and whenthe pH of the K6M soil was decreased from 5.5to 4.8, the growth rate increased from 1.11 ±

0.070 (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. nigerby S. marcescens, the clay mineralogy was alsoinvolved. At equivalent pH values, S. marces-

cens was a better inhibitor of A. niger in theK6M soil than in the K6K or Kitchawan soils,and inhibition was greater in the Kitchawanthan in the K6K soil at pH 5.5, although it wasequal at pH 4.8.

Sensitivity of spores versus mycelium ofA. niger to antagonism by S. marcescen&When both A. niger and S. marcescens were

inoculated into the same site, the fungus was

inoculated predominantly as spores, and theinitial interaction was between the bacteriumand spores, whereas the interaction was primar-ily between the bacterium and growing fungalmycelium when the fungus was inoculated intothe center of the soil dish and the bacterium wasinoculated near the periphery. When A. nigerwas inoculated with the bacterium as mycelialfragments, the fungus was still inhibited in all

soils, but the amount of inhibition in the Kitcha-wan and K6M soils was significantly less thanwhen the fungus was added as spores (Table 6).There was no significant difference in the levelof inhibition between spores and mycelium inthe K6K soil. In all soil-clay mixtures, thegrowth rate of the fungus, in either the absenceor presence of S. marcescens, was significantlygreater when A. niger was added as mycelial

TABLE 5. Influence ofpH on the growth rates ofA. niger alone and in combination with S. marcescens inK6K or K6M soil'Growth rate'

Clay added pHb Pd % of control'With bacterium Without bacterium

None 4.8 2.29 + 0.070 2.99 ± 0.184 <0.025 76.6 ± 2.33None 5.1f 2.09 ± 0.078 3.40 ± 0.113 <0.001 61.5 ± 2.44None 5.5 1.46 ± 0.121 2.64 ± 0.070 <0.005 55.3 ± 4.65

K6K 4.8f 2.29 ± 0.179 2.85 ± 0.070 <0.050 80.4 ± 2.44K6K 5.5 2.15 ± 0.0 3.06 ± 0.070 <0.001 70.3 ± 0.0

K6M 4.8 1.39 ± 0.001 2.85 ± 0.0 <0.001 48.8 ± 0.0K6M 5.5f 1.11 ± 0.070 3.26 ± 0.070 <0.001 34.1 ± 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 combinationwith S. marcescens, in K6K or K6M soir

Growth rate'System ~ Clay P fcnrlSystem added With bacte- Without bac- P % of control"

rium terium

A. niger added as None 2.09 ± 0.078 3.40 ± 0.113 <0.005 61.5 ± 2.44spores K6K 2.29 ± 0.179 2.85 ± 0.070 <0.050 80.4 ± 2.44

K6M 1.11 ± 0.070 3.26 ± 0.070 <0.001 34.1 ± 2.14

A. niger added as None 3.54 ± 0.0 4.10 ± 0.070 <0.005 86.3 ± 0.0mycelial frag- K6K 3.26 ± 0.0 4.10 ± 0.010 <0.001 79.5 ± 0.0ments K6M 3.47 ± 0.0 4.03 ± 0.010 <0.001 86.1 ± 0.0a 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.

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1126 ROSENZWEIG AND STOTZKY

fragments than as spores.Steiner and Lockwood (23) found that the

mycelial filaments of eight species of fungi, in-cluding three aspergilli, were less sensitive tosoil fungistasis than were the correspondingspores (conidia), and they concluded that my-celia represented the least sensitive stage of afungus to fungistasis. The increased sensitivityof A. niger to inhibition by S. marcescens whenboth were inoculated into the same site wasapparently a result of the greater sensitivity ofthe spores than of the mycelial filaments.These data indicate that the inhibition of

fungi by bacteria in soil is influenced by the claymineralogy, pH, nutrient levels, type of fungalpropagule present, and spatial relations of theorganisms. Furthermore, the results of thesestudies with model systems suggest that alteringthe physicochemical properties of soil in situ(e.g., incorporation of montmorillonite) mightstimnulate the naturally occurring antagonisticmicrobiota and, thereby, exert an indirect con-trol of soil-borne pathogenic fungi.

ACKNOWLEDGMENTSGratitude is expressed to A. L. Leaf for the soil analyses

and to American Colloid Co. and R. T. Vanderbilt Co. forproviding the clay minerals.

LITERATURE CITED

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3. Bashi, E., and N. J. Fokkema. 1977. Environmentalfactors limiting growth of Sporobolomyces roseus, anantagonist of Cochliobolus sativus, on wheat leaves.Trans. Br. Mycol. Soc. 68:17-25.

4. Broadbent, P., K. F. Baker, and Y. Waterworth. 1971.Bacteria and actinomycetes antagonistic to fungal rootpathogens in Australian soils. Aust. J. Biol. Sci. 24:925-944.

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19. Simonoff, P., and D. Gottlieb. 1951. The production androle of antibiotics in the soil. I. The fate of streptomycin.Phytopathology 41:420-430.

20. Skinner, F. 1956. The effect of adding clays to mixedcultures of Streptomyces albidoflavus and Fusariumculmorum. J. Gen. Microbiol. 14:393-405.

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22. Spiegel, M. R. 1961. Theory and problems of statistics.McGraw-Hill Book Co., New York.

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24. Stotzky, G. 1965. Replica plating technique for studyingmicrobial interactions in soil. Can. J. Microbiol. 11:629-636.

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27. Stotzky, G. 1971. Ecologic eradication offungi-dream orreality, p. 477-486. In M. L. Furcolow and E. W. Chick(ed.), Histoplasmosis. Charles C Thomas, Publisher,Springfield, Ill.

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