APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1977, p. 1059-1066Copyright © 1977 American Society for Microbiology

Vol. 33, No. 5Printed in U.S.A.

Effect of Cadmium on Fungi and on Interactions BetweenFungi and Bacteria in Soil: Influence of Clay Minerals and pH

H. BABICH AND G. STOTZKY*Laboratory ofMicrobial Ecology, Department ofBiology, New York University, New York, New York 10003

Received for publication 15 November 1976

Fungi (Rhizopus stolonifer, Trichoderma viride, Fusarium oxysporum f. sp.

conglutinans, Cunninghamella echinulata, and several species of Aspergillusand Penicillium) tolerated higher concentrations of cadmium (Cd) when grown

in soil than when grown on laboratory media, indicating that soil mitigated thetoxic effects of Cd. In soil amended with clay minerals, montmorillonite providedpartial or total protection against fungistatic effects of Cd, whereas additions ofkaolinite provided little or no protection. Growth rates ofAspergillus niger wereinhibited to a greater extent by 100 or 250 Ag ofCd per g in soil adjusted to pH 7.2than in the same soil at its natural pH of 5.1. However, there were no differencesin the growth rates ofAspergillus fischeri with 100 or 250 ,ug of Cd per g in thesame soil, whether at pH 5.1 or adjusted to pH 7.2. Growth ofA. niger and A.fischeri in a soil contaminated with a low concentration of Cd (i.e., 28 ,ug/g),obtained from a site near a Japanese smelter, did not differ significantly fromgrowth in a soil collected some distance away and containing 4 Ag of Cd per g.Growth ofA. niger in sterile soil amended with 100 ,g of Cd per g and inoculatedwith Bacillus cereus or Agrobacterium tumefaciens was reduced to a greaterextent than in the same soil containing 100 ,ug of Cd per g but no bacteria. Theinhibitory effects of Agrobacterium radiobacter to A. niger were slightly re-duced in the presence of 100 ,ug of Cd per g, whereas the inhibitory effects ofSerratia marcescens were enhanced.

The impact and long-term ecological ramifi-cations of pollution on the biosphere have stim-ulated research to evaluate the interactions be-tween pollutants, the environment, and thebiota. Of the numerous anthropogenic contami-nants emitted and deposited into the environ-ment, cadmium (Cd), an element of no knownbiological function, is of major concern (7, 8; H.Babich and G. Stotzky, in D. Perlman, ed.,Adv. Appl. Microbiol., vol. 22, in press). Therehas been relatively little research to evaluatethe response of microorganisms, especially soilmicroorganisms, to Cd. Although often ne-glected in environmental pollution studies, soilmicrobes are dynamically involved in manyecological processes, such as the biogeochemi-cal and decomposition processes necessary fornutrient cycling and maintenance of soil fertil-ity, energy transformations through the trophiclevels, and numerous microbe-microbe, mi-crobe-plant, and microbe-animal interactions.Many of these microbial processes and inter-actions, both positive and negative, may beeliminated or altered in polluted environments(5, 6, 32).Studies in pure culture have shown that var-

ious species of bacteria, including actinomy-

cetes, and fungi have differential sensitivitiesto Cd (7); that the toxicity of Cd to microbescould be lessened by the incorporation of cys-teine (33), zinc (17, 23), magnesium (1, 17),chelating agents (35), and the clay mineralsmontmorillonite and kaolinite (8); and that thetoxicity of Cd was greater under aerobic thananaerobic conditions (24) and was enhanced atalkaline pH levels (7). The toxicity of Cd to themicrobiota in natural habitats would, there-fore, be expected to be dependent on the physi-cochemical characteristics of the environmentinto which the pollutant is deposited. There is,however, little information on the interactionsbetween Cd and microorganisms in natural mi-crobial habitats. Consequently, this study eval-uated the influence of clay minerals and pH onthe toxicity of Cd to fungi in soil, inasmuch asthese two environmental factors have beenshown to affect the toxicity of Cd to microbes inpure culture (7, 8).Although some investigations have studied

the effects of Cd on virus-plant (36), fungus-plant (25), and bacteria-plant (12) interactions,there apparently have been no studies on theinfluence of Cd on interactions between bacte-ria and fungi in soil. Consequently, prelimi-

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1060 BABICH AND STOTZKY

nary studies on the influence of Cd on interac-tions between fungi and bacteria in a model soilsystem were also conducted.

MATERIALS AND METHODSSource and maintenance of microorganisms. Mi-

croorganisms were obtained from the culture collec-tion of the Laboratory of Microbial Ecology at NewYork University, the American Type Culture Col-lection, and the Midwest Culture Service. Bacteriawere grown and maintained on nutrient agar (Difco)amended with 1% glucose; fungi were grown andmaintained on Sabouraud dextrose agar (Difco). Thecultures were stored in a refrigerator at 4°C.

Description of soil. Soil, obtained from the Kit-chawan Research Laboratory of the Brooklyn Bo-tanic Garden at Ossining, N.Y., was amended witheither montmorillonite (Volclay, Panther Creek-Aberdeen, American Colloid Co., with a cation-ex-change capacity [CEC] of approximately 60 meq/100g [29]) or kaolinite (Continental, R. T. VanderbiltCo., with a CEC of approximately 6.5 meq/100 g [29])to yield approximate concentrations of 3, 6, 9, and12% (vol/vol). The clays and soil were mixed in an

electric cement mixer, and, after thorough mixing,the soil-clay mixtures were stored in 20-gallon (ca.75-liter) metal garbage cans containing plastic gar-

bage bags. Some chemical and physical properties ofthe soil, unamended and amended with clay, are

presented in Table 1. The soil contained a back-ground level of 0.014 ,ug of Cd per g, and X-ray dif-fraction analyses showed that it does not naturally

contain montmorillonite-type clay minerals but doescontain mica-illite, kaolinite, and vermiculite. Insome experiments, the pH ofthe soil was raised from5.1 to 7.2 by the addition of CaCO3, which was incor-porated into the water used to bring the soil to the1/3-bar water content.

Soils were also obtained from a site near a zincsmelter in Japan: the contaminated soil, obtained250 m east-southeast from the smelting factory, con-tained 28 ,ug of Cd, 1,460 ,ug of Zn, 90 ,ug of Cu, and334 jig of Pb per g and had a pH of 5.7; the controlsoil, obtained 750 m south of the smelter, contained4 jig of Cd, 243 ,ug of Zn, 50 ,tg of Cu, and 48 jig of Pbper g and had a pH of 6.1.

Experimental design and soil replicator appara-tus. The soil replica-plating technique (28, 31) wasused to study both the growth rates of fungi in soiland the influence of Cd on interactions betweenbacteria and fungi in soil. The soil replica-platingapparatus consisted of five components: a replicator(composed of an acrylic plastic square throughwhich stainless-steel nails were inserted in a squarepattern with 5 mm between nails, covered with an-other plastic square, and with a handle attached), atemplate, a humidifier-incubator, heavy-walled pe-tri plates containing soil, and petri plates contain-ing specific media for the differential isolation ofeither bacteria or fungi.

Soil was placed in plastic bags, and water was

added to bring the soil to 2% above the 1/3-bartension water content (determined with a 5-barpressure plate extractor, Soilmoisture Equipment

TABLE 1. Some chemical and physical characteristics ofKitchawan soil, unamended or amended with eitherkaolinite or montmorillonitea

SoilProperty

K K3K K6K K9K K12K K3M K6M K9M K12M

pH 5.1 4.8 4.8 4.7 4.6 5.4 5.5 5.6 5.7Organic matter 5.75 5.90 5.44 5.10 4.71 5.75 5.72 5.49 5.13

(%)CEC (meq/100 g) 8.15 8.38 8.88 8.99 9.61 10.77 11.27 13.67 14.67N (%) 0.132 0.131 0.129 0.124 0.115 0.128 0.127 0.124 0.119Sand (%) 56.8 58.2 54.8 52.2 52.8 57.8 52.2 52.8 55.8Silt (%) 33.8 31.2 33.6 31.8 27.6 30.6 35.8 31.6 26.6Clay (%) 9.4 10.6 11.6 16.0 19.6 11.6 12.0 15.6 17.6P (,ug/g) 27.6 28.2 24.4 24.0 32.4 30.0 33.0 39.0 43.4K (gg/g) 61.9 63.8 61.6 63.8 57.2 72.6 84.7 94.6 107.6Ca (,ug/g) 360.0 345.0 372.0 383.0 409.0 1,075.0 1,357.0 1,981.0 2,028.0Mg (IgIg) 63.1 55.9 59.8 61.0 62.4 109.2 111.6 132.6 140.4Na (,ug/g) 3.9 3.3 5.7 7.5 8.2 6.9 11.1 11.7 14.1Mn (gg/g) 5.8 6.7 7.1 7.3 7.7 4.5 4.6 4.5 5.4Fe (,ug/g) 1.5 1.4 0.9 1.1 1.0 1.3 1.0 0.9 0.9Zn (jAgIg) 0.8 0.6 0.7 0.8 0.8 0.5 0.5 0.5 0.5Pb (,Ag/g) BDL ND ND BDL ND ND ND 0.01 NDCu (,ug/g) 0.12 ND ND BDL ND ND ND 0.66 NDNi (,ug/g) 0.17 ND ND 0.18 ND ND ND 1.75 NDCr (,ug/g) 0.06 ND ND 0.06 ND ND ND 0.15 NDCd (,ug/g) 0.014 ND ND 0.062 ND ND ND 0.081 NDField capacity 20.7 21.1 21.7 21.8 22.0 21.8 22.3 23.4 24.4

(%) (1/3-barwater)

a K, Natural, unamended Kitchawan soil; K3K, Kitchawan soil + 3% kaolinite; K6K, Kitchawan soil + 6% kaolinite;K9K, Kitchawan soil + 9% kaolinite; K12K, Kitchawan soil + 12% kaolinite; K3M, Kitchawan soil + 3% montmorillonite;K6M, Kitchawan soil + 6% montmorillonite; K9M, Kitchawan soil + 9% montmorillonite; K12M, Kitchawan soil + 12%montmorillonite; BDL, below detectable limits; ND, not determined.

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EFFECTS OF Cd ON FUNGI: SOIL STUDIES 1061

Corp., Santa Barbara, Calif.), at which respirationand microbial activity of soil are at an optimum (see30). In soils amended with Cd, CdCl2 was incorpo-rated into the water used to bring the soil to the 1/3-bar tension. The soil and water were mixed thor-oughly, stored overnight in a refrigerator at 4°Cand then passed through a 2-mm sieve, and 40 g ofsoil, retained by a 0.5-mm sieve, was dispensed intoheavy-walled petri plates and leveled. The soilplates were then autoclaved for 0.5 h (121°C at 15 lb/in2), during which the extra 2% water was lost andthe sterilized soils were at their 1/3-bar tension wa-

ter content.Slants containing fungi, grown for 1 week at 25°C

on Sabouraud dextrose agar, or bacteria, grown for 2to 3 days at 25°C on nutrient agar amended with 1%glucose, were flooded with 0.85% saline and agitatedon a Vortex Genie. For studies on fungal growth, a0.1-ml inoculum of the spore-mycelial suspensionwas placed in a central depression, marked previousto autoclaving with the aid of the template, of steri-lized soil plates. In studies of interactions betweenbacteria and Aspergillus niger, 0.2 ml of bacterialinoculum was placed in the central depression and,after the soil plates were incubated at 25°C in thehumidifier-incubator for 2 to 3 days, 0.1 ml of fungalinoculum was placed in the same inoculation site.After inoculation, all soil plates were placed in thehumidifier-incubator, which was maintained at25°C. Three replicate soil plates were used for eachconcentration of Cd and clay, and experiments wereperformed twice.

Soil plates were usually replicated 1, 7, 14, and 21days after inoculation, but rapidly growing fungiwere replicated after 1, 3, and 6 days. For studies on

fungal growth, replications were made to Sabourauddextrose agar supplemented with 33.3 mg of rosebengal per liter. When both bacteria and fungi wereinoculated into the same soil plate, replicationswere made to Sabouraud dextrose agar containing33.3 mg of rose bengal and 80 mg of streptomycin(Sigma Chemical Co.) per liter for selection of fungi,

and to nutrient agar amended with 1% glucose and100 mg of cycloheximide (Acti-dione, Upjohn Co.)per liter for selection of bacteria.

Replicated agar plates were incubated at 25°C forseveral days, and measurements of the diameterspread of bacteria and fungi were made in fourdifferent directions. Maps were constructed showingthe spread of the microorganisms in different soils,and, based on these maps, growth rates were calcu-lated. Replications on day 1 were used to determinethe initial diameter of the inoculum in the soil.Growth rates, in millimeters per day, were calcu-lated by dividing the diameter of fungal growth atthe termination of the experiment, minus the initialdiameter of the inoculum, by the number of days ofgrowth.

RESULTSEffect of Cd on growth of fungi in soil.

Fungi were grown in soil unamended oramended with 10, 100, or 1,000 ,ug of Cd per g.For the majority of the fungi tested, 10 ,ug ofCdper g did not appreciably influence rates ofmycelial growth. In soil amended with 100 ,ugof Cd per g, growth of Penicillium vermicula-tum, Aspergillus flavipes, and A. janus wasreduced, but that ofA. niger, A. fischeri, Rhizo-pus stolonifer, Penicillium asperum, Tricho-derma viride, Cunninghamella echinulata, andFusarium oxysporum f. sp. conglutinans was

unaffected. In soil supplemented with 1,000gg of Cd per g, only P. asperum, T. viride, A.

janus, C. echinulata, and F. oxysporum f. sp.conglutinans grew, although at a reduced rate(Table 2).

Effect of Cd on growth of fungi in soilamended with kaolinite or montmorillonite.When fungi were grown in soil amended with 3,6, 9, or 12% kaolinite or montmorillonite, nei-

TABLE 2. Growth rates offungi in Kitchawan soil, with and without Cda

Mean growth rate (mm/day) + SEM at Cd concn (gg/g) of:Fungus

0 10 100 1,000Penicillium vermiculatum 3.4 ± 0.22 3.4 + 0.21 2.6 ± 0.08 0Aspergillus flavipes 3.0 ± 0.07 2.8 ± 0.08 2.2 ± 0.09 0Aspergillus fischeri 5.5 ± 0.18 5.5 ± 0.22 5.3 ± 0.34 0Aspergillus niger 5.4 ± 0.29 5.3 ± 0.31 4.8 ± 0.40 0Rhizopus stolonifer 11.9 + 0.17 12.2 ± 0.00 12.2 ± 0.00 0Penicillium asperum 3.3 ± 0.16 3.5 + 0.15 3.4 ± 0.09 1.9 ± 0.10Aspergillus janus 4.9 ± 0.34 4.3 ± 0.44 4.0 ± 0.23 2.8 ± 0.19Trichoderma viride 11.9 + 0.97 11.8 ± 0.15 11.8 ± 0.12 3.8 ± 0.21Cunninghamella echinulata 10.5 ± 0.06 10.1 ± 0.14 9.8 ± 0.30 4.3 ± 0.32Fusarium oxysporum f. sp. conglutinans 5.4 ± 0.14 5.6 ± 0.17 5.1 ± 0.16 4.3 ± 0.27

a Mean growth rate based on measurements of linear diameter extension. Growth rates for R. stoloniferand T. viride were determined after 7 days of growth, for A. fischeri, A. niger, and F. oxysporum f. sp.conglutinans after 14 days of growth, and forP. vermiculatum, P. asperum, A. flavipes, andA.janus after 21days of growth. Growth rates for C. echinulata in 0-, 10-, and 100-;Lg/g Cd-amended soil were measured after7 days, whereas growth rates in soil amended with 1,000 ,ug of Cd per g were determined after 14 days. SEM,Standard error of the mean.

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1062 BABICH AND STOTZKY

ther clay significantly influenced the growthrates ofA. fischeri or A. niger in the absence ofCd. In soil amended with 250 jig of Cd per g,montmorillonite completely eliminated the ad-verse effects of Cd on A. fischeri and reducedthe toxicity of Cd towards A. niger, whereasadditions of kaolinite did not appreciably influ-ence the toxicity of Cd towards either fungus.In the absence of Cd, kaolinite stimulatedgrowth of P. vermiculatum but not of P. as-

perum, whereas montmorillonite stimulatedgrowth of P. asperum but not of P. vermicula-tum. Montmorillonite completely eliminatedthe toxic effects of 100 gg of Cd per g to P.vermiculatum, whereas kaolinite only slightlylessened the inhibitory effects. Kaolinite didnot appreciably affect the toxicity of 1,000 ug ofCd per g to P. asperum, whereas montmorillon-ite provided protection against Cd; the additionof 3 or 6% montmorillonite provided partialprotection and 9 or 12% montmorillonite af-forded total protection. Kaolinite did not appre-ciably influence the growth rates of T. viride inthe absence of Cd, whereas montmorillonite, atconcentrations of 3 or 6%, but not at 9 or 12%,depressed growth. Both kaolinite and montmo-

rillonite, however, significantly reduced thetoxicity of Cd to T. viride in soil amended with1,000 lg of Cd per g, with montmorilloniteagain providing greater protection than equiva-lent concentrations of kaolinite (Table 3).

Effect of pH on growth of fungi in the pres-ence or absence of Cd. In the absence of Cd,growth of A. niger, but not of A. fischeri, wasmarkedly reduced by raising the pH of the soilfrom 5.1 to 7.2; growth of A. niger was de-creased by 26% and that of A. fischeri by 4%(Fig. 1). When grown on an agar medium, my-celial proliferation of A. niger was reduced by40%, and that ofA. fischeri by only 7%, at pH 7as compared to pH 5. A concentration of 10 pgof Cd per g did not significantly alter growth ofA. niger in the natural or in the pH-adjustedsoil. However, in the presence of 100 Ag of Cdper g, growth ofA. niger was reduced by 10% inthe natural soil and by 31% in the soil adjustedto pH 7.2. At 250 ug of Cd per g, growth of A.niger was decreased by 49% in natural soil andby 82% in pH-adjusted soil. Growth of A. fis-cheri in natural and pH-adjusted soils was notaffected by 10 pug of Cd per g and was onlyslightly decreased by 100 ug of Cd per g. At 250

TABLE 3. Growth rates of fungi in Kitchawan soil amended with Cd and either kaolinite (K),montmorillonite (M), or no claya

Mean growth rate (mm/day) + SEM at clay concn (%) of:Fungus Treatment (A,g/g)

0 3 6 9 12

Penicillium Cd, 0: K 3.5 ± 0.12 3.7 t 0.07 4.0 t 0.08 4.0 t 0.07 3.7 t 0.13vermiculatum Cd, 100: K 2.6 ± 0.09 2.8 t 0.10 3.1 t 0.17 2.8 + 0.06 2.8 t 0.06

Cd, 0: M 3.5 t 0.12 3.4 t 0.10 3.4 t 0.07 3.4 t 0.11 3.4 t 0.17Cd, 100: M 2.6 t 0.09 3.4 t 0.07 3.5 t 0.18 3.5 t 0.11 3.3 t 0.08

Aspergillus Cd, 0: K 5.5 t 0.18 5.4 t 0.47 5.2 t 0.36 5.3 t 0.15 5.1 t 0.28fischeri Cd, 250: K 3.9 t 0.28 3.5 t 0.11 3.6 t 0.33 3.4 t 0.23 3.4 t 0.17

Cd, 0: M 5.5 t 0.18 5.5 t 0.32 5.4 t 0.33 5.5 t 0.15 5.3 t 0.11Cd, 250: M 3.9 t 0.28 5.0 t 0.19 5.1 t 0.06 5.2 t 0.12 5.3 t 0.26

Aspergillus niger Cd, 0: K 5.4 t 0.29 4.8 t 0.40 4.9 t 0.37 4.9 t 0.23 4.9 t 0.31Cd, 250: K 2.7 t 0.35 2.7 ± 0.44 2.4 t 0.33 2.4 t 0.41 2.7 t 0.44Cd, 0: M 5.4 ± 0.29 5.2 t 0.44 5.4 t 0.37 5.3 t 0.32 5.2 t 0.66

Cd, 250: M 2.7 t 0.35 3.5 t 0.29 3.3 ± 0.31 3.4 t 0.41 3.5 t 0.41

Penicillium Cd, 0: K 3.4 t 0.14 3.2 t 0.08 3.2 t 0.11 3.3 t 0.14 3.2 t 0.12asperum Cd, 1,000: K 1.8 t 0.09 1.9 t 0.14 2.2 t 0.76 1.7 t 0.05 2.0 t 0.11

Cd, 0: M 3.4 t 0.14 3.6 + 0.08 3.8 t 0.20 4.0 t 0.15 3.9 t 0.18Cd, 1,000: M 1.8 t 0.09 2.6 t 0.05 2.8 t 0.16 4.2 t 0.11 4.0 t 0.12

Trichoderma Cd, 0: K 12.8 t 0.38 11.6 t 0.41 11.7 t 0.13 12.1 t 0.52 11.9 t 0.17viride Cd, 1,000: K 2.4 t 0.12 3.4 ± 0.50 3.2 t 0.67 3.5 t 0.31 5.3 t 1.03

Cd, 0: M 12.8 t 0.38 10.6 t 0.41 10.4 t 0.22 12.8 t 0.58 11.9 t 0.54Cd, 1,000: M 2.4 t 0.12 3.7 t 0.51 4.0 t 0.18 6.3 t 0.10 5.6 t 0.25

a Mean growth rate based on measurements of linear diameter extensions. Growth rates for P. vermicula-tum, P. asperum, A. fischeri, and A. niger were determined after 14 days of growth and for T. viride after 6days of growth. SEM, Standard error of the mean.

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CADMIUM CONCENTRATION (ppm)

FIG. 1. Effect of Cd on growth ofA. niger and A.fischeri after 14 days in natural (pH 5.1) and pH-adjusted (pH 7.2) soil. Mean + standard error of themean.

jig of Cd per g, the reductions in growth innatural and pH-adjusted soils were approxi-mately equivalent, i.e., 27 and 29%, respec-tively (Fig. 1).Growth of fungi in a Japanese soil contami-

nated with Cd. Growth of A. niger and A.fischeri in contaminated soil (containing 28 pgof Cd per g, pH 5.7) did not differ significantlyfrom growth in uncontaminated, control soil(containing 4 pg of Cd per g, pH 6.1): growthrates ofA. niger were 5.3 + 0.56 and 5.5 0.25mm/day and those ofA. fischeri were 5.6 0.15and 5.7 + 0.46 mm/day in the contaminated andcontrol soil, respectively.

Effect of Cd on interactions between fungiand bacteria in soil. A few studies evaluatedthe influence of Cd on interactions between A.niger and either Bacillus cereus, Agrobacte-rium tumefaciens, Agrobacterium radiobacter,or Serratia marcescens. Survival of the bacte-

ria, with the exception of some reduction inthat ofA. tumefaciens, was not affected after 2weeks ofincubation in soil amended with either10 or 100 jig of Cd per g.The end of the first week after inoculation of

the fungus, growth ofA. niger was essentiallyrestricted in all soil plates to an area within thesite where the bacteria had been inoculated 2 to3 days earlier. After 7 days of incubation in soilunamended or amended with 10 or 100 jig of Cdper g, growth ofA. niger was slightly inhibitedin the presence of A. tumefaciens or B. cereusand greatly inhibited in the presence of A. ra-diobacter or S. marcescens. In the presence ofA. radiobacter, growth ofA. niger after 7 dayswas greater in the soil amended with 100 ug ofCd per g than in soil amended with 0 to 10 ,ug ofCd per g, suggesting that, with 100 gg of Cd perg, the inhibitory effects ofA. radiobacter werereduced and, therefore, allowed for bettergrowth ofA. niger (Fig. 2).

In all fungus-bacterium systems, A. nigerhad overgrown the initial site of bacterial inoc-ulation after the second week of incubation. Insoil amended with 0 or 10 ,ug of Cd per g, A.niger grew approximately the same, whether inthe absence or presence ofA. tumefaciens orB.cereus. However, in soil containing 100 ,ug ofCd per g and A. tumefaciens or B. cereus,growth of the fungus after 2 weeks was reducedto a significantly greater extent than whengrown in soil containing 100 ,ug of Cd per g butno bacteria. Growth ofA. niger was reduced inthe presence of A. radiobacter in all soils ascompared to growth of the fungus in the ab-sence of the bacterium, and the presence of Cddid not significantly affect the interaction be-tween the fungus and the bacterium after 14days, although Cd did influence the inhibitoryeffects of A. radiobacter after 7 days when thefungus was restricted to the area also contain-ing bacteria. Growth of A. niger was greatlyreduced in all soils containing S. marcescens.In the absence of S. marcescens, 100 pg of Cdper g reduced growth of A. niger by 10%.whereas in the presence of S. marcescens, 100,ug of Cd per g reduced fungal growth by 30%,suggesting a synergistic interaction betweenCd and the bacterial antagonist on growth ofA.niger. With 10 ,ug of Cd per g, there was nosignificant effect on the growth of the fungus,either alone or in the presence ofS . marcescens(Fig. 2).

DISCUSSIONPrevious studies have evaluated the toler-

ance of a variety of fungi to Cd on a nutrientagar medium. The results ofthe present studies

|---*pH 5.1

A.-.opH 7.2

Aspergillus fischeri

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1064 BABICH AND STOTZKY

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FIG. 2. Effect ofCd on growth ofA. niger in soil in the absence and presence of bacteria. Mean + standarderror of the mean.

demonstrated that fungi generally toleratedhigher concentrations of Cd when grown in soilthan on agar (8). Several soil factors may havebeen responsible for this increased tolerance offungi to Cd in soil. The Cd ions may have beencomplexed with organic chelating agents (27)and may have been adsorbed to the clay min-erals and organic materials in soil (4, 14, 20,21). Studies with plants have shown that theuptake of Cd was inversely related to the or-ganic matter and clay mineral contents of soil(11, 13, 16, 26).The incorporation of montmorillonite into

soil reduced or totally eliminated the toxicity ofCd towards T. viride, A. fischeri, A. niger, P.asperum, and P. vermiculatum, whereas theincorporation of equivalent kaolinite concen-

trations provided little or no protection againstCd toxicity. Previous studies have shown thatthe ability of these clays to protect against fun-gistatic or fungicidal concentrations of Cd was

correlated with the CEC of the clay. Montomo-rillonite, with'the higher CEC, afforded greaterprotection against Cd than did equivalent con-

centrations of kaolinite (8). Incorporation ofmontmorillonite significantly increased theCEC of the soil, and the increased protection

afforded by these clay-amended soils was ap-parently related to their capacity to adsorb, bycation exchange, greater quantities of exoge-nous Cd. In previous studies (8) ofCd toxicity inan agar medium, where the background CECwas negligible, the addition of kaolinite pro-vided some protection to fungi against exoge-nous Cd, probably by the adsorption of small,but sufficient, quantities of Cd. Other studies(4) have also shown that kaolinite is a compara-tively poor exchanger of Cd.When A. niger and A. fischeri were grown in

soil or on a nutrient agar medium adjusted toapproximately pH 7.2, growth rates ofA. niger,but not ofA. fischeri, were less than in normalsoil (pH 5.1) or on agar medium of pH 5.0. Inthe presence of 100 or 250 jig ofCd per g, growthrates of A. niger were reduced to a greaterextent in pH-adjusted soil, suggesting either aninability of the fungus to tolerate an additionalheavy metal stress at alkaline levels or a syner-gistic interaction between Cd and the higherpH. A. niger was also more sensitive to Cd inbroth at pH 7, 8, or 9 than at pH 5 or 6 (7). Nodifferences in the extent of inhibition of growthof A. fischeri by Cd were noted in natural orpH-adjusted soil.

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EFFECTS OF Cd ON FUNGI: SOIL STUDIES 1065

Although there are no comparable studies inthe literature on the triple interaction betweenCd, soil pH, and microorganisms, there are afew studies, albeit with contradictory results,on the dual interaction between Cd and soil pHand on the triple interaction among Cd, soilpH, and plants. Cd has been suggested to bemost mobile in acidic soil ofpH 5 and below andimmobile in alkaline soil (37). The adsorption ofCd by illitic clay soil, organic peat soil, kaolin-ite (4), iron oxides (4, 10), and manganese ox-ides (10) was pH dependent, with Cd adsorptionincreasing as the pH was increased. The uptakeof Cd from soil by radishes (15, 16, 18), wheat(22), and fodder rape (4) was pH dependent,with the uptake being greater at lower soil pHlevels. Other studies, however, have shown theuptake ofCd by plants to be pH independent, inthat Cd was "taken up with ease by a greatnumber of plant species, in particular, grami-nae, regardless of soil pH" (19). Furthermore,radishes grown in a variety of garden soils con-taminated with Cd did not show any correlationbetween soil pH and the Cd content in theradishes (9).The growth of A. niger and A. fischeri in a

soil contaminated with heavy metals and ob-tained near a Japanese smelter was the sameas growth in non-contaminated soil obtainedsome distance away. The concentrations of Cdand other heavy metals in this contaminatedsoil were apparently not sufficient to inhibitgrowth of these fungi. In Kitchawan soil, 10 or100 ,ug of Cd per g did not adversely affectgrowth of A. niger or A. fischeri. In addition,other studies (17) have shown that the toxicityof Cd towards A. niger could be reduced by theaddition of zinc. The high zinc concentration(i.e., 1,460 Aglg) in the contaminated Japanesesoil may have lessened the toxicity of Cd to-wards these fungi, further demonstrating theimportance of the physicochemical characteris-tics of soil in the response of microbes to Cd.When interactions between A. niger and bac-

teria were studied in soil not amended with Cd,growth ofA. niger, at the end of 2 weeks, wasnot inhibited in the presence of eitherA . tume-faciens or B. cereus but was inhibited by eitherS. marcescens or A. radiobacter. Other investi-gations, in which A. niger was inoculated intothe center of soil plates and bacteria were inoc-ulated into peripheral sites on the same plate,have shown that S . marcescens or A. radiobac-ter inhibited growth of A. niger, whereas B.cereus did not affect its growth (W. D. Rosen-zweig, personal communication). A. radiobac-ter has also been shown to inhibit growth ofPenicillium jenseni in soil (28). S. marcescensproduces the red pigment prodigiosin, which

has antibiotic properties (2), and, when grownin synthetic medium, S. marcescens inhibitedgrowth of several fungi, e.g., Botrytis tulipaeand Gloeosporium affinae (3).Growth ofA. niger in soil containing 100 ,.g

of Cd per g and either B. cereus or A. tumefa-ciens was significantly less after 2 weeks thangrowth in soil containing an equivalent Cd con-centration but no bacteria, possibly reflectingthe effect of Cd on the inability of the fungus tocompete successfully with these bacteria in soil.At the end of 2 weeks, growth of A. niger wasgreater in soil amended with both A. radiobac-ter and 100 ,ug of Cd per g than in soil amendedwith only A. radiobacter, suggesting that A.radiobacter may have been more sensitive toCd than A. niger, thus perrnitting A. niger tocompete better. In the presence of S. marces-cens, growth of A. niger was greatly reducedwith 100 ,g of Cd per g as compared to growthin soil with 0 or 10 ,g of Cd per g, suggesting asynergistic interaction between higher concen-trations of Cd and S. marcescens. This syner-gistic effect may have involved an interactionbetween Cd and prodigiosin, similar to the po-tentiation by Cd of the antibacterial action ofbacitracin (38), spermine, spermidine (39), col-istin, erythromycin, streptomycin, and the tet-racyclines (34).The toxicity of Cd, as well as other particu-

late and gaseous pollutants (5, 6, 32), to mi-crobes is apparently dependent on the physico-chemical characteristics of the environmentinto which the pollutant is deposited. Thus, inassessing the impact of a pollutant on the bio-sphere, the influence of the abiotic physico-chemital factors on pollutant toxicity should beconsidered.

ACKNOWLEDGMENTSThis research was supported, in part, by grant R-800671

from the U.S. Environmental Protection Agency and by aPublic Health Service grant from the National Institutes ofHealth Biomedical Research Support Grant to New YorkUniversity.

Gratitude is expressed to W. D. Rosenzweig for the clayanalyses of the soil and for suggesting the use of A. radio-bacter and S. marcescens as antagonists to A. niger; to I.Watanabe for the soils from Japan; to T.J. Kneip for theheavy metal analyses of the soil-clay mixtures; to A. L. Leaffor the other soil analyses; and to American Colloid Co. andR. T. Vanderbilt Co. for providing the clay minerals.

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