Isolation Characterization Pseudomonas Strain That ...aem.asm.org/content/56/4/1053.full.pdf ·...

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Vol. 56, No. 4 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1990, p. 1053-1058 0099-2240/90/041053-06$02.00/0 Copyright C 1990, American Society for Microbiology Isolation and Characterization of a Pseudomonas Strain That Restricts Growth of Various Phytopathogenic Fungi RADHESHYAM K. JAYASWAL,* MARCEL A. FERNANDEZ, AND RALPH G. SCHROEDER III Department of Biological Sciences, Illinois State University, Normal, Illinois 61761 Received 15 November 1989/Accepted 23 January 1990 The characterization of a novel Pseudomonas strain exhibiting antagonism towards many important corn fungal pathogens is presented. This strain was isolated from the caryopses of the grass Tripsacum dactyloides and was identified as Pseudomonas cepacia. The antagonistic activity is due to the production of an antifungal compound. The chromatographic properties of this partially purified compound isolated from growth medium differ from those reported previously for other pseudomonads. The suppression of the growth of economically important phytopathogens by this strain and by the partially purified compound indicates a potential biocontrol agent. Maize, the third most important cereal crop in the world after wheat and rice, is prone to many soilborne plant pathogens that reduce the quality and quantity of grain production (32). Fungi are primary causes of grain loss, and some of them produce compounds that are toxic when consumed (31). Fungal diseases of plants are usually con- trolled by some combination of cultural practices, use of fungicides, and host plant resistance. Fungicides are the primary means of fungal disease control, but their use is currently controversial because investigations have indi- cated potentially undesirable environmental side effects (4, 25). One possibility for substantially increasing plant yields without imposing environmental threats is to make use of certain microorganisms that protect plants from the delete- rious microorganisms that occur in all agricultural soils. Recently, there has been an increasing interest among biol- ogists in using beneficial microorganisms as a solution to the overuse of potentially harmful pesticides (3, 6, 7, 9, 11, 26, 33, 35, 37, 38). Fluorescent pseudomonads, particularly Pseudomonas putida and Pseudomonas fluorescens, which are commonly isolated from the plant rhizosphere, have been shown to protect plants from fungal infection. Two factors have been cited as essential for biocontrol: coloniza- tion of the rhizosphere and production of antibiotics. The antagonistic activities are presumably due to the production of siderophores (18, 22). Siderophores are iron-chelating compounds produced by many microorganisms growing under iron-limiting conditions. It has been hypothesized that siderophores function as biostatic compounds by drastically reducing the amount of ferric ions available to certain rhizosphere microflora. However, other factors may be involved in the suppression of potential pathogens (35, 37). There are numerous reports of the production of antibiotic compounds by Pseudomonas spp. Some of these antibiotics have been characterized chemically (21). Howell and Sti- panovic (14, 15) provided evidence that different isolates of P. fluorescens were antagonistic to pathogens of cotton seedlings because of the production of the antibiotics pyr- rolnitrin and pyoluteorin. Another antibiotic, produced by a Pseudomonas strain that is able to suppress take-all in wheat, was identified as a dimer of phenazine-1-carboxylate (12). Other workers have reported a Pseudomonas isolate * Corresponding author. that produced an unidentified antibiotic able to inhibit the fungus that causes Dutch elm disease (20). The finding that mutants defective in the production of antibiotics are unable to protect plants against their pathogens indicates the role of antibiotics in plant disease suppression (7). Unfortunately, little information about the antagonistic organisms of corn pathogens is available. In 1968, fungal corn root infection was controlled biologically in the field by treating seed with the antagonistic organisms Bacillus sub- tilis and Chaetomium globosum (5). After a decade of field testing, these investigators concluded that the antagonists were frequently as efficacious as the application of the pesticide Captan (19). Circumstantial evidence indicated that these strains were antagonistic because of the production of an antibiotic. However, no further work was carried out to characterize either the strains or the mechanisms by which these bacteria were antagonistic. Isolation and partial char- acterization of a Pseudomonas strain that inhibited growth of several corn pathogens was briefly reported (1, 2), but the mechanism of antagonism was not investigated. We have isolated a Pseudomonas strain from the caryopses of Tripsacum dactyloides (eastern gama grass) which prelimi- nary tests indicate has antagonistic properties against impor- tant phytopathogens. In this paper, we describe the isolation and characterization of an antagonistic microorganism of fungal phytopathogens and present a preliminary character- ization of the mechanism of antagonism. MATERIALS AND METHODS Cultures and growth media. Fungi used in the study were chosen according to their economic importance in maize disease (32). Pure stock cultures of various fungi were obtained from Loral Castor (Funk Seed Co., Bloomington, Ill.) and Anthony Liberta (Department of Biological Sci- ences, Illinois State University, Normal) and maintained on potato dextrose agar (PDA) or corn meal agar (CMA) (Difco Laboratories, Detroit, Mich.) slants at 4°C. For petri plate assays, plugs were taken from cultures grown on CMA or PDA grown at 30°C. Isolation of antifungal bacteria. Both antagonistic and nonantagonistic strains of pseudomonads were isolated from the caryopses of T. dactyloides (eastern gama grass). Appro- priate serial dilutions from seed suspensions in sterile H20 were plated on CMA plates, and the plates were incubated at 30°C for 48 h. Single colonies were isolated and screened for 1053 on June 29, 2018 by guest http://aem.asm.org/ Downloaded from

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Vol. 56, No. 4APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1990, p. 1053-10580099-2240/90/041053-06$02.00/0Copyright C 1990, American Society for Microbiology

Isolation and Characterization of a Pseudomonas Strain ThatRestricts Growth of Various Phytopathogenic Fungi

RADHESHYAM K. JAYASWAL,* MARCEL A. FERNANDEZ, AND RALPH G. SCHROEDER III

Department ofBiological Sciences, Illinois State University, Normal, Illinois 61761

Received 15 November 1989/Accepted 23 January 1990

The characterization of a novel Pseudomonas strain exhibiting antagonism towards many important corn

fungal pathogens is presented. This strain was isolated from the caryopses of the grass Tripsacum dactyloidesand was identified as Pseudomonas cepacia. The antagonistic activity is due to the production of an antifungalcompound. The chromatographic properties of this partially purified compound isolated from growth mediumdiffer from those reported previously for other pseudomonads. The suppression of the growth of economicallyimportant phytopathogens by this strain and by the partially purified compound indicates a potential biocontrolagent.

Maize, the third most important cereal crop in the worldafter wheat and rice, is prone to many soilborne plantpathogens that reduce the quality and quantity of grainproduction (32). Fungi are primary causes of grain loss, andsome of them produce compounds that are toxic whenconsumed (31). Fungal diseases of plants are usually con-trolled by some combination of cultural practices, use offungicides, and host plant resistance. Fungicides are theprimary means of fungal disease control, but their use iscurrently controversial because investigations have indi-cated potentially undesirable environmental side effects (4,25).One possibility for substantially increasing plant yields

without imposing environmental threats is to make use ofcertain microorganisms that protect plants from the delete-rious microorganisms that occur in all agricultural soils.Recently, there has been an increasing interest among biol-ogists in using beneficial microorganisms as a solution to theoveruse of potentially harmful pesticides (3, 6, 7, 9, 11, 26,33, 35, 37, 38). Fluorescent pseudomonads, particularlyPseudomonas putida and Pseudomonas fluorescens, whichare commonly isolated from the plant rhizosphere, havebeen shown to protect plants from fungal infection. Twofactors have been cited as essential for biocontrol: coloniza-tion of the rhizosphere and production of antibiotics. Theantagonistic activities are presumably due to the productionof siderophores (18, 22). Siderophores are iron-chelatingcompounds produced by many microorganisms growingunder iron-limiting conditions. It has been hypothesized thatsiderophores function as biostatic compounds by drasticallyreducing the amount of ferric ions available to certainrhizosphere microflora. However, other factors may beinvolved in the suppression of potential pathogens (35, 37).There are numerous reports of the production of antibiotic

compounds by Pseudomonas spp. Some of these antibioticshave been characterized chemically (21). Howell and Sti-panovic (14, 15) provided evidence that different isolates ofP. fluorescens were antagonistic to pathogens of cottonseedlings because of the production of the antibiotics pyr-rolnitrin and pyoluteorin. Another antibiotic, produced by aPseudomonas strain that is able to suppress take-all inwheat, was identified as a dimer of phenazine-1-carboxylate(12). Other workers have reported a Pseudomonas isolate

* Corresponding author.

that produced an unidentified antibiotic able to inhibit thefungus that causes Dutch elm disease (20). The finding thatmutants defective in the production of antibiotics are unableto protect plants against their pathogens indicates the role ofantibiotics in plant disease suppression (7).

Unfortunately, little information about the antagonisticorganisms of corn pathogens is available. In 1968, fungalcorn root infection was controlled biologically in the field bytreating seed with the antagonistic organisms Bacillus sub-tilis and Chaetomium globosum (5). After a decade of fieldtesting, these investigators concluded that the antagonistswere frequently as efficacious as the application of thepesticide Captan (19). Circumstantial evidence indicated thatthese strains were antagonistic because of the production ofan antibiotic. However, no further work was carried out tocharacterize either the strains or the mechanisms by whichthese bacteria were antagonistic. Isolation and partial char-acterization of a Pseudomonas strain that inhibited growthof several corn pathogens was briefly reported (1, 2), but themechanism of antagonism was not investigated. We haveisolated a Pseudomonas strain from the caryopses ofTripsacum dactyloides (eastern gama grass) which prelimi-nary tests indicate has antagonistic properties against impor-tant phytopathogens. In this paper, we describe the isolationand characterization of an antagonistic microorganism offungal phytopathogens and present a preliminary character-ization of the mechanism of antagonism.

MATERIALS AND METHODSCultures and growth media. Fungi used in the study were

chosen according to their economic importance in maizedisease (32). Pure stock cultures of various fungi wereobtained from Loral Castor (Funk Seed Co., Bloomington,Ill.) and Anthony Liberta (Department of Biological Sci-ences, Illinois State University, Normal) and maintained onpotato dextrose agar (PDA) or corn meal agar (CMA) (DifcoLaboratories, Detroit, Mich.) slants at 4°C. For petri plateassays, plugs were taken from cultures grown on CMA orPDA grown at 30°C.

Isolation of antifungal bacteria. Both antagonistic andnonantagonistic strains of pseudomonads were isolated fromthe caryopses of T. dactyloides (eastern gama grass). Appro-priate serial dilutions from seed suspensions in sterile H20were plated on CMA plates, and the plates were incubated at30°C for 48 h. Single colonies were isolated and screened for

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antifungal activity by using the petri plate assay described byAnderson et al. (2). King medium B and minimal mediumwere prepared as described previously (17, 24).

Petri plate assay. All bacterial isolates were initiallyscreened for the ability to inhibit fungal growth on CMA(Difco) and PDA (Difco) plates by using Trichoderma virideas a preliminary test strain. Single colonies were selectedand patched along the perimeters of plates on which 30 jil ofa suspension of spores of Trichoderma viride (104 to 105spores in 0.9% NaCl) was placed at the center or spread overthe entire surface of the plate. The activity of the antifungalcompound from Pseudomonas sp. strain RJ2, purified byhigh-pressure liquid chromatography (HPLC), was similarlydetermined by using the petri plate assay. The plates wereincubated at 30°C for 48 to 72 h, and antifungal activity wasdetermined by measuring zones of fungal growth inhibition.The bacteria positive for antagonistic activity were thenselected from test plates and maintained in pure culture;various nonsuppressive bacteria were retained and served ascontrols.Those strains that inhibited Trichoderma viride were then

tested against other known phytopathogens on both CMAand PDA plates. Zones of inhibition indicated antifungalactivity, and the strains were ranked according to thediameter of the clear zone. The most active Pseudomonasstrain was designated RJ2 and used for further study. StrainRJ2 was selected for spontaneous rifampin resistance byplating the overnight culture of a single colony isolate onLuria-Bertani (LB) agar (23) containing rifampin (50 jig/ml).A strain, RJ3, was isolated from RJ2 by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis as described by Miller(24). Attempts were made to isolate plasmids from RJ2 by analkaline lysis method (23).

Classification of bacteria. The bacteria were classifiedaccording to Bergey's Manual of Determinative Bacteriol-ogy (10). Growth tests leading to differentiation betweenspecies were performed by the method of Hugh and Gilardi(16).

Isolation and purification of an antifungal compound. Anantifungal compound was isolated from strain RJ2 grown onCMA plates incubated at 30°C for 5 days. Plate cultures werecut into 1-cm squares and extracted with 3 volumes of 80%acetone with water. The combined extracts were filteredthrough cheesecloth to remove pieces of agar. Other partic-ulate matter was removed by centrifugation (9,OOOx g for 15min) at 4°C. The supernatant was collected and evaporatedunder a vacuum to remove the acetone. NaCl (5 g) wasdissolved in each 100 ml of the aqueous concentrate, whichthen was extracted three times with diethyl ether. The etherextracts were combined and evaporated by dryness under avacuum. The resulting residue was dissolved in acetone oracetonitrile and stored at -20°C until further purified.The relative mobility of antifungal compounds present in

the ether fraction was determined by using silica gel (thin-layer chromatography silica gel 60, 20 by 20 by 0.25 cm;Merck & Co., Inc., Rahway, N.J.) in various solvent sys-tems. The crude extract (30 [lI) was spotted, and the solventfront was allowed to run for approximately 16 cm. Therunning lane was then dried thoroughly and cut into portions(1 by 2.5 cm). These portions were scraped into microcen-trifuge tubes and extracted with 100% acetone. The silicaresidue was removed by centrifugation, and the supernatantwas transferred to a second set of microcentrifuge tubes.Each fraction was concentrated by evaporating off theacetone and tested for antifungal activity by using the petriplate assay described above. The Rf values were determined

for each fraction showing activity against fungi. Furtheranalysis of the crude compound was performed by HPLC ona high-pressure liquid chromatograph (model 112; BeckmanInstruments, Inc., Fullerton, Calif.) with a Beckman Ultra-sphere C18 reverse-phase column containing 2 ,uM Li-Chrosorb RP-18 (Merck). Elution was done by using increas-ing concentrations of acetonitrile-H20 (50 to 100%) solventat a flow rate of 0.5 ml/min. Purified fractions were collectedand stored at -20°C until further analysis.

RESULTS

Isolation and characterization of antagonistic bacteria. Allthe antifungal bacteria isolated from unshed caryopses of T.dactyloides had similar phenotypes. They were short rods(approximately 1 by 1.6 ,um) that mainly grew in pairs or inclusters, and they were gram negative, lophotrichous (Fig.1), and motile. Strain RJ2 was used for further characteriza-tion. This strain grew at 20 to 40°C but not at 41°C. Coloniesgrown on LB agar or King B medium at 30°C for 16 h wereyellow pigmented, nonfluorescent, raised, circular, smooth,and metallic. The strain was aerobic, failed to grow underacidic conditions, did not require organic growth factors,grew on acetate as the sole carbon source, was oxidase andcatalase positive but indole negative, and did not producegas from sugars. This strain was tested for its utilization ofseveral carbon sources (Table 1). Because these character-istics are the main features of bacteria belonging to the genusPseudomonas (10), it was concluded that strain RJ2 belongsto this genus.The species level identification of the strain was attempted

with the key tests published by Doudoroff and Palleroni (10)and Hugh and Gilardi (16). The strain showed urease,L-lysine decarboxylase, and L-ornithine decarboxylase ac-tivities; no activity in L-arginine dehydrolase reaction; yel-low diffusible nonfluorescent pigment; failure to grow onstarch, rhamnose, maltose, and deoxycholate; inability toproduce hydrogen sulfide; and ability to utilize ribose,arabinose, trehalose, adonitol, and m-hydroxybenzoate as acarbon source for growth. These observations suggested thatthe organism belonged in the species Pseudomonas cepacia.However, in contrast to many cepaciae, it utilized tartrate,did not utilize lactose as a carbon source, and failed to growat 41°C. Strain RJ2 was resistant to several antibiotics (Table1). Resistance to these antibiotics did not appear to bedetermined extrachromosomally, because plasmids couldnot be isolated by either of two different methods describedby Maniatis et al. (23). In order to isolate a strain amenableto genetic manipulation, we mutagenized strain RJ2 withN-methyl-N'-nitro-N-nitrosoguanidine and obtained strainRJ3, which retained antifungal activity and was sensitive totetracycline, neomycin, chloramphenicol, ampicillin, genta-micin, kanamycin, erythromycin, novobiocin, streptomycin,and penicillin.Mechanism of antagonism. The antagonistic properties of

Pseudomonas strain RJ2 against various important patho-gens of corn tested in the petri plate assay are shown inTable 2 and Fig. 2. It is important to note that some of thesephytopathogens were inhibited more on CMA than on PDA,and vice versa. Although the significance of this observationis not understood, it suggests that growth conditions orenvironmental factors may structurally modify the antifungalcompound and thus affect the antagonistic properties of thePseudomonas strain.To determine whether siderophores or antibiotics are

responsible for the antagonistic properties of strain RJ2, we

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FIG. 1. Photomicrograph of Pseudomonas strain RJ2 under a transmission electron microscope (magnification,with 1% phosphotungstic acid. The arrow points to the tufts of flagella.

tested for antifungal activity in the presence of 10 to 100 ,uMFeCl3. Because this Pseudomonas strain inhibited thegrowth of fungi in ferric ion-rich medium, siderophores maynot be involved in the antagonistic response. Next, weinvestigated the possibility of the production of antibioticsubstances by this strain. Crude extracts from a 5-day-oldculture of a Pseudomonas strain grown on CMA plates wereprepared by partitioning with 80% acetone. Extracts weretested by using the petri plate assay. The results indicatedthat the antagonistic activity was due to the production ofantifungal compounds and that the compounds could beextracted from growth medium by partitioning with organicsolvents.

Partial characterization of an antibiotic. The partially pu-rified compound is stable to heat treatment (boiling water for10 min), acid and alkali treatment (0.1 M), and proteasetreatment. The active substance was partially characterizedby thin-layer chromatography. The Rr values for the activefractions are different from the Rf values reported for otherantibiotic-producing strains of Pseudomonas (12, 14, 15)(Table 3). A fraction containing yellow pigment did not showactivity, indicating that the compound may not be phena-zine. A compound purified by thin-layer chromatography orHPLC showed activity against all the fungal pathogens thatwere inhibited by strain RJ2 (Table 2). This indicated that theantagonistic property of strain RJ2 is due to this compound.Attempts were made to further purify the antifungal com-pound by HPLC with a C18 reverse-phase column. A singlepeak had the entire antifungal activity, as determined by thepetri plate assay (data not shown).

x 15,500) after staining

DISCUSSION

Disease suppression by microorganisms in agriculturallyimportant crops has been studied in detail (3, 9, 11, 14, 15,20, 22, 26-29, 34, 35, 37), but few reports of organismsantagonistic to corn pathogens are available (1, 2, 5, 19). Themechanisms by which microorganisms colonize the host orantagonize the pathogens have not been fully elucidated. Wehave initiated a study of screening and investigating theantagonistic microorganisms of corn pathogens. This paperpresents the isolation and characterization of a novel Pseu-domonas strain (RJ2) that displays antagonism toward adiverse group of pathogenic fungi of corn. On the basis of allthe physiological and biochemical tests, we find that thecharacteristics indicate the species P. cepacia, althoughsome of the tests described for P. cepacia in Bergey'sManual ofDeterminative Bacteriology were negative (10).The strain restricted the growth of these fungi by produc-

tion of an antifungal compound but probably not by produc-tion of siderophores. The antifungal compound is synthe-sized and secreted by strain RJ2 on PDA and CMA media.Although the growth of strain RJ2 on CMA and PDA wasequal, the antagonistic activities against various fungi wereusually different on the two media (Table 2). The differencesin the antagonistic activities on the two media are partiallydue to the inability of the fungi to grow or may be due to thechemical modification of the antifungal compound. On thebasis of the chromatographic properties of this compoundand its comparison in the same solvent systems with otherknown antifungal compounds of Pseudomonas strains, itappears different from those reported in the literature (12,

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TABLE 1. Morphological and biochemical characteristicsof Pseudomonas strain RJ2

Test or characteristic Result

Gram stain reaction .............................. -

Cell type ............................... Rod (many in pairs)Size .............................. 1 ,m wide, 1.6 ,m

longMotility .............................. + (weak)Flagella.............................. Polar tuft (3 to 5)Colony color.............................. + (yellow, nonfluores-

cent)

FermentationGlucose ............ .................. +Lactose, sucrose, mannitol, starch ........-

HydrolysisGelatin liquefaction ............................. + (weak)Casein ........... ................... +Fat.............................. +

Indole.............................. -

Voges-Proskauer ............................... -Citrate utilization............................... +Nitrate reduction .............................. -

H2S production ............................... -

Urease............................... -

Catalase .......... .................... +Oxidase .............................. + (weak)Litmus milk.............................. PeptonizationNaCl tolerance .............................. 2%Lysine decarboxylase ............................. +

Arginine dihydrolase .............................. -

Ornithine decarboxylase ......................... +Egg yolk agar .............................. Lipolysis (weak)Deoxycholate agar ............................... -

Brain heart infusion broth ....................... + (pH 7.0), - (pH 4.5)Carbon sources for growth

Acetate, pyruvate, succinate, tartrate,malate ............... ................ +

Adonitol ............. ................. +D,L-Arabinose, D-ribose, D-xylose .........+Cellulose, amylose, amylopectin, starch .. -

m-Hydroxybenzoate ........................... +Glycerol, inositol, mannitol, sorbitol ......+Lactose, maltose, rhamnose ................. -

D-Mannose, sucrose, trehalose .............+Salicin ........... ................... +Ethanol, methanol .............................. -

Carbon and nitrogen sources for growthAlanine, arginine, aspartate, glutamate ... +Histidine, proline, phenylalanine,

tryptophan ........................... ... +Glycine, glutamine, methionine .............-

Antibiotic sensitivityChloramphenicol, novobiocin,

tetracycline .............................. SensitiveAmpicillin, erythromycin, gentamicin,kanamycin .............................. Resistant

Methicillin, neomycin, penicillin,streptomycin ............................... Resistant

Antibiotic production ............................. + (antifungal)

14, 15). The antifungal compound is not affected by protease,heat, or acid or alkali, indicating that it is not a proteinmolecule. The partially purified compound was activeagainst all of the fungi that were inhibited by strain RJ2,

TABLE 2. Antifungal activities of Pseudomonas strain RJ2against various corn pathogens in the petri plate assay

Antifungal activity ina:Pathogen

CMA PDA

Aspergillus flavus + +Cercospora zeae-maydis - + +Collectrichium graminicolum - + +Diplodia maydis + + +Exserohilum turcicum ++Fusarium moniliforme + +Fusarium roseum ++ +Helminthosporium coarbonum ++ +Helminthosorium maydis + + +Macrophomina phaseolina ++ +Penicillium chrysogenum + +Rhizopus stolonifer + ++Trichoderma viride + + +Ustilago maydis ++Erwinia stewartii

a + +, Good inhibition; +, poor inhibition; -, no inhibition.

indicating that the compound can be readily extracted fromthe growth media.We are currently analyzing the chemical composition of

the HPLC active peak. Once a pure compound is obtained,its chemical structure will be determined by using a combi-nation of gas chromatography-mass spectrometry, FT-in-frared, and nuclear magnetic resonance. The identification ofthe compound is important for several reasons. (i) Thepartially purified fraction showed excellent activity againstmost of the important corn root rot, stalk rot, and seed rotpathogens (Table 2). These results indicate the potential useof this compound(s) in the control of fungal diseases, whichare of considerable economic importance. (ii) The activefraction also protected maize seeds from Aspergillus andFusarium strains, which produce several mycotoxins. Thus,identification of the compound could lead to the chemicalsynthesis of this compound and analogs for use in thesuppression of the destructive activities of pathogens in thefield as well as in grain storage. On the basis of preliminarycharacterization, this compound seems different from thosereported in the literature.On the basis of these data, it is difficult to say whether the

antifungal compound is directly gene encoded or encodedindirectly via the production of a secondary metabolite.Recently, attempts were made to clone the bacterial genesresponsible for antibiotic production (13, 20, 34). Thesereports of cloning genes for antifungal compounds in otherPseudomonas strains (13, 20) suggest that our antifungalcompound may also be a gene-encoded product. In order toclone genes responsible for the production of the compound,we have constructed a clone bank of RJ3 DNA in a wide-

TABLE 3. Silica gel thin-layer chromatographic migration of theantifungal compound produced by Pseudomonas strain RJ2

Solvent system Rf

CH2CI2 (100%)................................. 0.66CH2Cl2-methanol (9:1) ................................. 0.78CH2Cl2-acetone (4:0) ................................. 0.81CH2Cl2-ethyl acetate (9:1)................................. 0.80Chloroform (100%) ................................. 0.57Chloroform-acetone (9:1) ................................. 0.63Chloroform-ethyl acetate-formic acid (5:4:1) ........................ 0.93

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FIG. 2. Petri plate assay for antifungal activity. (a) Growth inhibition of Trichoderma viride by different isolates of Pseudomonas spp. (b)Growth inhibition of Trichoderma viride by a partially purified antifungal compound. The arrows point to various isolates of Pseudomonasspp. exhibiting differing antifungal activities (a) and the site at which the antifungal compound was spotted (b).

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host-range cosmid vector, pCP13 (8). We are performingtransposon mutagenesis of strain RJ3 by pGS9 (30), pME12(36), and other transposon vectors to isolate mutants defec-tive in the synthesis or secretion of an antifungal com-

pound(s). The emphasis of this study will be to identify andclone the DNA sequences responsible for the production andregulation of the compound(s).

ACKNOWLEDGMENTS

We thank A. E. Liberta and R. C. Anderson for helpful discus-sions; J. W. Webb and M. E. Kurz for help with the HPLC analysis;M. Nadakavukaren for electron microscopy; L. Castor, Funk SeedInternational, for providing fungal strains; and H. Brockman and K.Miller for suggesting improvements in the manuscript.R.G.S. is thankful to the Illinois State University Honors Program

for the M. J. Scholarship. This work was supported by the ResearchGrant Program from Illinois State University.

LITERATURE CITED1. Anderson, R. C., and A. E. Liberta. 1986. Occurrence of

fungal-inhibiting Pseudomonas on caryopses of Tripsacum dac-tyloides L. and its implication for seed survival and agriculturalapplication. J. Appl. Bacteriol. 61:195-199.

2. Anderson, R. C., A. E. Liberta, and M. E. Neville. 1980.Inhibition of selected fungi by bacterial isolates from Tripsacumdactyloides L. Plant Soil 56:149-152.

3. Baker, R. 1985. Biological control of plant pathogens: defini-tions, p. 25-39. In M. A. Hoy and D. C. Herzog (ed.), Biologicalcontrol in agricultural IPM systems. Academic Press, Inc., NewYork.

4. Carlile, W. R. 1988. Fungicides, p. 57-79. In W. R. Carlile (ed.),Control of crop diseases. Edward Arnold Publisher, London.

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