Control of Rhizoctonia solani on Cotton Seedlings with Pseudomonas fluorescens and With an Antibiotic Produced by the Bacterium

HOWELL, C. R., and R. D. STIPANOVIC. 1979. Control of Rhizoctonia solani on cotton seedlings with Pseudomonasfluorescens and with an antibiotic produced by the bacterium. Phytopathology 69:480-482. A strain of Pseudomonasfluorescens antagonistic to Rhizoctonia solani Alternaria sp., and Verticillium dahliae. A Fusarium sp. was only partially was isolated from the rhizosphere of cotton seedlings. An antibiotic inhibited and Pythium ultimum was unaffected. Treating cottonseed with strongly inhibitory to R. solani was isolated from P. fluorescens cultures P. fluorescens or pyrrolnitrin at time of planting in R. solani-infested soil and identified as pyrrolnitrin (3-chloro-4-[2'-nitro-3'-chlorophenyl]increased seedling survival from 30 to 79% and from 13 to 70%, respectively. pyrrole). The antibiotic also inhibited growth of other fungi associated with Pyrrolnitrin persisted for up to 30 days in moist 'nonsterile soil with no the cotton seedling disease complex including: Thielaviopsis basicola, measurable loss in activity. Additional key words: seed treatment, biological control. Fluorescent pseudomonads appear frequently among isolates portion was extracted twice with 100-ml volumes of ethyl ether. from plant rhizospheres (11) and often comprise an important The ether extracts were combined and taken to dryness in vacuo. component of the bacterial population (8,12). Pseudomonas spp. The residue was dissolved in chloroform at a concentration 1OOX were ineffective for control of damping-off by Rhizoctonia solani that in the original culture volume, and l-ml samples were streaked and Pythium spp. in nonsterile soil (3). However, Pseudomonas on thin-layer plates coated with silica gel 7GF. The plates were spp. have been used successfully to protect carnation cuttings developed in cyclohexane/chloroform/diethylamine (50:40:10, against Fusarium roseum f. sp. cerealis (9), potato seedpieces v/v) and observed under long and shortwave UV light to detect against certain pathogenic bacteria and fungi (4), and soybean fluorescing and adsorbing bands. Bands and blank areas were seedlings against Phytopthora megasperma var. sojae (13). scraped separately from the plates and eluted with acetone. In our work on seedling diseases of cotton we have isolated Acetone eluates diluted 1:10 with sterile water were assayed for fluorescent pseudomonads from the seedling rhizosphere and activity against R. solani by placing 100-/t1 aliquots in 7 mm report here: the isolation and identification of a fluorescent diameter wells cut in PDA plates inoculated simultaneously on the pseudomonad antagonistic to R. solani; the isolation and surface with agar plugs of the fungus. After 2 days of incubation the identification of an antibiotic produced by the isolate; and the plates were examined for mycelium-free zones around the wells. efficacy of that isolate or its antibiotic as a seed treatment to The eluate that contained active material again was streaked on prevent damping-off of cotton seedlings by R. solani. silica gel 7GF plates and developed with chloroform and the procedure for locating bands and detecting zones with antibiotic MATERIALS AND METHODS activity then was repeated. The eluate from the band showing inhibitory activity was concentrated in hot cyclohexane, and the Isolation and identification of an antagonistic bacterium from antibiotic was crystallized from it after cooling. Mass and NMR rhizosphere soil. Dilutions (10-6 and l0 , w/v) of rhizosphere soil spectra of the purified crystaline antibiotic were made and from 7-day-old cotton seedlings were spread in 0. l-ml samples on compared with standard spectra for authentic pyrrolnitrin plates of medium 523 in agar (7). Plates were incubated at 24 C for 4 obtained from the Fujisawa Pharmaceutical Co., Osaka, Japan. days then inoculated on the surface with PDA plugs of R. solani Antifungal spectrum of the antibiotic. Purified antibiotic (strain W-18 from cotton). After 3 days of incubation at 24 C the dissolved in 10% aqueous acetone was diluted to 25 vg/ml with plates were examined for bacterial colonies antagonistic to the sterile water. The solution then was assayed (by the methods growth of R. solani. Colonies with putative antibiotic activity were described previously for the chromatogram eluates) against the isolated and challenged with the fungus after 3 days. The most following fungi that have been associated with cotton seedling active isolate, a fluorescent pseudomonad, was subjected to diseases: Thielaviopsis basicola, Alternaria sp., Vertici/lium standard staining (10) and physiological tests (I), and identified to dahliae, Fusarium sp., and Pythium ultimum. species according to Bergey's manual (2). Efficacy of cottonseed treatment with bacterial cultures or Isolation and identification of an antibiotic from the antibiotic to control damping-off. Nonsterile soil containing 33 g of antagonistic bacterium. Plates containing medium 523 in agar were R. solani inoculum per 9 kg of soil was prepared and its level of spread with a bacterial suspension, incubated for 10 days at 24 C, infestation was determined according to methods described cut into 1-cm squares, and extracted with 200 ml of 80% aqueous previously (5). Dilutions of purified antibiotic in acetone were acetone for each 10 plates of culture medium. The extracts were prepared at 0, 20, 40, 60, 80, 100,200, and 400 pg/ml. One-milliliter filtered through cheesecloth and centrifuged at 12,000 g for 10 min samples were added to 0.2-g lots of diatomaceous earth carrier, and to remove particulate matter. The supernatant fluids were the acetone was removed in vacuo. Each of the 0.2-g samples then combined and condensed in vacuo at 40 C. Five grams of NaC1 was mixed with methyl cellulose, and coated onto 30 cottonseed per were added to each 100 ml of the aqueous concentrate and each sample, and assayed for optimum protective effect against R. solani This article is in the public domain and not copyrightable. It may be freely by the soil tube method described previously (5). reprinted with customary crediting of the source. The American PhytopathCottonseed treated with 200 /Ag/ml antibiotic were planted in ological Society, 1979. lots of 30 seed per flat in R. solani-infested or noninfested