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Pseudomonas Stutzeri YPL-1 Genetic Transformation and Antifungal APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1991, p. 510-516 Vol. 57, No. 2 0099-2240/91/020510-07$02.00/0 Copyright © 1991, American Society for Microbiology Pseudomonas stutzeri YPL-1 Genetic Transformation and Antifungal Mechanism against Fusarium solani, an Agent of Plant Root Rot HO-SEONG LIM, YONG-SU KIM, AND SANG-DAL KIM* Department of Applied Microbiology, Yeungnam University, Gyongsan 713-749, Korea Received 24 July 1990/Accepted 4 December 1990 An actively antagonistic bacterium that could be used as a biocontrol agent against Fusarium solani, which causes root rots with considerable losses in many important crops, was isolated from a ginseng rhizosphere and identified as a strain of Pseudomonas stutzeri. In several biochemical tests with culture filtrates of P. stutzeri YPL-1 and in mutational analyses of antifungal activities of reinforced or defective mutants, we found that the anti-F. solani mechanism of the bacterium may involve a lytic enzyme rather than a toxic substance or antibiotic. P. stutzeri YPL-1 produced extracellular chitinase and laminarinase when grown on different polymers such as chitin, laminarin, or F. solani mycelium. These lytic extracellular enzymes markedly inhibited mycelial growth rather than spore germination and also caused lysis of F. solani mycelia and germ tubes. Scanning electron microscopy revealed degradation of the F. solani mycelium. Abnormal hyphal swelling and retreating were caused by the lysing agents from P. stutzeri YPL-1, and a penetration hole was formed on the hyphae in the region of interaction with the bacterium; the walls of this region were rapidly lysed, causing leakage of protoplasm. Genetically bred P. stutzeri YPL-l was obtained by transformation of the bacterium with a broad-host-range vector, pKT230. Also, the best conditions for the transformation were investigated. Antagonistic microorganisms, by their interactions with serial dilutions were plated on nutrient agar. Each isolate various soil-borne plant pathogens, play a major role in was tested for its inhibition of F. solani, a pathogenic plant microbial equilibrium and serve as powerful agents for fungus, as described below. The fungus F. solani was biological disease control (2, 5, 8, 15, 39). The interactions provided by the Korea Ginseng and Tobacco Research between biocontrol agents and plant pathogens have been Institute and was grown on potato dextrose agar (PDA). The studied extensively, and the application of biocontrol agents most efficient antagonistic bacterium was selected and iden- in the protection of some commercially important crops is tified according to criteria in Bergey's Manual ofSystematic promising (38, 47). Biocontrol of plant pathogens provides Bacteriology (22). an alternative means of reducing the incidence of plant In vitro antifungal activity tests. Two different techniques disease without the negative aspects of chemical controls were used for testing the antagonistic effect against F. like pesticides (6). Chemical fungicides are costly, can cause solani. In the first, which assayed the antifungal activity of environmental pollution, and may induce pathogen resist- bacterial strains on plates, samples (5 ,ul, containing approx- ance (18, 24). Additionally, they can cause stunting and imately 106 cells) from overnight cultures of bacterial strains chlorosis of young seedlings (18). Fusarium solani, a patho- in nutrient broth were inoculated 1 cm from the edge of petri genic plant fungus, causes root rots, which results in con- plates and allowed to soak into the agar. A small plug (about siderable economic losses in many important crops (7, 9). 5 mm square) of F. solani inoculum from the leading edge of The use of antagonists to control diseases incited by F. a culture of F. solani grown at 28°C for 3 days on PDA solani is being intensively studied (31, 47), but the mecha- nism involved in lysis of the fungus by bacteria is not well containing 0.2% chitin was placed in the center of the plate. established. Plates were incubated at 28°C and scored after 4 or 5 days by The objectives of the present study were to (i) isolate, measuring the distance between the edges of the bacterial select, and identify potentially useful bacterial antagonists colony and fungal mycelium. In the second assay, which for biocontrol of F. solani; (ii) determine its antifungal tested the antifungal activity in broth culture, bacterial mechanism with biochemical tests, mutational anialyses, and cultures were grown at 30°C for 84 h with aeration. Cells microscopic observations; and (iii) attempt transformation of were removed by centrifugation at 12,000 x g for 20 min. the antagonist to create a model system for the further The culture supernatants were then filtered aseptically genetic development of multifunctional biocontrol agents. through 0.45-,um-pore-size membrane filters. The resulting filtrates were stored at 4°C. Petri plates were filled with molten PDA with 1% culture filtrate. After the plates were MATERIALS AND METHODS cooled, the F. solani inoculum was placed on the agar Isolation and identification of an antagonistic bacterium. surface, and the plates were incubated for 4 or 5 days. The Antagonistic bacteria were isolated from rhizospheres in diameters of the F. solani colonies were recorded, and the ginseng root rot-suppressive soils in Yeungpung-gun, Korea. inhibition ratios were calculated relative to that of a control To isolate bacteria, the rhizosphere soils were suspended in without incorporated culture filtrate. Small plugs taken from 0.01 M phosphate buffer (pH 7.2) with a mortar, and then 2- to 3-day-old cultures of F. solani and added to 250-ml Erlenmeyer flasks containing 2.64% potato dextrose broth (PDB) were incorporated aseptically with 5% culture super- * Corresponding author. natants and incubated on a rotary shaker at 28°C for 5 days. 510 VOL. 57, 1991 ACTION OF P. STUTZERI AGAINST F. SOLANI 511 Fungal mycelia were collected on oven-dried preweighed distilled water. Fifty spores per well were counted for each paper (Toyo filter paper no. 2) and dried at 90°C, and dry of the three replicates per treatment. Germination rates of weights were determined. The inhibition ratio was expressed the chlamydospores and lysis of germ tubes were examined relative to a control (H20). under a light microscope at x400 magnification. Enzymatic activity tests. The cell wall-degrading enzymes Scanning electron microscopy. Microscopic observations such as exo-1,3-0-D-glucosidase (,B-1,3-glucanase) and ,B-N- were made in the interacting regions of F. solani grown with acethyl-D-glucosaminidase (chitinase) were assayed in cul- P. stutzeri YPL-1 in dual culture. The samples were fixed ture filtrates of Pseudomonas stutzeri YPL-1. For the prep- with 3% glutaraldehyde in 0.2 M phosphate buffer (pH 6.5) aration of crude chitinase, the bacterium was grown at for 3 h, washed with the same buffer for 15 min, fixed with 30°C for 84 h on a rotary shaker in chitin-peptone medium 2% OS04 for 2 h, and finally washed again with the buffer. (pH 6.8) containing 0.5% glucose, 0.2% peptone, 0.2% The material was dehydrated with ethanol at 4°C by using a chitin, (from crab shells; Sigma), 0.1% K2HPO4, 0.05% series of steps for 10 min each. The specimens were dried in MgSO4 7H2O, and 0.05% NaCl. For the preparation of a Hitachi HCP-2 critical point drier with CO2 as the carrier crude laminarinase, bacteria were grown at 30°C for 72 h on gas. The dried specimens were mounted on stubs with a rotary shaker in peptone medium containing laminarin Television Tube Koat to prevent charging. The specimens (from Eisenia arborea; Tokyo Chemical Co.). The cultures were sputter coated with gold palladium in a Ion Coater Giko were centrifuged aseptically at 12,000 x g for 20 min at 4°C. IB-5 and observed with a scanning electron microscope (ISI The lytic enzymes were prepared by salting them with SS 103). ammonium sulfate and dialyzing them with buffer. Genetic transformation. The transformation procedure The activities of chitinase and laminarinase were deter- was based on the method of Bagdasarian and Timmis (1). mined by measuring the release of reducing sugar by the Bacterial cells were grown in LB broth at 30°C for 24 h on a method of Nelson (36). One unit of chitinase (laminarinase) rotary shaker. A portion (0.5 ml) of such a culture was activity was determined as 1 ,umol of glucose per mg of reinoculated in 50 ml of fresh LB broth and grown to an protein per h. The reaction mixture of chitinase contained optical density at 660 nm of 0.25, which equals 7 x 105 0.3 ml of 1 M sodium acetate buffer (pH 5.3), 0.5 ml of 0. 1% CFU/ml. The cells were then chilled, harvested by centrifu- colloidal chitin prepared by the method of Bemiller (4) or F. gation at 12,000 x g for 3 min, and washed once with 25 ml solani mycelium prepared by the method of Morrissey et al. of 10 mM MOPS (morpholinepropanesulfonic acid) (pH (34), and 0.25 ml of enzyme solution. The reaction was 7.0)-10 mM RbCI-100 mM CaCl2. The cells were again carried out at 50°C for 4 h. The reaction mixture of lamina- centrifuged and suspended in 25 ml of MRC (100 mM MOPS rinase contained 0.3 ml of 1/15 M phosphate buffer (pH 5.5), [pH 6.5], 10 mM RbCl, 100 mM CaCl2). The cells were kept 0.5 ml of 0.2% soluble laminarin or F. solani mycelium, and on ice for 45 min, harvested by centrifugation, and sus- 0.25 ml of enzyme solution. The reaction was carried out at pended in 5 ml of MRC.
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