In vitro Antifungal Activity of Streptomyces griseus Against Phytopathogenic Fungi of Tomato Field

In vitro tests of interactions between Streptomyces griseus strains and some soil borne plant pathogens like Fusarium oxysporum; Alternaria alternate; Rhizoctonia solani and Fusarium solani of tomato were studied on PDA medium. Strains tested produced a metabolite that inhibited growth of plant pathogenic fungi on PDA medium (Dual Culture Test). When grown in liquid cultures containing colloidal chitin or fungal cell walls as sole carbon sources, Streptomyces griseus produced chitinase enzyme in the medium. Higher levels of this enzyme were induced by Rhizoctonia solani and the crude chitinase enzyme extracted showed zone of inhibition on pathogens inoculated PDA plates of all tested concentrations. When lytic enzyme produced by Streptomyces griseus were incubated with hyphal wall of the test fungi treated with 2M NaOH and Chloropharm: Ethanol showed significantly increased the release of glucose and N acetyl glucosamine than untreated one. This result suggests that proteins in the cell walls of pathogens may make these walls more resistant to degradation by the extracellular lytic enzymes. Pretreatment with alkali or proteolytic enzyme increases their susceptibility for lysis. Ionic strength optimum concentration of NaOH for lytic activity was tested. The enzyme lysed fungal cell wall best at ionic concentration of 2.0 M treatment. In vitro lytic activity of treated cell wall, ionic strength concentrations provide an appropriate condition and the effect of biocontrol organism in field level treatment. Key word: Streptomyces griseus, anti fungal activity, lytic enzyme production, NaOH treatment INTRODUCTION resistance of the pathogens. In order to tackle these The production of tomato is of worldwide use of antagonistic microbes seems to be one of the agricultural importance. Tomato (Lycopersicon promising approaches. It is accepted as a suitable and esculentum) is one of the most popular and important environmentally friendly alternative [2]. commercial vegetable crops grown throughout the During the last decade, -1, 3-glucanase and world. It is rich in vitamins A, B and C. In India, it chitinase as key enzymes responsible for fungal cell and occupies an area of 0.54 million ha with a production of wall lysis and degradation has been reported [3]. These 7.60 million tonnes. Many diseases and disorders can enzymes have been shown to be produced by several affect tomato during the growing season. Fusarium fungi and bacteria and may be an important factor oxysporum f. sp. lycopersici (FOL) is a highly destructive in biological control [4, 5]. Many biological control pathogen of both greenhouse and field grown tomatoes agents in the last few years are being tested and in warm vegetable production areas. The disease caused are not commercially available. However, there is still by this fungus is characterized by wilted plants, yellowed considerable interest in finding more efficient strains, leaves and minimal or absent crop yield. There may be a which differ considerably with respect to their biocontrol 30 to 40% yield [1]. effectiveness [6]. The purpose of this study is to evaluate The usage of fungicides has resulted in the the antagonistic activity of strain Streptomyces griseus accumulation of toxic compounds potentially hazardous under the biological control of various tomato plant to humans and environment and also in the buildup of pathogens in vitro. national and global problems, effective alternatives, Academic J. Plant Sci., 2 (2): 119-123, 2009 120 MATERIALS AND METHODS 15,000 rpm for 10 min at 4°C. (Remi-C 24) The collected Organism, source of plant pathogenic fungi: Chitinase activity [7]. enzyme producing Streptomyces griseus (MTCC-*4734) stain purchased from Microbial Type Culture Collection, Chitinase assay: Colloidal chitin was used as a substrate Chandigarh, India was used for the current enzyme with reference to chin et al. [8]. 0.3 ml of 1% colloidal extraction, purification and antagonistic activity studies. chitin in acetate buffer (50 mM, pH 6.0) was taken to that The following plant pathogenic fungi of tomato plant: 1 ml of enzyme was added and incubated at 30°C for AFC -Fusarium oxysporum; AFC -Alternaria alternate; 30 min. The hydrolysis reaction was terminated by 1 2 AFC3-Rhizoctonia solani and AFC4-Fusarium solani adding 0.6 ml of dinitrosalicylic acid (DNS) reagent. The were kindly obtained from Central Institute for Cotton mixture was kept in a boiling water bath for 15 min, Research, Coimbatore. The spores were maintained in chilled and centrifuged to remove the insoluble chitin. The PDA slants and stored at 4°C until use. resulting adduct was measured in UV double beam Dual culture test of Streptomyces griseus: Antagonistic unit of enzyme activity was defined as the amount of activity was also observed directly on plates of YMA medium, using modification of the hyphal extensioninhibition assay, where actively growing Streptomyces griseus were streaked on the one edge and the phytopathogenic fungal strains on the opposite side of a petri dish containing YMA medium. After the desired incubation time at 28°C, an inhibitory effect of growing Streptomyces griseus against growth of all the test pathogenic fungi was measured and tabulated. (Zone of inhibition) Percent inhibition of test pathogen by antagonistic strain was calculated. Preparation of hyphal wall: All the isolated plant pathogenic fungal cultures were inoculated into 50 ml of YM broth and incubated at 30°C for 5 days. After incubation the mycelia was collected by filtration. The mycelia were thoroughly washed with autoclaved distilled water and homogenized on ice, with a homogenizer for 5 min. The mycelia suspension was centrifuged at 15,000 rpm for 20 min at 4°C. The pellet was resuspended in distilled water and sonicated on ice 4 times for 5 min. The suspension was centrifuged at 8000 rpm for 10 min at 4°C. to precipitate the coarse particle. Then precipitate was washed, air dried and stored at 4°C [4]. Induction of extra cellular lytic enzyme: Enzyme assays filtrate: For assessing the hydrolytic activity, the reaction were made under conditions that were similar to those mixture (1 ml) containing 1 mg/ml of cell wall with 0.3 ml of existing in the in vitro antagonism test, namely in MS crude enzyme was incubated at 38°C for 24 h. Each test medium that contained the following (g/L); 1 g each tube was amended with 10 μl of toluene to prevent fungal cell wall, 5 g peptone, 5 g Yeast, 0.7 g KH PO , 0.3 contamination. The released total reducing sugars [9]; 2 4 g K HPO , 4 g NaCl, 5 g MgSO • 7H O, 1 mg FeSO •7H O, glucose [11] and the GlcNAc [12] was measured in treated 2 4 4 2 4 2 0.1 mg ZnSO •7H O, 0.1 mg MnSO •7H O and 1 g NH SO . and untreated fungal cell walls. 4 2 4 2 3 4 1ml of spore suspension was inoculated and incubated for seven days at 30°C at 125 shakes/min (Remi-R 8 C). Effect of ionic strength of NaOH on lytic activity: The At the end of incubation the culture was centrifuged at effect of ionic strength on lytic activity of Streptomyces supernatant was tested directly for chitinase enzyme spectrophotometry (Systronics; 2101) at 540 nm [9]. One enzyme that catalyzed the release of 1 ìmol of N-acetyl D-glucosamine per ml per min. Inhibition of fungal growth by crude enzyme extract: The possible involvement of actinomycetes chitinase in the antagonism towards other pathogenic fungi was determined indirectly in YMA medium uniformly spread with pathognes. Filter paper discs were laid on the swabbed plates, in to each disk 50 μl of the purified lyophilized enzyme at the concentration of 5 U, 50 U and 100 U extracted from Streptomyces griseus was added. For control 50 μl of distilled water was added. Fungal growth was observed over 4 days of incubation at 30°C. The radial diameter of the zone formation was measured [10]. Modification of hyphal cell walls: For modification process, 4 mg of cell walls of isolated fungal cultures were suspended and kept in a rotatory shaker for 1 h at 50 rpm (Remi-R 8C) in 6 ml of (a) 2 M NaOH at 25°C and (b) chloroform/methanol (2:1, v/v) at 37°C. After each treatment the hyphal walls were washed thoroughly by centrifugation at 10000 rpm for 10 min at 4°C. The pellet was suspended in acetate buffer (50 mM, pH 5.0) [4]. Hydrolytic activity of Streptomyces griseus culture