Control of Fusarium Wilt of Tomato by Bioformulation of Streptomyces griseus in Green House Condition

Ability of biocontrol agent Streptomyces griseus to control the F. oxysporum f. sp. lycopersici. (FOL) induced fusarium wilt disease in tomato was studied. Talc-based formulation of Streptomyces griseus either single or with or without chitin were developed and tested under greenhouse conditions. Self life of S. griseus in talk based formulation showed stable up to 105 day with 122 x 10 , 118 x 10 cfu/g after storage at 30°C th 7 7 and 4°C correspondingly. The various formulations of S. griseus were assessed for their efficiency in controlling F. oxysporum incidence in greenhouse conditions. The treatments were imposed as seed treatment and seedlings dip. A significant lowest disease severity on treatment of self fusant (SFSg 5) S. griseus suspension (root dipping) and chitin amended S. griseus (root dipping) was recorded compared with chitinase enzyme preparation of the same. For effective disease control, S. griseus introduced to root system before Fusarium oxysporum infestation than that of seed treatment. Key word: Tomato Fusarium wilt and Streptomyces griseus formulation INTRODUCTION alternative treatments for control of plant diseases are The production of tomato and pepper fruit is of pathogens, known as biological control is now in practice worldwide agricultural importance. Tomato (Lycopersicon [2]. It is accepted as a suitable and environmentally esculentum) is one of the most popular and important friendly alternative or a supplemental way of reducing the commercial vegetable crops grown throughout the world. use of chemicals in agriculture against plant disease It is rich in vitamins A, B and C. In India, it occupies an management [3]. Biocontrol preparation of both fungi and area of 0.54 million ha with a production of 7.60 million bacteria have been applied to seeds, seedlings and tonnes. Many diseases and disorders can affect tomatoes planting media to reduced tomato wilt disease in the green during the growing season. Fusarium oxysporum f. sp. house and field condition with various degrees of lycopersici (FOL) is a highly destructive pathogen of success. The purpose of the present work was designed both greenhouse and field grown tomatoes in warm to evaluate the effect of Streptomyces griseus on the vegetable production areas. The disease caused by this growth of tomato wilt pathogens as well as their effect on fungus is characterized by wilted plants, yellowed leaves tomato wilt disease incidence under greenhouse and minimal or absent crop yield. There may be a 30 to condition. 40% yield [1]. Agricultural production in over the past few decades, MATERIALS AND MATHODS producers became more and more dependent on agrochemicals, as a reliable method of crop protection Microbial Culture: Antagonistic bacteria of S. griseus with economic stability of their operations. However, used in this study was isolated from Prawn cultivated increasing use of chemical inputs causes several negative pond soil collected from Peddapuram village; East effects, i.e., development of pathogen resistance to the Godavari district. A survey was conducted to assess the applied agents and their environmental impact. Therefore, intensity of Fusarium wilt disease Coimbatore District of needed. The use of microorganisms to control plant African J. of Basic & Appl. Sci., 1 (1-2): 9-14, 2009 10 Tamil Nadu. The infected plant material collected and broth medium. The culture of S. griseus was inoculated in plated in a PDA and incubated at RT for 5 days. The the nutrient broth and incubated for seven days at 30°C pathogenic colonies raised were subcultured in a PDA and at 125 shakes/min (Remi C24) respectively [8]. Broth slant and it was identified. The micro conidia and macro containing 9 x 10 cfu/ml was formulated in talc powder. conidial structures showed that it was belonging to The talc-based formulations of S. griseus amended with F. oxysporum f. sp. lycopersici. The pathogenicy was chitin were prepared as mentioned previously. proved as per standard method. Preparation of Pathogenic Fungi: The respective isolates For seed treatment, the seeds were initially surface of F. oxysporum f. sp. lycopersici, were multiplied on sterilized with 1% sodium hypochlorite and soaked in sand maize medium [4]. Sand and ground maize seeds double volume of sterile distilled water containing were mixed in the ratio of 20:1, moistened to 50% moisture talc-based formulation (S. griseus 10 g/kg of seeds). content. Then it was filled in 500ml conical flask After 12 h, the bacterial suspension was drained off and and autoclaved for two hours. Cell suspensions of the seeds were dried under shade for 30 min and sown [9]. F. oxysporum f. sp. lycopersici was prepared by culturing Another set of seed were treated with chitin amended the fungus in Czapek broth medium on a rotary shaker at talc-based formulations following the same procedure 25°C for 5 days [5]. The resulting culture was filtered stated above [10]. through cheesecloth to remove mycelial fragments, washed by centrifugation (10,000 rpm for 15 min) and Preparation of Antagonist Suspension of S. griseus for resuspended in sterile distilled water. microconidial suspension of F. oxysporum f. sp. lycopersici, final density of 10 microconidia/ml was inoculated in to sand 5 maize medium and it was incubated at room temperature for 14 days. For green house experiment, the substrate at 5 percent (w/w) was mixed with sterilized soil and infested into the pot ten days prior to transplanting the 60-day-old seedling transplantation. The population of F. oxysporum f. sp. lycopersici in the substrate was estimated as number of colony forming units (CFU) per gram of substrate, as previously described [6]. Approximately 2 x 10 cfu/g of innoculum of F. oxysporum f. sp. lycopersici 8 was used for pot experiments. Preparation of Talk Based Formulation of Biocontrol Agent: A loop full of S. griseus was inoculated in to the nutrient broth medium and incubated for seven days at 30°C and at 125 shakes/min. (Remi C 24). After incubation the broth containing 9 x 10 cfu/ml was used 8 for the preparation of talc based formulation. To 400 ml of mycelial or spore suspension 1 kg of talc powder, calcium carbonate 15 g (to adjust the pH to neutral and carboxy methyl cellulose 10 g (as additive) were mixed under sterile condition following the method described by Vidhyasekaran and Muthamilan [7]. The product was shade dried to reduce the moisture content to 20% and then packed in polypropylene bags and sealed [8]. Chitin Amendment with Talc-based Formulations: Colloidal chitin was prepared by the procedure stated in 3.2.1.1. Colloidal chitin was incorporated into and nutrient 8 Preparation of Seeds Using Talk Based Formulation: Root Application: The isolated S. griseus was grown on MS with 1 g colloidal chitin. 1ml of the bacterial spore suspension was inoculated into flasks containing the production medium and incubated for seven days at 30°C and at 125 shakes/min. (Remi C24) At the end of incubation the culture was the culture was harvested, filtered and it was centrifuged at 10,000 rpm for 10 min at 4°C and used as crude enzyme. The cell suspension of S. griseus were re suspended in sterile distilled water at the rate of 9 x 10 cfu/ml was used to control the fusarium 8 wilts of tomato [11, 12]. Preparation of Purified Chitinase Enzyme: Purification of chitinase enzyme from S. griseus was described previously. The crude enzyme and partially purified enzyme of S. griseus was used to control the fusarium wilts of tomato [13]. Preparation of Suspension of Self Fusants S. Griseus: Higher percent inhibition showed self fusant (SFSg 5) of S. griseus was inoculated in to MS medium and incubated for 7 days at 30°C and at 125 shakes/min (Remi C 24). After incubation cells of S. griseus were re suspended in sterile distilled water. The suspension containing the rate of 9 x 10 cfu/ml was used to control the fusarium wilts of 8