MODE OF ACTION OF BACILLUS SUBTILIS EPCO16 AGAINST TOMATO FUSARIUM WILT SA. Ramyabharathi and T. Raguchander Dept. of Plant Pathology, Centre for Plant Protection Studies, Tamilnadu Agricultural University, Coimbatore- 641 003, India. e-mail: ramyabharu@gmail.com (Accepted 12 March 2014) ABSTRACT : Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici has been known to cause severe yield losses in tomato. Hence, attempts were made to develop an effective ecofriendly strategy to manage the disease using endophytic and plant growth promoting rhizobacteria. Accordingly, the strains of Bacillus subtilis was tested for its effectiveness against tomato Fusarium wilt under in vitro. Under in vitro conditions B. subtilis EPCO16 was found to effectively inhibit the mycelial growth of the pathogen (46.04%) and promote the growth of tomato seedlings (vigour index: 2311.46) when compared to control. Further, B. subtilis EPCO16 produced a large orange halo in Chrome Azurol Sulphonate (CAS) agar plate assay and the nature of siderophore was Hydroxymate type. The same strain was found to be positive for hydrogen cyanide production and produced volatile compound which effectively inhibited the growth of F. oxysporum f. sp. lycopersici to an extent of 60.78 per cent in vitro. B. subtilis EPCO16 is positive for chitinase, β-1,3-glucanase and Proteolytic enzyme activity. These findings suggest that B. subtilis EPCO16 can be successfully employed as an eco-friendly strategy for the management of Fusarium wilt of tomato. Key words : Bacillus subtilis, siderophore, HCN, lytic enzymes. INTRODUCTION Tomato is one of the most versatile vegetable with wide usage in Indian culinary tradition. It is a protective supplementary food because of its special nutritive value. One of the most important diseases of tomato in the warm regions of the world is Fusarium wilt. It is caused by the vascular wilt pathogen, F. oxysporum f. sp. lycopersici (Sacc.) Snyder and Hansen. Biocontrol by use of Bacillus subtilis represents a potentially attractive alternative disease management approach since B. subtilis are known for growth promotion and disease reduction in crops (Jetiyanon and Kloepper, 2002). Competition for iron by siderophore production has long been recognized as an important antagonistic trait found in many of the bacterial biocontrol agents against plant pathogens (De Boer et al, 2003). Therefore, siderophore-producing microorganisms may have promise as biological control agents. Knox et al (2000) reported that the antifungal volatile of B. subtilis had strong inhibitory effect on pathogenic fungi. The lytic enzymes viz., chitinase, protease, β-1,3-glucanase have direct antifungal properties by their ability to degrade isolated fungal cellwalls (Boller et al, 1983) and to inhibit growth of fungi in vitro (Priest, 1993). In the present study the biocontrol potential of B. subtilis exhibited the strong inhibition against Fusarium pathogen in vitro. MATERIALS AND METHODS Biocontrol agent – Bacillus subtilis Rhizosphere soil from healthy tomato plants were collected from different locations. The bacterial antagonist Bacillus spp. was isolated by serial dilution technique using nutrient agar media. Bioagents B. subtilis EPCO16 was obtained from the Culture Collection Section, Department of Plant Pathology, Tamilnadu Agricultural University, Coimbatore, Tamil Nadu, India. Isolation of Fusarium oxysporum f. sp. lycopersici Infected tissues from collar and root regions of tomato cultivar (PKM 1) showing wilt symptoms were collected separately from farmer’s field. Tissue bits were surface sterilized with 10 per cent sodium hypochlorite for 5-10 min. and subsequently three washings with sterile distilled water. Then, they were placed on potato dextrose agar (PDA) medium separately and incubated at the laboratory conditions at 25 ± 2 0 C for five days. The fungi were purified separately by transferring the tip of the mycelia into PDA slants and maintained as stock cultures for further studies. Pathogenicity The fungi viz., F. oxysporum f.sp. lycopersici was mass multiplied separately in sand maize medium. Sand and ground maize were mixed at 9:1 ratio (w/w), moistened to 50 per cent moisture content, filled in polypropylene bags at 3/4 th level and stream sterilized at 1.4 Kg per cm 2 for two hour. After sterilization, nine mm mycelial disc from each fungal culture was inoculated in sand maize medium separately and incubated at laboratory conditions as described earlier for fifteen days. The pathogenicity of fungi was tested separately on tomato Biochem. Cell. Arch. Vol. 14, No. 1, pp. 47-50, 2014 ISSN 0972-5075