Jae Seok Yang, Hyun Goo Lee and Youn Su Lee* Greenhouse Evaluation of the Rhizobacteria for the Biological Control of Plant Pathogenic Fungi Mahesh Adhikari, Sang Woo Kim, Dil Raj Yadav, Anam Giridhar Babu, Young Hyum Um, Eun BI Gim, Jae Seok Yang, Hyun Goo Lee and Youn Su Lee* Department of Applied Plant Sciences, Kangwon National University, Chuncheon 200-701, Korea ABSTRACT INTRODUCTION Five rhizobacterial isolates (Paenibacillus polymyxa PJ-1, Alcaligenes faecalis PJ-2, Bacillus pumilus PJ-3, Bacillus megaterium PJ-4 and Bacillus amyloliquefaciens PJ-5) isolated from different soil samples were evaluated against Fusarium wilt of tomato, anthracnose of pepper and white mold of lettuce under greenhouse condition. The results showed that isolate Bacillus amyloliquefaciens PJ-5 significantly inhibited Fusarium wilt caused by Fusarium oxysporum and 70% of the disease suppression was recorded. Bacillus pumilus PJ-3 and Bacillus megaterium PJ-4 reduced anthracnose disease on foliar and fruits of pepper as compared to pathogen-inoculated control and disease suppression rates of 74.8 and 67.5% were recorded in Bacillus pumilus PJ-3 and Bacillus megaterium PJ-4, respectively. In lettuce the percentage of diseased plants was significantly lower in Paenibacillus polymyxa PJ-1 and Bacillus amyloliquefaciens PJ-5 as compared to other isolates. The use of microorganism to control crop pest and disease is an exciting and rapidly advancing branch of applied biology. Biological control of plant pathogen can be defined as the condition or practice under which survival or activity of the plant pathogen is reduced through the use of any other living organism, with the result that there is reduction in the evidence of the disease caused by pathogen. (Rivera et al., 1974). Relationship between two microorganisms in which each other adversely affected is termed as antagonism (Ulloa and Hanlin, 2000). By screening this antagonistic character against target pathogen biological control agent are selected. Biological control of plant pathogens is now an established sub-discipline in the science of plant pathology (Paulitz and Belanger, 2001). Thus PGPR acts upon the various plant disease and promotes the growth through the different modes of action. MATERIALS AND METHODS Isolation of bacteria: Soil dilutions were prepared with each gram of soil sample suspended in 99 ml of sterile distilled water and shaken vigorously for 2 min. Then the soil samples were serially diluted in sterile distilled water, then 1 ml of each 10-5 and 10-6 dilution was pipette out and poured into petri dishes and plated on sterile tryptic soy agar (TSA) medium. The plates were incubated at 28℃ for 12-14 days. Then colonies with different morphological appearances were selected from the countable plates and re-streaked on a new plate of the TSA media to obtain pure colonies. Preparation of plant pathogen inoculum; Fusarium wilt of tomato (Fusarium oxysporum) (KACC 4005), anthracnose of pepper (Colletotrichum gloesporoides) (KACC 40003) and white mold of lettuce (Scleretonia sclerotiorum) (KACC 40457) were obtained from Korean Agriculture Culture collection (KACC) and grown on PDA medium at 28℃ for 12 days. Seed sowing: Tomato, pepper and lettuce seeds were surface sterelized for 20 min in 20% sodium hypochlorite followed by a brief rinse with 50% methanol before planting in sterile water. Seeds were sown in a plastic pot filled with commercial soil (Baroker , Seoul Bio Co., Ltd). Preparation and application of bacterial inoculum: A loop-full of a bacterial isolate was inoculated into tryptic soy broth (TSB) and incubated in a rotary shaker at 150rpm for 72 hr at room temperature (28℃). After incubation , the bacterial suspension was diluted in sterelized distilled water (SDW) to give final concentration of 108- 109 CFU/ml (OD 0.1-0.50 at 550 nm using a haemocytometer and optical density measurement . RESULTS Table 1. List of selected fungal pathogens used in the study and their host plants. Table 2. Effect of rhizobacterial isolates on suppression of Fusarium wilt of tomato caused by Fusarium oxysporum in greenhouse conditions. Fig. 1. Percentage of disease (anthracnose) incidence on pepper in greenhouse trials. Control refers plants inoculated with pathogens only. Bars with the same letters indicate no significant differences between means as determined by LSD test (P≤0.05) . Errors bars indicates ±SD. Fig. 2. Percentage of disease (anthracnose) suppression on pepper in greenhouse trials. Control refers plants inoculated with pathogens only. Bars with the same letters indicate no significant differences between means as determined by LSD test (P≤0.05) . Errors bars indicates ±SD. Fig. 3. Percentage of disease (white mold) incidence on lettuce in greenhouse trials. Control refers plants inoculated with pathogens only. Bars with the same letters indicate no significant differences between means as determined by LSD test (P≤0.05) . Errors bars indicates ±SD. Fig. 4. Percentage of disease (white mold) suppression on lettuce in greenhouse trials. Control refers plants inoculated with pathogens only. Bars with the same letters indicate no significant differences between means as determined by LSD test (P≤0.05) . Errors bars indicates ±SD. DISCUSSION In this study isolates, Paenibacillus polymyxa PJ-1, Alcaligenes faecalis PJ-2, Bacillus pumilus PJ-3, Bacillus megaterium PJ-4 and Bacillus amyloliquefaciens PJ-5 showed significant suppression of tested plant pathogenic fungi . Isolate Bacillus amyloliquefaciens PJ-5 inhibited Fusarium wilt in tomato by 70%. Like wise other isolates also exhibited significant reduction in pepper anthracnose and white mold in lettuce. The results of the present study suggested that the rhizobacterial isolates can be utilized as biofertilizer and bicontrol agents for vegetable production in sustainable and ecological agricultural systems. REFERENCES Paulitz TC and Belanger RR. Biological control in green house systems. Annu. Rev. Phytopathol. 2001;39:103-33. Rivera JR, Gonalez MA, and Cuevas-Ruiz J. Sprout inhibition in yam by gamma irradiation. J. Agric. Univ. P. rico. 1974;17:319-39. Ulloa M and Hanlin RT. Illustrated Dictionary of Mycology. The American Phytopathological scoiety, St. Paul, USA: APS Press; 2000. 448pp.