Biological control of Verticillium black spot of Japanese radish using Bacillus spp. and genotypic differentiation of selected antifungal Bacillus strains with antibiotic marker

Bacillus species can form endospores and have broad-spectrum activity of their antibiotics. For that reason, they offer several advantages over other bacteria for protection against root pathogens. This work was intended to determine the ability of Bacillus spp. to inhibit Verticillium dahliae. First, antagonistic activities of Bacillus spp. were studied in greenhouse trials. These results suggest that the bacterium can control soil-borne diseases. Secondly, to investigate the situation in a culture environment, Bacillus spp. were examined for their activity to control Verticillium black spot of Japanese radish and plant-growth promotion rhizobacteria (PGPR) effects in a field. After 2 months, these bacteria reduced Verticillium black spot of Japanese radish. The disease severity of those Biological Control Agent (BCA) treatments was less than in the pathogen control treatment. Furthermore, the plants treated with KB-3 showed the best PGPR effect. Field conditions show that Bacillus spp. isolate KB-2, KB-3 is a potential BCA against V. dahliae. Subsequently, to select bacteria that are able to more control disease and more colonize in the plants and which facilitate recovery from soil and plants, Bacillus spp. were isolated from roots and were made resistant to ampicillin using UV irradiation. In all, 17 ampicillin-resistant mutants were tested for inhibition V. dahliae and F. oxysporum in vitro. Five bacteria isolates displayed inhibitory effects on FOM. All bacteria isolates indicated high antibiotic activity on V. dahliae. Then, to investigate the molecular characterization of all isolated strains of Bacillus spp., PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS-PCR), restriction fragment length polymorphism of the ITS-PCR (ITS-PCR RFLP), and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) were conducted. The ITS-PCR and ITS-PCR RFLP results suggest that KB-1 and strains of isolated from KB-1 were identical. Furthermore, those of the strains of KB-2 were also identical. However, KB-3 and all strains of isolates from KB-3 were different by ITS-PCR RFLP. Results suggest that the strains of isolates from KB-3 resembled those of KB-2. Using ERIC-PCR, 20 strains of Bacillus spp. could be distinguished.