Analysis of the active components of Bacillus amyloliquefaciens HAB-2 against Colletotrichum gloeosporioides

Abstract:【Objective】Recently, biocontrol by using microorganisms has successfully been carried out for the suppression of some soil-borne plant pathogens. Simultaneously, these microorganisms may be environmentally friendly in controlling plant pathogens. Bacilllus spp. is the most widely studied biocontrol agent and has been extensively used in the development of biopesticides like fungicides and bactericides. Bacilllus spp. play vital roles in controlling plant diseases due to their antimicrobial metabolites like lipopeptides. The strain HAB-2, isolated from the rhizospheric soil of Xinjiang cotton fields, has a strong antimicrobial activity. Clarifying its taxonomic status and the stability of its antimicrobial active substance is of great significance for the research and development of biological pesticides.【Methods】The β-mannanase gene (gumG or ydhT) was used as specific primers (Bsu-man, Bli-man and Bam-man) to identify the cloned molecules. The PCR product was TA cloned, transformed and sequenced after gel purification and recycle. The obtained sequences were analyzed by BLAST program of NCBI. A loop of strain HAB-2 was inoculated in 100 mL lysogeny broth (LB) liquid medium at 180 r· min^-1 and 28 ℃ for 48 h. The activated strain HAB-2 was inoculated on AYDA medium, BPY medi-um, LB medium, nutrient agar (NA) medium, basal medium and fermentation medium, respectively.The colony morphology of HAB-2 strain was observed on six different media. The HAB-2 strain was cultured in LB liquid medium broth, shaking at 28 ℃ and 180 r· min^-1 for 48 h. The fermentation liquid was centrifuged at 10 000 r· min^-1 for 10 min. Crude extracts can be obtained from the supernatant of HAB-2 by the organic solvent extract, ammonium sulfate saturation precipitation and acid precipitation, respectively. The crude extracts were respectively dissolved in a little methanol, and then serially diluted to 5 mg· mL^-1. Extract diluent was filter-sterilized by using 0.22 μm micro-filters. The strain Colletotrichum gloeosporioides was inoculated in a triangular flask containing 100 mL Potato Dextrose Agar (PDA) liquid medium, shaking at 28 ℃ for 72 h. The fermentation liquid was filtrated to remove mycelia by the sterile gauze, and after that, agar petri plates were prepared by mixing 50 mL PDA medium with C. gloeosporioides spore suspensions. The extract diluent was added on paper disks and then placed on the agar petri plate. The diameters of growth inhibition zones were measured after 5 days.The stability of antimicrobial activity affected by UV, pH and heat were studied. The N-butanol extract was irradiated for 0, 30, 60, 90, 120 min respectively at a distance of 40 cm from UV. The pH of n-butanol extract was adjusted to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, respectively. The n-butanol extract was respectively treated at the temperatures of 40, 50, 60, 70, 80, 90 and 100 ℃ for 20 min. These diameters of growth inhibition zones were measured. By plate confrontation culture method, it was observed that the pathogenic mycelia were affected by the n-butanol extract. Mango fruits were smeared with n-butanol extract and aseptic water respectively, and then inoculated with C. gloeosporioides and preserved at25 ℃ for 72 h. The experiments were performed in triplicates.【Results】An 1 275 bp band was generated when Bam-man was used as a specific primer, but no band appeared when Bli-man and Bam-man were used. The sequence from the band amplified with Bam-man was highly homologous with B. amyloliquefaciens. The morphology of HAB-2 was different on the six media. Only the n-butanol extract, ammonium sulfate precipitation extract and acidification extract showed strong antifungal activity. The diameters of inhibition zone were (22.01±0.3) mm, (16.21±0.3) mm and (21.88±0.3) mm, respectively.The n-butanol extract retained 93.64% after 120 min exposure to UV, and 81.12% at 100 ℃ for 20 min.The optimum pH levels ranged from 5 to 6. The mycelia of C. gloeosporioides treated with n-butanol extract was distorted, swollen and deformed, effectively being inhibited. The application of n-butyl alcohol can reduce the development of C. gloeosporioides on the mango.【Conclusion】It could be concluded that HAB-2 was B. amyloliquefaciens with strong antimicrobial activity. The antimicrobial activity of the n-butanol extract was stable under the UV irradiation, or at different temperatures and pH levels.The extract of strain HAB-2 can effectively prevent mango from infecting with C. gloeosporioides.