Screening,identification and antagonistic mechanism in vitro of biocontrol bacteria against tobacco black shank pathogen

【Objective】Strains with high antagonistic activity against the pathogen of tobacco black shank were isolated from tobacco rhizosphere soil in the high incidence area of tobacco black shank disease in Yunnan. The taxonomic status of strains was clarified and the in vitro antagonism mechanism was preliminary studied to provide biocontrol resources and reference basis for the prevention and control of tobacco black shank disease.【Method】Strains antagonistic to tobacco black shank pathogen were isolated from tobacco rhizosphere soil using the plate standoff method. The strains with strong antagonistic activity were identified by physiological and biochemical characteristics and 16S r DNA sequence analysis.The biocontrol activity and mode of action of biocontrol bacteria against Phytophthora nicotianae in vitro were determined.【Result】Six bacterial strains with antagonistic activity against P. nicotianae were isolated from the collected tobacco rhizosphere soil samples, among which strain CD-1 showed the strongest antagonistic activity, with a radius of inhibition zone of 10.12 mm. According to physiological and biochemical characteristics and 16 S rDNA sequence analysis, the strain was identified as Bacillus licheniformis. Strain CD-1 had broad-spectrum antifungal properties, and both intracellular and extracellular antifungal substances existed. Among them, the antifungal substance extracted using dichloromethane as bacterial fermentation liquid extractant had the best inhibitory effect. When pH was in the range of 6-8 and the temperature was between 40-80 ℃, it showed relatively stable inhibitory activity. Strain CD-1 cultured in NB medium showed strong inhibitory activity against P. nicotianae in both culture supernatant extracts and cell extracts, with inhibition rates of 76.91% and 67.23%, respectively. The antifungal substances secreted by strain CD-1 could destroy the spore plasma membrane of P. nicotianae and inhibit spore germination. Among them, the spore membrane loss rate after treating P. nicotianae spores with the antifungal crude extract 0.4 mg/mL for 6 h reached 53.62%, and the spore germination was completely inhibited, while obvious ion and nucleic acid leakage was detected.【Conclusion】The metabolite produced by B. licheniformis CD-1 has a broad antimicrobial spectrum and strong bacteriostatic activity, which has the potential to be developed into antifungal agents.