Isolation,identification and optimization of fermentation conditions of antagonistic bacteria against tobacco black shank

【Objective】 To clarify classification of bacterial strain with antagonism effect on tobacco black shank from the intestinal tract of Periplaneta americana，to optimize fermentation conditions and to determine indoor control effects of tobacco black shank，so as to provide bacterial resources for biological control of tobacco black shank.【Method】 Intestinal tract of P.americana was used as material，and the antagonistic strains against tobacco black shank were screened by dilution coating plate method and plate confrontation method.The strains were identified according to their morphological characteristics，physiological and biochemical characteristics，16S rDNA and gyrB gene sequence analysis.Single factor and orthogonal test were carried out on the fermentation medium and fermentation conditions of bacterial fermentation broth with OD₆₀₀ value as the index.Control effect of the strain on tobacco black shank was determined by indoor pot experiment.【Result】 200 bacterial strains were isolated from the intestinal tract of P.americana by dilution coating method.A strain MC2-1 screened by plate confrontation method，with an average inhibitory rate of 62.87% and an antibacterial bandwidth of 8.00 mm，was strong antagonistic against tobacco black shank.Considering the results of colony morphology，physiological and biochemical detection，and phylogenetic evolutionary tree of 16S rDNA and gyrB，the strain MC2-1 was identified as Bacillus velezensis.The optimum medium formula was：beef extract 8 g/L，yeast extract 5 g/L and maltose 10 g/L；the optimum culture condition was：inoculation amount 10%，liquid volume 30 mL，initial pH value of medium 7，rotation speed 180 r/min，culture temperature 36℃，culture time 60 h.The bacterial solution concentration of optimized culture medium and fermentation condition（OD₆₀₀ were 2.72 and 2.81，respectively）were significantly higher than that the original culture medium and fermentation condition（OD₆₀₀ were 2.31 and 2.56，respectively） （P<0.05）.The results of pot experiment showed that the average control effect of the optimized strain MC2-1 on tobacco black shank was 63.92%，was significantly higher than the 53.72% before the optimization.【Conclusion】The optimized scheme can increase the bacterial mass of MC2-1 and enhance the control effect of tobacco black shank.Strain MC2-1 is potential to be developed as a biocontrol bacterium against tobacco black shank.