Isolation,Identification And Active Component Analysis Of Bacillus ZW-10 Inhibiting Rice Blast

Rice blast is caused by rice blast fungi which infect rice.Because of its wide distribution,many physiological races of pathogenic fungi,strong breaking power and variability,rice blast affects rice production worldwide.At present,it is mainly through spraying chemical pesticides and breeding resistant varieties to prevent and control rice blast.Chemical control brings about environmental pollution and resistance of pathogenic bacteria.Resistant varieties also have problems such as long breeding cycle and difficulty in coping with pathogenic bacteria variation.The application of biological control methods to control rice blast has attracted more and more attention.In this paper,a bacterial strain named ZW-10 was screened from the soil,which had strong antagonistic effect on rice blast.A series of follow-up experiments on antagonistic bacteria were carried out.Morphological observation under optical microscopy and scanning electron microscopy,testing the physiological and biochemical indicators of antagonistic bacteria,comparing their 16 S rDNA sequencing and phylogenetic tree analysis,the antagonistic bacteria ZW-10 was identified as Bacillus velezensis.Then the fermentation conditions of ZW-10 were explored.It was determined that when the fermentation medium of strain ZW-10 was Landy medium,the fermentation time was 84 h,and the fermentation temperature was 37 ?,the yield of antimicrobial active substances was the highest.Qualitative detection of antagonistic bacteria showed that ZW-10 could produce proteinase and cellulase which had antagonistic effect on fungi.Antagonistic bacteria were sensitive to 18 common antibiotics such as tetracycline,but only resistant to neomycin and penicillin.ZW-10 fermentation broth can keep its activity under acidic condition and low temperature,but under alkaline condition and high temperature condition,its activity decreases gradually,especially when pH is greater than or equal to 12 and temperature is greater than 100 ?.The results of in vitro leaf test and field test showed that antagonistic bacteria ZW-10 and its fermentation broth had a strong control effect on rice blast.The preventive effect of ZW-10 was better than that of the therapeutic effect.The control effect of 1-fold concentration of ZW-10 sterile fermentation broth on rice blast was slightly worse than that of 0.1mg/mL carbendazim,but the control effect of 1-fold concentration of ZW-10 bacterial broth on rice blast was similar to that of 0.1mg/mL carbendazim for production.Its living bacteria preparation and metabolites had development and application value.We used MCI column chromatography to isolate the active substance initially,then used high performance liquid chromatography to further isolate and purify the substance.Finally,a single substance was detected by mass spectrometry.The results showed that the molecular weight of the substance was 541.25 which provides the possibility for the development of new biological pesticides in the future.