Identification and antagonistic mechanism of biocontrol bacteria against Rhizoctonia solani

【Objective】To screen antagonistic bacteria with biocontrol potential against Rhizoctonia solani,to provide strains for biocontrol on tobacco target spot.【Method】The dilution coated plate method was used to isolate and purify the bacteria from the rhizosphere soil of healthy flue-cured tobacco in Guangxi,with R. solani as the target pathogen. The plate confrontation method was used to screen the strains with good antagonistic effect,and the antibacterial effect was determined. Scanning electron microscopy and ultra depth of field 3D microscope was used to observe the morphological changes of mycelia of R. solani that treated with antagonistic strains and colony morphology,and measured its physiological and biochemical characteristics. PCR amplification was performed on the DNA of antagonistic strains using universal bacterial primers 16S rDNA sequences and gyrA gene. The sequencing results were compared and analyzed for homology using BLAST from NCBI. A phylogenetic tree was constructed based on sequences and type strains with close genetic relationships. The antibacterial effect of volatile organic compounds(VOCs)of antagonistic strains was preliminarily explored by double-plate buckle method. The volatile compounds of antagonistic strains were identified by headspace solidphase microextraction(HS-SPME)and gas chromatography-mass spectrometry(GC-MS). Identified the control effect of antagonistic strains on tobacco target spot disease through pot experiments on K326 tobacco variety.【Result】An antagonistic strain GXLL3319 with biocontrol activity was screened,and combined with the colony morphology,physiological and biochemical characteristics,and phylogenetic analysis results,the strain was identified as Bacillus subtilis. The inhibition rate of this strain on three kinds of tobacco pathogenic fungi,namely R. solani,Colletotrichum nicotianae and Fusarium oxysporum,was above 50%. The inhibition rate of this strain and its volatile substances on R. solani was 69.70% and 59.22%,respectively. The results of scanning electron microscopy showed that the mycelial growth of R. solani treated with GXLL3319 was obviously inhibited,and the mycelia were fractured,swollen and dissolving. Thirty-one antibacterial active substances was identified from VOCs,including ketones,alcohols,aldehydes,heterocycles,sulfides, organic acids,esters,alkanes and phenols. The results of pot experiment showed that GXLL3319 bacterial fluid could effectively inhibit the occurrence of tobacco target spot,and the relative control efficacy reached 66.07%,which was not significantly different(P>0.05)from that of 15% Jinggangmycin A soluble powder.【Conclusion】B. subtilis GXLL3319 can effectively inhibit the mycelial growth of R. solani,its VOCs contain a variety of antibacterial active substances. GXLL3319 has a good control efficacy on tobacco target spot and has the potential to be developed as a biocontrol bacteria against tobacco target spot.