Screening,Identification And Application Of Biocontrol Bacteria Against Tobacco Bacterial Wilt

Tobacco bacterial wilt,making bad effect on the quality and harvest of tobacco and a great loss to tobacco industry,is a vascular soil-borne disease caused by Ralstonia solanacearum,which distributes widely and spreads rapidly.In this study,strain YH-22 with effective antagonistic activity against Ralstonia solanacearum was screened from 36 bacteria isolated from the rhizosphere soil and tobacco tissue in vitro by the dual culture.According to morphological and physiological characteristics and nucleotide sequence of 16S rDNA,strain YH-22 was identified as Bacillus amyloliquefaciens with a broad anti-fungal activity.The fermentation medium and cultivation conditions were optimized for B.amyloliquefaciens YH-22 in flasks.The composition of optimized medium(g/L)are:corn starch 15,soybean meal 50,K2HPO4·3H2O 1.00,MgSO4·7H2O 0.75,MnSO4·H2O 0.010.The optimized fermentation conditions:initial pH 7.0,the inoculum amount 5%(v/v),the broth content 20 mL/250 mL flask,temperature 37?,rotation speed 180 r/min,incubation period 48 h.Under the condition of optimized(?)medium,the biomass of strain YH-22 was up to 150.5×108 CFU/mL and the percentage of spores production was more than 95%.In the field trials,the incidence rate of tobacco bacterial wilt in early stage was 5.0%-14.2%and the control efficiency of bactericide on bacterial wilt in tobacco was not obvious while the control efficiency of chemicals for testing field and adjacent field were 30.0%and 16.3%.During the period of high-incidence of the disease,31.7%-56.7%in the test field,62.4%-95.8%in the adjacent field,the corresponding control efficiency were 14.7%-21.8%and 60.6%-76.1%for strain YH-22,while 0.0%and 36.2%-69.4%for chemicals.As it can be seen,the control efficiency of strain YH-22 on bacterial wilt was not obvious at the early stage of bacterial wilt,but in the period of high-incidence of disease,chemical agents could not prevent the development of disease while strain YH-22 could repress it to a certain degree.The biocontrol mechanism of disease resistance to bacterial wilt was studied through the detection of colonization ability,partial extracellular enzyme as well as some physicochemical property.The colonization ability of strain YH-22 resistant to 400 ?g/mL rifampicin in the rhizosphere soil and tobacco tissue were detected in the pot tests.The content of resistant strain YH-22 retained 105 CFU/g in the rhizosphere soil and 103 CFU/g in tobacco tissue.Strain YH-22,whose capacity of phytate solubilization was 31.25 mg/L,could form obvious transparent circle on protein plate and cellulose plate.The antibacterial ability of antagonistic substances of strain YH-22 had certain heat stability and stayed over 95%after treantment with different pH between 4.0-10.0.What?s more,the antibacterial ability remained 93.0%under exposure of 8 watts Ultraviolet(UV)10 cm apart for 30 min.Treated by neutral protease,trypsin and pepsase,the antibacterial ability kept more than 50%.Chloroform and ethyl acetate could not resolve the antagonism substances.The antibacterial ability of acid precipitation of culture filtrate which could display oil displacement and droplet collapse maintained 76.3%.In conclusion,strain YH-22,secreting cellulase and protease and solubilizing phosphate,had definite colonization ability in the rhizosphere soil and tobacco tissue and could synthetize antibacterial components conjectured as a mixture of lipopeptide and protein.