Isolation,Identification And Biocontrol Effect Of An Antagonistic Bacterium WK1 Against Rice Sheath Blight

Rice sheath blight is one of the three major rice diseases,which seriously affects yield and quality of rice.In this study,an antagonistic bacterium against rice sheath blight was isolated and identificated on the basis of tissue isolation method.and biological characteristics of the antagonistic bacteria was studied.The control effect of the antagonistic bacterium fermentation liquid on rice sheath blight was tested by field plot experiment.Moreover,the antagonistic components in the fermentation liquid were preliminarily separated and the stability of the antagonistic substances was studied,the results provided a basis for field control of rice sheath blight using the antagonistic bacteria.The main findings are as follows:A strain of endophytic antagonistic bacteria against rice sheath blight was isolated from healthy leaves of rice by tissue isolation method,which was named WK1.Based on morphological feature,physiological-biochemical characteristic,16 S rDNA sequence homology and phylogenetic tree construction,WK1 strain was identified as Bacillus velezensis.The biological characteristics of antagonistic strain WK1 against Rhizoctonia solani were studied.The results showed that optimal culture conditions for WK1 were as follows:temperature of 25-30℃,pH of 6-8,shaking speed of 140-180 rpm,culture medium lording of below 120 mL(250mL flask),carbon resource of glucose,nitrogen resource of beef extract and tryptone.Under optimal culture conditions,strain WK1 was at lag phase within12 h,logarithmic phase between 12 h to 24 h,stable phase between 24 h and 36 h,and turned into decline phase after 36 hThe bacteriostatic effect of strain WK1 measured by different methods is different.The effect of WK1 fermentation broth with different concentration on mycelial growth of R.solani was measured.When the dilution ratio of WK1 was 5 times,10 times and 20 times,the inhibition rate of mycelial growth of R.solani was 84.42%,62.57%,and 52.25%,respectively.Meanwhile,the control effect of WK1 fermentation broth against rice sheath blight was determined at greenhouse condition.The results showed that the disease index of blank control was 29.71,and the disease index of WK1 treatment was only 14.19,the control effect was 52.23%,which worked equally with 5% jinggangmycin AS,a regular fungicide for Rice Sheath Blight control.Disease index of 30% difenoconazole.Propiconazole SC treatment was 7.48,and the control effect reached 74.82%.The antagonistic substances in the fermentation broth of WK1 strain were separately extracted by ethyl acetate,dimethylbenzene,n-butyl alcohol extraction,hydrochloric acid sedimentation and ammonium sulfate precipitation,and their antagonistic effect on R.solani were measured.The results showed that the organic solvent extracts from the fermentation broth of WK1 strain had no antagonistic effect on R.Solani,and the lipopeptide extracts had a certain antagonistic effect,the best antagonistic effect was obtained by precipitation of50% saturated ammonium sulfate,and its inhibition rings reached 11.8mm.The stability of bacteriostatic protein was studied.It was found that the bacteriostatic protein had better activity between 30-70 °C and pH between 6-8.The antimicrobial protein was sensitive to Proteiase K and Proteiase E,which were treated at 37 °C for 30 min and then inactivated.