Isolation,identification and functional study of antagonistic endophytic bacteria against Pinellia ternata damping off (Rhizoctonia solani)

【Objective】An endophytic bacterium with antagonistic activity against Pinellia ternata damping off(Rhizoctonia solani)was isolated from the medicinal plant P. ternata,in order to provide theoretical and practical basis for biological control of P. ternata damping off.【Method】The main diseases of P. ternata in the greenhouse of Nanzhangpu in Qianjiangof Hubei were investigated and isolated. The pathogenicity test was conducted by Koch's rule to screen out the most pathogenic pathogens and identify their categories. Endophytic bacteria were isolated from healthy and disease-free P. ternata by tissue separation. The strain with the strongest antagonistic activity against R. solani was screened by plate confrontation,and the antifungal spectrum of the strain was determined by this method. The inhibitory activity against R. solani of aseptic fermentation broth and volatile substances from the strain were measured. The acid and alkali resistance, extracellular enzyme and the biofilm formation activity of the strain were detected. The effect of protease K and different temperature on the activity of the sterile fermentation fluid of this strain against R. solani was determined by plate test. The lipopeptide active substances were extracted by ethyl acetate extraction method and the main components of active substances were detected by liquid chromatography-mass spectrometry(LC-MS).【Result】The main diseases of P. ternata in the greenhouse of Nanzhangpu were tuber rot,root rot,leaf spot and viral disease,with the incidence of 20.87%, 7.03%,3.57% and 4.08%. The highest incidence of tuber rot was caused by R. solani. Five strains of bacteria isolated from P. ternata seed ball tissue had inhibitory activity to against P. ternata damping off pathogenic fungi. The strain named B2-7 had the highest inhibitory rate with 79.60%,which was identified as Bacillus velesiensis. The results of antifungal spectrum showed that the strain B2-7 had antagonistic activity against 12 different plant pathogenic fungi,and its inhibitory rate ranged from 61.11% to 89.47%. The volatile substances of strain B2-7 and the sterile fermentation solution had antagonistic effect on R. solani. The inhibitory activity against R. solani of volatile substances was 41.18%. When the concentrations of sterile fermentation fluid from B2-7 were 2.5%,5.0%,10.0% and 20.0%,the inhibitory rates for R. solani were 39.40%,56.06%,73.89% and 77.09%. Strain B2-7 had strong acid resistance,but alkaline resistance was weak. The strain B2-7 could produce cellulase,amylase,protease and secrete siderophores. The ability to form biofilm was strong,which increased first and then decreased with the increase of the concentration of bacterial solution. The sterile fermentation solution from B2-7 was sensitive to temperature and protease K,and the inhibitory activity decreased to 23.45% at 75℃ and decreased by 48.60% treated with protease K. The lipopeptide active substances of strain B2-7 could inhibit the growth of R. solani with an inhibition bandwidth of 2.06 mm. The results of LC-MS analysis showed that there were at least 16 known organic compounds in lipopeptide active substances,including surfactin,bacillaene,microlactin, difficidin and other antagonistic substances reported.【Conclusion】Endophytic bacterium B2-7 of P. ternata has good inhibitory activity against P. ternate damping off pathogenic fungi. It is a potential strain to control P. ternata damping off and has a good application prospect