Isolation, Identification Of Antagonistic Strain Bacillus Amyloliquefaciens S20Against Eggplant Bacterial Wilt And Studies On Its Antimicrobial Substances

The bacterial wilt of eggplant, one of the main diseases of eggplant, is the soil-borned desease that is caused by Ralstonia solanacearum and threatens production of eggplant. It is difficult to effectively control the disease by routine control methods, such as chemical fungicides.Compared with the routine control,rhizobacteria-mediated biocontrol is a more effective alternative.This paper presents the screening and identification of antagonistic bacteria strains against bacterial wilt of eggplant.Lipopeptides synthesis-related genes were detected by PCR. Fermentation conditions for antimicrobial substance production and their characteristics were tested respectively. Antimicrobial substances were identified by HPLC and HPLC-MS. The main results of this work were as following:(1)40strains were isolated in this study which inhibited Ralstonia solanacearum. Six strains were screened by inhibitory circle tests, among them strain S20showed the best inhibition to Ralstonia solanacearum. Results in potted trial showed that the control efficiency of strain S20against bacterial wilt of eggplant reached68.6%.(2) Based on morphological observation, physiological and biochemical characteristics and16S rRNA gene sequence analysis strain S20was primarily identified as Bacillus amyloliquefaciens.(3) Genes sfp, fenB, bamA and ituA, responsible for synthesis of antibiotics Surfactin, Fengycin,Bacillomycin, and Iturin were detected in strain S20.Lipopptides synthesis-related genes such as sfp,fenB,bamA and ituA were detected by PCR in strain S20.(4) The optimal conditions for producing antimicrobia substance by strain S20were:60mL NA medium with initial pH7in250mL flask, incubation at30℃with170r/min for84h. (5) Crude lipopeptides were extracted from the Bacillus amyloliquefaciens S20culture by6mol/L HC1precipitation, and dissolved in100%methano. The crude extract could inhibit the growth of Ralstonia solanacearum. The crude extract was heat-stable, which still maintained inhibition against Ralstonia solanacearum after100℃treatment for30min.The crude extract was very stable after treatment at pH from2to9. Antimicrobial substance was sensitive to trypsin, but was very stable to proteinase K and pepsin. The antimicrobial substance was also very stable to UV.(6) The lipopeptide substrates in crude extract was separated by reversed-phase high performance liquid chromatography (HPLC). The results showed there were surfactin and iturin A in crude extract based on the standard surfactin and iturin A. Different components were collected and tested their the inhibitory activity against Ralstonia solanacearum. The results showed that IturinA was the main antagonistic substances of strain S20.(7) HPLC-MS analysis showed three series of ion peaks present in the crude extract. Further electrospray ionization/collision-induced dissociation spectra revealed that the three series ion peaks represented different Iturin, Fengycin and Surfactin homologues. The first series of ion peaks representec IturinA homologues with C13-C16fatty acids carbon chains. The second series of ion peaks represente fengycinA homologues with C14-C19fatty acids carbon chains and fengycinB homologues with C14-C17fatty acids carbon chains. The third series of ion peaks were C13-C15Surfactin homologues.