Purification And Characterization Of Antimicrobal Substances Produced By Bacillus Subtilis Strain Bv10

Base on optimizing the fermentation of Bacillus subtilis strain Bv10, which could inhibit the growth of Athelia rolfsii, the Antimicrobal substances were purified and identified. The control efficacy of the Antimicrobal substances from strain Bv10against the sesame southern blight was also tested. The primary results are summarized as follows:1. The optimized conditions for growth of strain Bv10were at temperature30℃, initial pH8.0,3%inoculums volume, medium volume100mL/250mL, rotation speed170rpm and incubation time5d.The composition (w/v) of the optimized medium for the growth and the productivity of Antimicrobal substances of strain Bv10was3.0%sucrose,0.5%NH4Cl,0.5%beef extract,3.0%K2HP04and0.5%MgSO4·7H2O.2. The Antimicrobal substances were stable to heat. However, their activities were reduced slightly with time after being treated at121℃. The activities of Antimicrobal substances were relatively stable at pH7-9, and not affected by irradiation of fluorescent light or natural light for5d as well as by the treatments of enzymes such as trypsin, pepsin protease, papain and pectinase. In addition, the Antimicrobal substances could be easily dissolved in methanol, phosphate buffer solution (PBS) and water, but not in acetone, acetonitrile and dichloromethane.The Antimicrobal substances showed suppressive activities against growth of14plant pathogenic fungi (Alternaria alternata, Pestalotiopsis foedans, Botryosphaeria berengeriana f. sp. piricola, Trichothecium roseum, Pythium aphanidermatum, Pseudocercospora musae, Botryosphaeria dothidea, Botryotinia fuckeliana, Athelia rolfsii, Gibberella zeae, Fusarium equiseti, Gibberella moniliformis, Fusarium proliferatum, Colletotrichum gloeosporioides) and two non-plant-pathogenic fungi (Chactomium globosum, Gelasinospora brevispora) as well as5plant pathogenic bacteria(Xanthomonas oryzae pv. oryzicola, Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, Xanthomonas campestris pv. citri and Pseudomonas syringae pv. tomato), and no inhibition to Pythium ultimum and Phytophthora sp. as well as Pectobacterium carotovora.3. Two Antimicrobal substances (A and B) were extracted and purified from the sterile fermentation broth of Bacillus subtilis strains Bv10by acid precipitation, methanol extraction and reversed-phase high performance liquid chromatography (RP-HPLC), with a strong suppressive activity against Athelia rolfsii. MALDI-TOF-MS and MALDI-TOF-MS/MS CID spectrum analysis showed that the two purified substances had the molecular mass of1042Da and1057Da, respectively. In addition, the peptide ring sequence was identified as Pro-Asn-Ser-βAA-Asn-Tyr-Asn-Gln. The substance A was identified as iturin A2, while the substance B as iturin A4.4. The germination of sclerotia of Athelia rolfsii was completely inhibited by both the Antimicrobal substances A and B at a concentration of200mg-L-1. The growth inhibition rates of sclerotia by the substance A were82.22%and73.33%at the concentrations of100mg-L-1and50mg-L-1, respectively. The growth inhibition rates of sclerotia by the substance B were77.78%and68.89%at the concentrations of100mg-L-1and50mg-L-1, respectively. Under microscopic observation, the hyphae of Athelia rolfsii developed abnormally in the presence of the antimicrobal substances. The antimicrobal substances caused development of deformed hyphae, such as constrictions at hyphal branches and fragmentation of the branches. They also turned the hyphal cells into bigger sacs, concentrated their protoplasm and made the hyphal tips into vesicules.5. The median effect concentration (EC50) of the purified Antimicrobal substance A and B against Athelia rolfsii were36.7866mg-L-1and43.0329mg-L-1respectively.On the sesame plants treated with ethanol extracts and sterile fermentation broth prior to pathogen inoculation, the effects of the two treated methods on the disease were77.42%and39.82%, respectively. On the sesame plants treated with ethanol extracts and sterile fermentation broth after pathogen inoculation, the effects of the two treated methods on the disease were68.69%and30.30%, respectively. The results showed that the ethanol extracts and sterile fermentation broth could control sesame southern blight caused by Athelia rolfsii, and the protective effect of the antimicrobal substances was superior to their treatment effect.