解淀粉芽孢杆菌B1619脂肽类抗生素的分离鉴定及其对番茄枯萎病菌的抑制作用

doi:10.3864/j.issn.0578-1752.2016.15.008

Abstract

Abstract: 【Objective】 Bacillus amyloliquefaciens, biocontrol strain B1619, can effectively prevent and control the occurrence and damage of tomato fusarium wilt. In order to study and analyze theantifungal substancesproduced by strain B1619, the lipopeptide antibiotics were isolated from the supernatant of fermented broth.【Method】Three primers designed based on the known lipopeptide genes sfp, ituA and fenB were used to amplify the corresponding genes from the genome of B1619 strain. The crude lipopeptides extracts were extracted by acid precipitation and methanol. The supelclean^TM LC-18 column and PHASE HF-NH2 column were used to separate lipopeptide antibiotics. All the samples were analyzed by HPLC and MALDI-TOF-MS techniques. Inhibition rates of the three lipopeptide antibiotics against Fusarium oxysporum f. sp. lycopersici were tested by dual culture on plate. 【Result】The PCR products with three primer pairs were cloned and sequenced. The result showed that the sfp, ituA and fenB genes existed in the genome of B1619. The HPLC results showed that the peak time of 60% methanol eluent isolates were at 12,16.5 and 18 min, the secretion was 18.5 mg·L^-1, and the retention times were consistent with bacillomycin L standards. The peak time of 70% methanol eluent isolates were at 22, 34, 37, 51 and 53 min, the secretion was 5.1 mg·L^-1, and the retention times were consistent with fengycins standards. The peak time of 100% methanol eluent isolates were at 27, 37, 41, 51 and 53 min, the secretion was 74.3 mg·L^-1, and the retention times were consistent with surfactins standards. The further verification of MALDI-TOF-MS results showed that 60% methanol eluent isolates which m/z were 1 043.4, 1 057.5 and 1 071.4 Da, was C13-C15 bacillomycin L, 70% methanol eluent isolates which m/z were 1 463.9, 1 477.9, 1 491.9 and 1 505.9 Da, was C15-C17 fengycins, 100% methanol eluent isolates which m/z were 1 008.6, 1 022.7 and 1 036.7 Da, was C13-C15 surfactins. The mycostatic tests showed that the antifungal activities of 1 mg·mL^-1fengycins were higher than 1 mg·mL^-1bacillomycin L, and the 1 mg·mL^-1surfactins had the weakest effect onF. oxysporum. However, when the concentration of surfactins was increased to 3 and 6 mg·mL^-1, the antifungal activities didn’t change obviously. 【Conclusion】B1619 strain secreted the three kinds of lipopeptide antibiotics in which fengycins and bacillomycin L played an important role in the inhibitory effect on F. oxysporum, while the surfactins had no obvious effect on it.

Key words: Bacillus amyloliquefaciens B1619, lipopeptide antibiotics, antifungal activities; Fusarium oxysporum f. sp. lycopersici

XIANG Ya-ping, ZHOU Hua-fei, LIU Yong-feng, CHEN Zhi-yi. Isolation and Identification of Lipopeptide Antibiotics Produced by Bacillus amyloliquefaciens B1619 and the Inhibition of the Lipopeptide Antibiotics to Fusarium oxysporum f. sp. lycopersici[J].Scientia Agricultura Sinica, 2016, 49(15): 2935-2944.

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Isolation and Identification of Lipopeptide Antibiotics Produced by Bacillus amyloliquefaciens B1619 and the Inhibition of the Lipopeptide Antibiotics to Fusarium oxysporum f. sp. lycopersici

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