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

摘要/Abstract

摘要： 【目的】从解淀粉芽孢杆菌（Bacillus amyloliquefaciens）生防菌株B1619发酵液上清中分离脂肽类抗生素，分析其抑菌活性相关成分,为应用解淀粉芽孢杆菌B1619菌株防控番茄枯萎病提供依据。【方法】利用特异性引物对B1619中3种脂肽类抗生素合成相关基因sfp、ituA和fenB进行检测；通过盐酸沉淀、甲醇抽提法等方法提取脂肽类抗生素粗提物；用Supelclean^TM LC-18柱及PHASE HF-NH2柱对3种脂肽类抗生素粗提物进行分离；通过MALDI-TOF-MS和HPLC分析、鉴定3种脂肽类抗生素；采用平板对峙培养分别测定3种脂肽类抗生素粗提物对番茄枯萎病菌(Fusarium oxysporum f.sp.lycopersici)的抑菌活性。【结果】从解淀粉芽孢杆菌B1619的基因组DNA中扩增sfp、ituA和fenB等脂肽类抗生素合成基因,扩增产物经克隆、测序分析，表明B1619菌株中含有这3个基因。通过HPLC分析，发现60%甲醇洗脱液分离物分别在12、16.5、18 min处出现相应峰值，保留时间与bacillomycin L标准品一致，分泌量为18.5 mg·L^-1；70%甲醇洗脱液分离物分别在22、34、37、51及53 min处出现相应峰值，保留时间与fengycins标准品一致，分泌量为5.1 mg·L^-1；100%甲醇洗脱液分离物分别在27、37、41、51及53 min处出现相应峰值，保留时间与surfactins标准品一致，分泌量为74.3 mg·L^-1。经液质联用进一步验证分析3种脂肽类抗生素的质子化峰值，60%甲醇洗脱液分离物峰值范围在m/z=1 043.4、1 057.5和1 071.4 Da，是C13—C15的bacillomycin L；70%甲醇洗脱液分离物峰值范围在m/z= 1 463.9、1 477.9、1 491.9和1 505.9 Da，是C15—C17的fengycins；100%甲醇洗脱液分离物峰值范围在m/z=1 008.6、1 022.7和1 036.7 Da，是C13—C15的surfactins。平板对峙法试验结果表明1 mg·mL^-1 bacillomycin L和fengycins对番茄枯萎病菌有较强的抑菌效果，而1 mg·mL^-1surfactins对番茄枯萎病菌的抑菌活性较弱，再将surfactins的浓度提高至3和6 mg·mL^-1，发现其抑菌效果没有明显变化。【结论】B1619菌株分泌3种脂肽类抗生素，其中对番茄枯萎病菌生长起重要抑制作用的抗生素是bacillomycin L和fengycins，surfactins对番茄枯萎病菌的抑制活性较弱。

关键词: 解淀粉芽孢杆菌B1619, 脂肽类抗生素, 抑菌活性, 番茄枯萎病菌

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

向亚萍，周华飞，刘永锋，陈志谊. 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[J]. 中国农业科学, 2016, 49(15): 2935-2944.

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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