芽孢杆菌的抑菌活性与其产脂肽类抗生素的相关性

The Antifungal Activities of Bacillus spp. and Its Relationship with Lipopeptide Antibiotics Produced by Bacillus spp.

【目的】明确55株芽孢杆菌(Bacillus spp.)菌株对3个植物病原真菌(小麦纹枯病菌Rhizoctonia cerealis、小麦赤霉病菌Fusarium graminearum、西瓜枯萎病菌Fusarium oxysporum f.sp.niveum)抑菌活性及与其产生的脂肽类抗生素种类的定性定量关系。【方法】采用平板对峙培养分别测定55株芽孢杆菌活体菌及其脂肽类抗生素粗提物对3个真菌的抑菌活性;根据脂肽类抗生素合成相关基因序列srf A、itu A和fen A对55株芽孢杆菌进行PCR扩增;采用质谱及液相色谱分析55株芽孢杆菌脂肽类抗生素的种类、数量。【结果】55株芽孢杆菌菌株及其脂肽类抗生素粗提物对3个病原真菌均具有一定的抑菌活性,其中16.5%菌株脂肽类粗提物的抑菌活性比活体菌的强,55.8%菌株粗提物的抑菌活性比活体菌的弱,27.7%菌株粗提物的抑菌活性基本没有变化。55株芽孢杆菌基因组PCR扩增结果表明,89.1%菌株含有srf A,87.3%菌株含有itu A,只有56.4%菌株中含有fen A。55株芽孢杆菌粗提物质谱检测结果表明,53株菌株检测到surfactins,48株检测到iturins,只有菌株Bs916(Bacillus subtilis 916)检测到fengycins,粗提物中没有检测到3种抗生素的菌株其抑菌活性较弱或者没有。同时发现,在检测到iturins的48株菌株中,48株均检测到bacillomycin D,2株检测到bacillomycin L,12株检测到bacillomycin F、iturin A或mycosubtilin。对55株芽孢杆菌粗提物的液相色谱检测结果表明,53株菌株检测到surfactins;51株检测到iturins;只有菌株Bs-916检测到fengycins;有2株菌株未检测到上述3种抗生素。根据iturins的色谱峰出峰时间及峰型分为4大类,命名为1、2、3和4型,4种类型的iturins对病原菌的抑菌活性强度顺序为1型>2型>3型>4型。在3株菌株JCC-1、JCC-9和JCC-18中检测到2个未知的脂肽类化合物(命名为Unknowns:Unknown1、2)的质谱峰。【结论】芽孢杆菌脂肽类抗生素是抑制植物病原真菌的主要成分,并具有多样性;iturins结构的变化及其分泌量与抑制植物病原真菌活性有一定的相关性。芽孢杆菌还产生一些未知的脂肽类化合物。

【Objective】 The objective of this study is to clear the inhibition effect of 55 Bacillus spp. strains to pathogenic fungi, Rhizoctonia cerealis, Fusarium graminearum and Fusarium oxysporum f. sp. niveum, and the qualitative and quantitative relationship with lipopeptide antibiotics produced by the Bacillus strains.【Method】Inhibition rates of the 55 Bacillus spp. strains and their crude lipopeptides extracts against three pathogenic fungi were tested, respectively, by dual culture on plate. Three pairs of primers designed based on the known lipopeptide genes were used to amplify the corresponding genes, srf A, itu A and fen A, from the genome of the 55 strains. The lipopeptides antibiotics were identified by high performance liquid chromatography（HPLC） and mass spectrum（MS） techniques.【Result】 The 55 strains of Bacillus spp. and their crude lipopeptides extracts had certain biological activities to the tested pathogenic fungi. The crude lipopeptides extracts of 16.5% strains had higher antifungal activities than the Bacillus strains in vivo, the antifungal activities against the pathogenic fungi of crude lipopeptides extracts of 55.8% strains were weaker than living cells of the strains in vivo, while the antifungal activities of 27.7% strains did not change basically. The results of PCR amplification showed that among the 55 Bacillu strains, the genomes of 89.1% Bacillus strains contained srf A, 87.3% strains contained itu A, only 56.4% strains contained fen A. Among the 55 Bacillus strains, 53 Bacillus strains were identified with surfactins in the crude lipopeptides extracts by the MS analysis, 48 strains were identified with iturins, only Bs916（B. subtilis 916） was identified having fenfycins in the crude lipopeptides extracts. Those strains that were not identified having the three kinds of lipopeptides had weaker or no antifungal activity. There were 48 strains which were found to have iturins all identified with bacillomycin D, only 2 strains were identified with bacillomycin L, 12 strains were identified with bacillomycin F, iturin A or mycosubtilin. Among the 55 Bacillus strains, 53 strains were detected with suefactins by HPLC, 51 strains were detected with iturins, only Bs916 strain was detected with fengycins and 2 strains were not detected with the 3 kinds of lipopeptide antibiotics. According to the time and type of chromatographic peak, iturins were divided into 4 types, named as types 1, 2, 3, and 4. The antifungal activities intensity of 4 types of iturins were type 1〉type 2〉type 3〉type 4. There were two unknown lipopeptides（named as Unknowns： Unknown1, 2） which were speculated as new lipopeptides were detected with MS analysis in 3 strains JCC-1, and JCC-9 and JCC-18. 【Conclusion】 Lipopeptides antibiotics produced by Bacillus spp. were the main composition against plant pathogenic fungi and had diversity characteristics. The structure changes and secretion of iturins had certain relevance to antifungal activities. There were some unknown new resisted resources in lipopeptides compounds from Bacillus spp..