小檗内生放线菌H21的鉴定及抑菌活性成分分析

doi:10.3864/j.issn.0578-1752.2015.06.06

摘要/Abstract

摘要： 【目的】从药用植物小檗（Berberis thunbergii）中筛选抗菌活性内生放线菌，对活性菌株的分类地位及抑菌活性进行研究，分离并鉴定候选菌株发酵液中活性成分的化学结构及其抑菌活性，为农用杀菌剂的创制提供依据。【方法】采用选择性培养基从小檗叶片分离内生放线菌，16S rDNA法进行菌株鉴定；采用管碟法测定发酵液对烟草青枯病菌（Ralstonia solanacearum）等7种供试菌的抗细菌活性，采用抑制菌丝生长速率法测定发酵液对番茄灰霉病菌（Botrytis cinerea）等7种植物致病菌的抗真菌活性；依次采用大孔树脂吸附、硅胶柱层析及反相高效液相色谱（HPLC）等技术分离纯化活性组分；采用核磁共振波谱（NMR）和质谱（MS）技术对分离到的活性成分进行结构鉴定；采用微量肉汤稀释法测定化合物对烟草青枯病菌等7种细菌的最低抑菌浓度，抑制孢子萌发法和菌丝生长速率法测定化合物对番茄灰霉病菌的抗菌活性。【结果】分离到一株活性菌株H21，该菌株经16S rDNA序列分析鉴定为链霉菌（Streptomyces sp.）；除铜绿假单胞菌（Pseudomonas aeruginosa）外，H21菌株发酵液对烟草青枯病菌、猕猴桃溃疡病菌（Pseudomonas syringae pv. actinidiae）、枯草芽孢杆菌（Bacillus subtilis）、蜡状芽孢杆菌（Bacillus cereus）、金黄色葡萄球菌（Staphylococcus aureus）、大肠埃希氏菌（Escherichia coli）等多种病原细菌有明显的抑菌活性；H21发酵液对番茄灰霉病菌表现出较强的抑制菌丝生长作用，对其他供试病原真菌无明显的抑菌活性；从H21菌株发酵液中分离鉴定了3个化合物，分别为N-乙酰基-2-(4-羟苯基)乙胺、环-（L-亮氨酸-L-精氨酸）和二硫吡咯类抗生素—全霉素；全霉素为广谱抗生素，其对烟草青枯病菌、猕猴桃溃疡病菌、蜡状芽孢杆菌、枯草芽孢杆菌、大肠埃希氏菌和金黄色葡萄球菌的最低抑菌浓度（MIC）分别为0.156、0.313、0.078、0.313、0.156和0.313 µg·mL^-1；对番茄灰霉病菌菌丝生长和孢子萌发的抑菌中浓度（IC₅₀）分别为13.56和7.89 µg·mL^-1。【结论】小檗内生菌放线菌H21可能是一种新的全霉素产生菌，其对植物致病细菌烟草青枯病菌和猕猴桃溃疡病菌有抗菌活性，全霉素对于开发针对致病细菌的农用杀菌剂具有重要的参考价值。

关键词: 小檗, 内生放线菌, 全霉素, 抑菌活性

Abstract: 【Objective】The objectives of this experiment are to screen bioactive endophytic actinomycete from Berberis thunbergii, a traditional Chinese medicinal plant, study the taxonomic status and antimicrobial activity of the active strain, investigate the chemical structures and antimicrobial activity of active ingredients in the fermentation, and to provide a basis for the development of agricultural fungicide.【Method】 The endophytic actinomycete was isolated from the leaf of B. thunbergii by using selective culture techniques, and the obtained strain was identified by using 16S rDNA sequencing. The antibacterial and antifungal activities of the fermentation broth against seven species of tested bacteria (such as Ralstonia solanacearum) and plant pathogenic fungi (such as Botrytis cinerea) were tested by cylinder-plate method and mycelial growth method, respectively. The active ingredients were obtained by the techniques of macroporous resin adsorption, silica gel column chromatography and preparative reverse phase high performance liquid chromatography. Structures of the obtained compounds were elucidated by NMR and ESI-MS techniques. Minimum inhibitory concentrations (MICs) of active ingredients against tested bacteria were determined by micro-broth dilution method. Antifungal activity of H2101 against B. cinerea was tested by the methods of mycelial growth and spore germination, respectively.【Result】Strain H21, an active species, was isolated from the plant, and it was identified as Streptomyces sp. by the method of 16S rDNA sequencing. Except Pseudomonas aeruginosa, the fermentation broth of H21 showed strong antibacterial against the tested bacteria, such as R. solanacearum, P. syringae pv. actinidiae, Bacillus subtilis, B. cereus, Staphylococcus aureus and Escherichia coli. However, the fermentation broth could only inhibit the mecelial growth of B. cinerea. No inhibitory effect was observed against other tested fungi. Holomycin, a member of the pyrrothine class of antibiotics, as well as N-acetyl-2-(4-hydroxyphenyl)-ethylamine and cyclo (L-Leu-L-Arg), was isolated from the broth of strain H21. Holomycin exhibited broad antimicrobial activity, and the MIC values against R. solanacearum, P. syringae pv. actinidiae, B. cereus, B. subtilis, E. coli and S. aureus were 0.156, 0.313, 0.078, 0.313, 0.156 and 0.313 µg·mL^-1, respectively. Meanwhile, holomycin had strong antifungal activity against B. cinerea, and its IC₅₀ values against mycelial growth and spore germination were 13.56 and 7.89 µg·mL^-1, respectively.【Conclusion】Streptomyces sp. is a newly discovered producer of holomycin, and it has the antibacterial holomycin against R. solanacearum and P. syringae pv. actinidiae, two species of plant pathogenic bacteria. In general, holomycin is a valuable lead compound for the development of agricultural fungicides with those acts on bacteria.

Key words: Berberis thunbergii, endophytic actinomycete, holomycin, antimicrobial activity

魏少鹏，国政，姬志勤. 小檗内生放线菌H21的鉴定及抑菌活性成分分析[J]. 中国农业科学, 2015, 48(6): 1095-1102.

WEI Shao-peng, GUO Zheng, JI Zhi-qin. Identification and Antimicrobial Ingredients of an Endophytic Actinomycete Strain H21 from Berberis thunbergii[J]. Scientia Agricultura Sinica, 2015, 48(6): 1095-1102.

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