云南嵩明大哨天然虫生真菌及其内生真菌的分离鉴定与抑菌活性分析

Isolation, identification, and antimicrobial activities analysis of natural entomogenous fungi and the endophytic fungi from Dashao village, Songming county, Yunnan province

摘要:

【背景】云南存在着丰富的虫草资源，天然虫草群落中蕴藏多样的真菌资源，是挖掘新型抑菌活性化合物的潜在来源。【目的】了解云南嵩明大哨天然虫生真菌及其内生真菌的物种多样性，并从中筛选具有抑菌活性的菌株。【方法】采用组织分离法对所采集的野生虫草样本进行虫生真菌及其内生真菌的分离，并通过形态观察和ITS联合nrSSU、nrLSU、tef-1α、rpb1、rpb2多基因测序进行物种鉴定及多样性分析；通过平板对峙法以7株病原细菌、5株植物病原真菌为病原指示菌进行抑菌活性测试。【结果】共采集86份天然虫草样本，经鉴定隶属于3科5属7种，包括虫草科（Cordycipitaceae）虫草属（Cordyceps）（1种）、白僵菌属（Beauveria）（2种）、鳞翅虫草属（Samsoniella）（2种）、麦角菌科（Clavicipitaceae）泛普可尼亚属（Metapochonia）（1种），以及线虫草科（Ophiocordycipitaceae）线虫草属（Ophiocordyceps）（1种）。同时，从采集到的虫草样本中分离得到26株内生真菌，分属于9科9属，其中木霉属（Trichoderma）（38%）和镰刀菌属（Fusarium）（19%）为此次分离得到的优势菌属。对所分离保存的真菌按其分离源及种类归属，从虫生真菌和内生真菌菌株中共挑取20株代表性菌株进行抑菌活性筛选，有11株真菌对2株及以上病原指示细菌具有不同程度的抑菌活性，13株真菌对1株以上植物病原真菌具有不同程度拮抗能力；其中Trichoderma sp.Y3-1和Fusarium sp.WZ3-1具有广谱抑菌活性。【结论】云南嵩明大哨分布有丰富的虫生真菌及内生真菌资源，分离所得的真菌对多种病原菌具有不同程度的抑制作用。本研究丰富了云南虫草资源多样性，为云南省虫草及其内生真菌资源的开发利用提供了数据支持，也为下一步从虫草及其相关真菌资源中挖掘抑菌活性物质提供了菌株资源。

关键词:虫生真菌;内生真菌;纯培养分离;物种多样性;抑菌活性

Abstract:

[Background] Yunnan boasts abundant Cordyceps species resources and the natural Cordyceps species community contains diverse endophytic fungi, which can be used for mining antimicrobial.[Objective] To investigate the diversity of entomogenous fungi and the endophytic fungi in Dashao village, Songming county, Yunnan province, and screen strains with antimicrobial activities. [Methods] Entomogenous fungi and the endophytic fungi were isolated from Cordyceps species samples with tissue separation method. They were identified and the diversity was analyzed based on morphological observation and sequencing of ITS, nrSSU,nrLSU, tef-1α, rpb1, and rpb2. The activities against 7 pathogenic bacteria and 5 plant pathogenic fungi were tested by plate confrontation assay.[Results] A total of 86 natural Cordyceps species samples were collected, which belonged to 7 species in 5 genera in 3 families:Cordyceps (1 species), Beauveria (2 species), and Samsoniella(2 species) in Cordycipitaceae, Metapochonia (1 species) in Clavicipitaceae, and Ophiocordyceps (1 species) in Ophiocordycipitaceae. Meanwhile, 26 strains of endophytic fungi in 9 genera of 9 families were isolated from the samples, among which Trichoderma(38%) and Fusarium (19%) dominated. In addition, 20 strains, which were selected from entomogenous fungi and endophytic fungi according to their origins and taxonomy, were tested for the antimicrobial activities. Results demonstrated that 11 strains showed activities against two or more pathogenic bacteria, and 13 strains against more than one plant pathogenic fungi. Trichoderma sp. Y3-1 and Fusarium sp. WZ3-1 demonstrated broad-spectrum antimicrobial activities. [Conclusion] The Dashao village in Songming county of Yunnan province enjoys rich resources of entomogenous fungi and endophytic fungi and the isolated fungi show activities against diverse pathogens. This study enriches the diversity of entomogenous fungi in Yunnan province, providing not only data support for the development and utilization of entomogenous fungi and the endophytic fungi in Yunnan province but also strains for mining active substances from them.

Key words:entomogenous fungi;endophytic fungi;culture isolation;species diversity;antimicrobial activities