杨帆 刘春来 刘亮 王爽 蒋希峰 李敏 曹大为 李新民

摘要 為挖掘生防新资源，采用组织分离法从21种野生药用植物不同组织部位分离纯化内生真菌，以6种植物病原菌为靶标菌筛选拮抗菌株，并根据形态学及分子生物学对菌株进行鉴定，在此基础上研究了高效拮抗菌株对靶标菌菌丝生长及孢子萌发的影响。结果表明：从分离纯化的478株内生真菌中筛选出11株高活性拮抗菌株，分属于青霉属Penicillium、平脐蠕孢属Bipolaris、棘壳孢属Pyrenochaeta、镰孢属Fusarium、粒毛盘菌属Lachnum、垫壳孢属Coniella和Neonectria 7个属，其中青霉属占比最高，达36.36%。平皿对峙试验表明，

棘壳孢菌12-R-5对灰葡萄孢表现出高效性和专一性，其对菌丝抑制率达66.67%。含药平皿试验表明，菌株12-R-5发酵滤液10倍稀释液对灰葡萄孢菌丝生长抑制率达100%，与其他处理差异显著。菌株12-R-5 10倍发酵滤液处理灰葡萄孢10 h，孢子不萌发;100倍稀释液处理对孢子萌发抑制率为91.01%。显微镜观察发现，棘壳孢菌使灰葡萄孢菌丝扭曲变形，膨大肿胀及断裂，部分菌丝发生缢缩、消融现象。说明棘壳孢菌12-R-5菌株具有很好的生防潜力及应用前景。

关键词 药用植物; 内生真菌; 植物病原菌; 拮抗作用

中图分类号： S 572

文献标识码： A

DOI： 10.16688/j.zwbh.2020499

Isolation of endophytic fungi from medicinal plants and screening for antagonistic strains

YANG Fan1，2，3， LIU Chunlai2，3， LIU Liang2，3， WANG Shuang2，3，

JIANG Xifeng2，3， LI Min2，3， CAO Dawei2，3， LI Xinmin1，2，3*

（1. Postdoctoral Research Workstation， Heilongjiang Academy of Agricultural Sciences， Harbin 150086， China;

2. Institute of Plant Protection， Heilongjiang Academy of Agricultural Sciences， Harbin 150086， China;

3. Scientific Observation and Experimental Station of Crop Pests in Harbin， Ministry of

Agriculture and Rural Affairs， Harbin 150086， China）

Abstract

In order to explore new biocontrol resources， endophytic fungi were isolated and purified from different tissues of 21 wild medicinal plants by using tissue isolation method. Antagonistic strains were screened against six common pathogenic fungi， and the strains were identified based on morphology and molecular biology. The effects of antagonistic strains against target pathogens on mycelial growth and spore germination were further studied. The results showed that 11 highly active antagonistic strains were screened out from 478 strains of endophytic fungi， which belonged to Penicillium， Bipolaris， Pyrenochaeta， Fusarium， Lachnum， Coniella and Neonectria. Penicillium accounted for the highest proportion， up to 36.36% of the total. The inhibition rate  of the strain 12-R-5 against Botrytis cinerea hyphae was 66.67% by the plate confrontation test. The inhibition rate of 10-fold fermentation filtrate of 12-R-5  against pathogen hyphae growth reached 100%， which was significantly higher than other treatments. Pathogen spores treated 10-fold the fermentation filtrates of the strain 12-R-5 for 10 h could not germinate. When the spores were treated with 100-fold diluent， the inhibition rate of spore germination was 91.01%. According to microscopic observation， the strain 12-R-5 could cause the hyphae of B.cinerea to expand and twist， and some hyphae shrunk and melted. The results indicated that the strain 12-R-5 had a good biocontrol potential and application prospect.

Key words

medicinal plants; endophytic fungi; phytopathogens; antagonistic activity

Petrini于1991年将内生菌定义为在其生活史中的某一段时期生活在植物组织内，且没有引起植物组织明显病害症状的微生物[1]。同时植物内生菌的挖掘被认为是寻找具有潜在农业、医药和工业应用价值的天然产物的新领域[2-3]。药用植物是具有生物活性的天然化合物的丰富资源，且药用植物内生真菌能够产生与寄主植物相同或相似的活性成分[4-5]，而且在很大程度上仍未被开发[6]。……