李岚岚，戴利铭，蒋桂芝，刘一贤，施玉萍，蔡志英

摘  要：目前化学农药的缺点日益突显，研发环保的生防菌剂是当前绿色安全的一种方法，筛选具有抑制橡胶樹炭疽病的生防菌株，为研发新型生防菌剂储备资源。本文采用组织培养法分离纯化橡胶树内生菌，平板对峙筛选拮抗菌株;通过菌株培养特征、生理生化、根据16S rRNA和gyrA基因序列构建系统发育树确定其分类地位;采用平板对峙法研究内生菌株的抗菌持久性、稳定性、广谱性;采用牛津杯法研究拮抗内生菌对杀菌剂的敏感性。从橡胶树健康的根组织中分离得到4株对橡胶树炭疽病具有抑制作用的细菌（Bac RZS3D4-1、Bac RZS3D4-2、Bac RZS3D4-3、Bac RZS3D4-4），系统发育树分析结果显示这4株细菌与解淀粉芽孢杆菌（Bacillus amyloliquefaciens）亲缘关系很近，且培养性状和生理生化特征也与解淀粉芽孢杆菌相似。这4株内生菌的抗菌持久性强，抗菌稳定性好;对尖孢炭疽菌（Colletotrichum acutatum）DFMP1E和MLZZP3、小孢拟盘多毛孢（Pestalotiopsis microspora）MF375898均有较强的抑菌能力，对茄类镰刀菌（Fusarium solani）XJ160901的抑制作用较弱;对多菌灵、异菌脲、百菌清、苯醚甲环唑4种杀菌剂均不敏感，有望开发为生防菌剂。

关键词：橡胶树炭疽病;解淀粉芽孢杆菌;生防内生菌;分离鉴定

中图分类号：S763.7;S794.1      文献标识码：A

Isolation， Identification and Bacteriostasis Study of Endophytic Bacteria to Control Colletotrichum Leaf Disease on Rubber Tree

LI Lanlan， DAI Liming， JIANG Guizhi， LIU Yixian， SHI Yuping， CAI Zhiying*

Yunnan Institute of Tropical Crops， Jinghong， Yunnan 666100， China

Abstract： At present， the disadvantages of chemical pesticides are prominent and the development of environmental friendly biocontrol agent is needed. The screening of biocontrol strains with inhibition of rubber anthracnose can reserve resources for the development of new biocontrol agents. The endophyte bacteria of rubber trees were isolated and purified by tissue culture， and growth plate-confrontation was used to screen antagonistic strains. Strain Bac RZS3D4-1， Bac RZS3D4-2， Bac RZS3D4-3， Bac RZS3D4-4 were identified based on morphological， physiological， biochemical characteristics and the phylogenetic tree was constructed with 16S rRNA and gyrA gene sequence. The growth plate-confrontation was used to study the antimicrobial persistence， stability and broad spectrum of endophytic bacteria. The Oxford cup method was used to study the sensitivity of antagonistic endophytic bacteria to fungicides. Four strains of bacteria （Bac RZS3D4-1， Bac RZS3D4-2， Bac RZS3D4-3， Bac RZS3D4-4） were isolated from the healthy root tissues of rubber trees. Phylogenetic tree analysis showed that the four strains were closely related to Bacillus amyloliquefaciens， and the culture traits and physiological and biochemical characteristics were similar to those of B. amyloliquefaciens. The results showed the strains had strong antibacterial persistence and good antibacterial stability， strong antibacterial activity against Colletotrichum acutatum （DFMP1E and Mlzzp3） and Pestalotiopsis microspora （MF375898）， and weak inhibitory effect on Fusarium solani （XJ160901）. They were not sensitive to carbendazim， isocarb， chlorothalonil and difenoconazole， so it is expected to be developed as a biocontrol agent.

Keywords： Colletotrichum leaf disease on rubber tree; Bacillus amyloliquefaciens; biocontrol endophytic microorganism; isolated identification

DOI： 10.3969/j.issn.1000-2561.2021.10.028

炭疽病是橡膠树上的重要病害，严重发生时会导致橡胶树叶片落光，胶乳产量下降[1]。橡胶树炭疽病在世界各国植胶区均有发生，广泛分布于南美洲、非洲中部、亚洲南部和东南部等地[2]。主要是由胶孢炭疽菌（Colletotrichum gloeosporioides）和尖孢炭疽菌（Colletotrichum acutatum）引起的，近年来在国内外广泛流行，发病趋势日益严重，导致橡胶产业损失逐年增加[3-5]。……