一株防治香蕉枯萎病的短密木霉筛选及代谢物木霉素作用评价

Screening and evaluation of the biocontrol efficacy of a Trichoderma brevicompactum strain and its metabolite trichodermin against banana Fusarium wilt

摘要:

由尖孢镰刀菌古巴专化型热带四号小种(Fusarium oxysporum f. sp. cubense tropical race4, FocTR4)引起的香蕉枯萎病(banana Fusarium wilt, BFW)是全世界范围内难以防治的真菌病害，给香蕉产业造成巨大的经济损失。本研究旨在筛选高效拮抗FocTR4的木霉生防菌株，并对其发酵代谢产物进行分离、提纯和鉴定，为香蕉枯萎病的高效生物防治提供重要生防菌株和活性化合物资源。从作物根际土壤中分离出木霉菌株，通过平板对峙培养、发酵液对病原菌孢子萌发及菌丝生长抑制，测试筛选出高效抑制FocTR4的生防木霉菌株；通过构建系统发育树明确生防菌株的分类地位；通过柱色谱法分离纯化菌株发酵液中活性成分，通过核磁共振波谱法(nuclear magnetic resonance spectroscopy, NMR)解析活性成分的结构；通过香蕉苗感病盆栽实验检测生防木霉菌株对香蕉枯萎病的防治效果。结果表明，本研究筛选到了1株拮抗FocTR4的菌株JSHA-CD-1003，平板对峙抑制率为60.6%；发酵液在24 h内能完全抑制FocTR4孢子萌发，7 d内对FocTR4菌丝生长的抑制率为52.6%；基于内转录间隔区(internal transcribed spacer, ITS)和tef1-α基因串联序列构建系统发育树，该菌株鉴定为短密木霉(Trichoderma brevicompactum)，通过柱色谱法分离提纯和NMR鉴定单一活性化合物为木霉素(trichodermin)，最小抑菌浓度(minimum inhibitory concentration, MIC)为25 μg/mL；盆栽生防实验表明，菌株JSHA-CD-1003发酵液对香蕉枯萎病的叶片黄化防治率为47.4%，球茎褐化防治率为52.0%。因此，JSHA-CD-1003通过产生木霉素有效抑制FocTR4孢子萌发和菌丝生长，对FocTR4引起的香蕉枯萎病具有良好的生物防治效果，是一株具有生防潜力的菌株。

关键词:香蕉枯萎病;生物防治;短密木霉;柱色谱法;木霉素

Abstract:

The banana Fusarium wilt (BFW) caused by Fusarium oxysporum f. sp. cubense tropical race4 (FocTR4) is difficult to control worldwide, which causes a huge economic losse to banana industry. The purpose of this study was to screen Trichoderma strains with antagonistic activity against FocTR4, to isolate and purify the active compound from the fermentation broth, so as to provide important biocontrol strains and active compound resources. In this work, Trichoderma strains were isolated and screened from the rhizosphere soil of crops, and the strains capable of efficiently inhibiting FocTR4 were screened by plate confrontation, and further confirmed by testing inhibition for the conidial germination and mycelial growth of FocTR4. The phylogenetic tree clarified the taxonomic status of the biocontrol strains. Moreover, the active components in the fermentation broth of the strains were separated and purified by column chromatography, the structure of the most active component was analyzed by nuclear magnetic resonance spectroscopy (NMR), the BFW control effect was tested by pot experiments. We obtained a strain JSHA-CD-1003 with antagonistic activity against FocTR4, and the inhibition rate from plate confrontation was 60.6%. The fermentation broth of JSHA-CD-1003 completely inhibited the germination of FocTR4 conidia within 24 hours. The inhibition rate of FocTR4 hyphae growth was 52.6% within 7 d. A phylogenetic tree was constructed based on the ITS and tef1-α gene tandem sequences, and JSHA-CD-1003 was identified as Trichoderma brevicompactum. Purification and NMR identification showed that the single active compound was trichodermin, and the minimum inhibitory concentration (MIC) was 25 μg/mL. Pot experiments showed that the fermentation broth of strain JSHA-CD-1003 was effective against BFW. The control rate of leaf yellowing was 47.4%, and the rate of bulb browning was 52.0%. Therefore, JSHA-CD-1003 effectively inhibited FocTR4 conidial germination and mycelium growth through producing trichodermin, and showed biocontrol effect on banana wilt caused by FocTR4, thus is a potential biocontrol strain.

Key words:banana Fusarium wilt;biological control;Trichoderma brevicompactum;column chromatography;trichodermin