内生特基拉芽孢杆菌7PJ-16在桑树中定殖特征及对桑椹菌核病菌拮抗机制的研究

Sclerote disease of mulberry fruit is disaster to the healthy development of mulberry fruit related industry. Currently, control of the sclerote disease of mulberry fruit mainly depends on the application of synthetic agricultural chemical. However, over used synthetic agricultural chemical undoubtedly result in environmental pollution, and threaten the safety of mulberry fruit as food. It is a promising biocontrol strategy that controlling plant diseases with antagonistic endophytes. Using Scleromitula shiraiana as target, antagonistic endophyte B. tequilensis 7PJ-16 strain was screened out in the previous research. Biocontrol effect of endophyte was depending on the colonization of endophyte in mulberry and its inhibitory mechanisms to the pathogens. This project will explore the colonization characteristics of B. tequilensis 7PJ-16 strain in Mulberry through infecting mulberry seedlings with green fluorescent protein tagged strains. Then, the research will optimize the fermentation conditions of B. tequilensis 7PJ-16 strain, purify and identify the main active components from fermentation supernatant of B. tequilensis 7PJ-16 strain. Finally, the antagonistic mechanisms of mulberry endophytic B. tequilensis 7PJ-16 strain on Scleromitula shiraiana (pathogen of sclerote disease of mulberry fruit) will be revealed through analysis the effect of the main active component on the cell growth, cell structure and protein synthesis of the pathogenic fungi. The research project will build theoretical basis and experimental foundation for the biological control of sclerote disease of mulberry fruit. Meanwhile, this project will provide important reference for the biological control of other mulberry diseases.

桑椹菌核病是影响果桑产业健康发展的严重病害。目前该病害主要依靠化学农药进行防治，然而化学农药的过量使用不仅会造成环境污染，亦会威胁桑椹食用安全。利用植物内生拮抗菌防控植物病害是近年来备受青睐的生防新策略。项目组前期研究以桑椹缩小性菌核病菌（桑椹核地杖菌）为靶标，筛选获得一株桑树内生拮抗菌特基拉芽孢杆菌7PJ-16。拮抗菌在桑树体内的定殖能力及其对菌核病菌的拮抗机制是决定防效的关键。故本项目拟利用绿色荧光蛋白标记的7PJ-16菌株侵染桑苗，研究其在桑树中的定殖能力及定殖特征。同时，优化7PJ-16菌株产生抑菌活性物质的发酵条件，分离纯化活性发酵液的主要抑菌成分，解析其分子结构，进而通过分析主要抑菌成分对桑椹核地杖菌菌体生长、细胞结构及蛋白合成等的影响阐明拮抗机制。本项目的实施将为桑椹菌核病生物防治奠定理论依据和实验基础，亦为其他桑树病害的生物防治提供重要参考。

桑椹菌核病是影响果桑产业健康发展的严重病害，过量使用化学农药防治该病，不仅会造成环境污染，亦会威胁桑椹食用安全。项目组前期从健康桑树中寻获一株对菌核病具有生防潜能的内生芽孢杆菌7PJ-16菌株，本项目首先研究了该菌株在桑树中的侵染定殖规律，及芽孢杆菌在健康桑树中的分布特征；通过对该菌株的全基因组测序分析，推测了抑菌活性物质的主要类型，分离纯化了主要抑菌活性组分，并解析其分子结构；进而通过研究7PJ-16菌株的田间防效和对病原菌生长的影响，以及对桑苗生长的促进作用及机制，系统阐明该菌株的生物防治机制。绿色荧光蛋白标记的Bacillus sp. 7PJ-16可在桑树根、茎以及叶部稳定定殖，健康桑树内生细菌种群结构分析结果亦显示，芽孢杆菌菌群能够长期稳定存在于不同品种的果桑体内；基于全基因组的近缘物种进化分析结果，将前期鉴定为Bacillus tequilensis 7PJ-16菌株更新分类为Bacillus subtilis 7PJ-16；从7PJ-16菌株抑菌活性发酵液中分离获得环二肽cycle (Pro-phe) 等6个与抑菌活性相关的小分子物质，并通过全基因组序列分析和LC-MS检测结果发现，该菌株还可产生表面活性素和丰原素两种脂肽类抗生素；该菌株除可通过提高宿主防御酶活，抑制病原菌丝生长、菌核萌发及子实体生长发育等功能而对桑椹菌核病具有防治能力外，还可通过产生IAA、铁载体等促进桑种萌发和桑幼苗生长。本项目的研究结果为桑椹菌核病生物防治奠定理论依据和实验基础，亦为其他桑树病害的生物防治提供重要参考。