草果内生菌多样性及其对果腐病菌的抗性机制研究

Amomum tsao-ko (Zingiberaceae) with region restriction, more than 95% at home is produced in Yunnan. Fructus taso-ko (the dry fruits of A. tsao-ko) is one of the major medicinal herbs and spices with medical and edible functions. The annual product income was about 20 billion Yuan. However, fruit rot disease of amomum seriously affects the yield and quality. Therefore, it is imperative to take strategies for reducing disease incidence. Endophytes widely distributed in host plant tissues, have abundantly richness. They can prenvent the infenction of phytopathogens by the mechanisms such as induced resistance and production of secondly metabolic compouds with anti-pathogen activties. However, The diversity of endophytes from Amomum tsao-ko and its resistance to pathogens are still unclear. In the present project, the diversity and composition of endophytes (bacteria, fungi, actinobacteria) in fruits of both diseased and healthy Amomum tsao-ko in Yunnan and Guangxi will be analyzed by dependent-culture and metagenomic pyrosequencing, to reveal the relationship of endophytes diversity and community structure with fruit rot disease of A. tsao-ko. Then the mechanism of resistance to fruit rot disease of endophytes will be detected by screening the existance of functional genes including PKS, NRPS genes, as well as identification of antagonistic secondly metabolic compouds and detecting induced resistance against fruit rot disease of A. tsao-ko by potted experiments. The results would be used to discuss the mechanism of endophytes and provide scientific foundation to control fruit rot disease of A. tsao-ko.

草果生长具有严格的地域限制性，云南产量占我国95%以上；其干果是药食两用的大宗中药材、香辛料之一，年产值约22亿元。然而果腐病的频发严重影响草果的产量和品质。因此，寻找应对策略，减少病害对草果生产的影响已迫在眉睫。内生菌广泛分布于植物组织内，具有丰富的多样性，可通过诱导抗性、产抗菌次生代谢产物等机制抵御植物病原菌入侵。然而，我们对草果内生菌的多样性及其抗病性知之甚少。因此，本项目拟以云南、广西重发病和健康种植区的草果为研究材料，利用纯培养和宏基因组测序技术，分析其内生菌（细菌、真菌、放线菌）多样性和群落结构差异性，揭示自然条件下内生菌与果腐病的关系；利用对峙生长法筛选果腐病菌的拮抗内生菌，继而通过PKS、NRPS等功能基因检测、抗菌次生代谢产物鉴定及盆栽实验检测诱导抗性，揭示内生菌对果腐病菌的抗性机制。本研究将揭示草果内生菌多样性及其对果腐病菌的抗性机制，为果腐病的生物防治提供科学依据。

调查并收集了文山州富宁县、马关县，红河州金平县、元阳县、个旧市与怒江州福贡县等地发病区与不发病区的果实样品，通过高通量测序发现内生细菌有22门45纲126目229科446属549种1958个ASV、内生真菌有11门42纲111目277科662属1133种4993个ASV，样地发病与否、发病程度以及地区差异都对样地菌群结构组成的相似性具有显著影响，样地发病及发病程度是影响内生细菌菌群结构组成的主要因素，而地区差异是影响内生真菌菌群结构组成的的主要因素。通过多种培养基分离、纯化、合并获得146株内生细菌，其中136株隶属于4门7纲13目18科28属47个种级分类单元，从95株内生细菌筛选到38株对3株导致果腐病真菌具有拮抗作用的菌株；分离、纯化、合并获得302株内生真菌，其中296株隶属于3门7纲16目40科55属108个种级分类单元，从146株真菌筛选到对4株导致果腐病的真菌具有拮抗作用的菌株75个，其中29株为相对抑菌率大于50%的强拮抗菌株，7株功能基因NRPS检测阳性，5株功能基因PKSⅠ检测阳性。以拮抗菌株Nigrospora Chinensis 153发酵液为研究样品，经微萃取-顶空进样-气质联用法（ME-HS-GC-MS）分析发酵后产生的挥发性成分，发现36个化合物，其中2个具有抗真菌作用。进而，以粉红粘帚霉、哈茨木霉加芽孢杆菌进行田间防效测试，防病率分别达25.41%、25.67%，低于生防菌淡紫拟青霉的54.89%和噁霉灵芽孢杆菌的42.65%。