东乡野生稻内生放线菌多样性及农用活性化合物挖掘

Plant endophytic actinomycetes have been widely used in plant growth and antangonism of plant pathogens, and become as a vital resource with new species and natural metabolic production during their long-term adapting to the estreme environment. Dongxiang wild rice (DW) in Dongxiang county, Jiangxi province, one of the most northern species of Oryza rufipogon Griff. in the world, possess strong tolerance to cold, drought, barren and disease, etc. As an endemic plant with strong stress-tolerance characteristics, it would be destined to have unique resources of endophytic actinomycetes, which can carried out by their metabolites or the action of signaling role to promote the stress-tolerance of host. Our previous studies revealed that the abundance of endophytic actinomyces and antimicrobial activity may be closely related with the stress-tolerance characteristics (disease resistance). The community diversity and structural dynamic of endophytic actinomycetes from DW will be scientifically investigated by culture-dependent and high-throughput sequencing technologies during their different growth periods. Multiple isolation methods will be designed to obtain the culturable actinomycetes. Meanwhile, the antimicrobial bioactivity of endophytic actinomycetes will be tested basing on some rice pathogens. Then the active metabolites of endophytic actinomycetes strains with the most potential antimicrobial activity will be scientificly separated for antibiotics,and the structures of the obtained active compounds would be determined by Mass spectrum and Spectroscopic techniques. This implementation of this project will not only elucidate the community diversity, but also lay the foundation for mining novel agricultural active compounds and demonstrating the relationship between host plant and microbe.

植物内生放线菌所处生境特殊，物种新颖多样，在促生、抑制病害等方面应用前景广阔，已成为新菌种和天然活性化合物的重要来源。东乡野生稻（东野）作为中国特有（仅残存于江西东乡）、世界分布最北且耐寒、抗旱、抗病害等抗逆性丰富的野生稻，不仅是水稻育种的优良基因库，也是内生放线菌资源和活性化合物勘探的宝库。我们前期研究发现，东野的抗逆性与其蕴含内生微生物的多样性和抗菌活性有关，本研究拟采用培养法并结合高通量测序技术，揭示东野内生放线菌群落多样性及动态变化规律，建立东野内生放线菌资源库；以水稻常见病原菌为靶标，筛选拮抗水稻常见病害的放线菌；利用现代色谱、质谱和波谱技术对高活性放线菌化合物进行分离鉴定并获得活性化合物资源。本研究不仅能系统揭示东野内生放线菌群落多样性，丰富放线菌资源；而且可为发掘利用新型农用生物活性化合物及阐明内生放线菌与宿主间的相互作用奠定基础。

项目采用可培养与高通量测序相结合方法开展东乡野生稻不同生育期不同组织部位内生放线菌动态分布规律和多样性的研究，发现东乡野生稻内生放线菌随着生育期和组织部位的不同呈现一定的规律性变化，成熟期、抽穗期到秧苗期分离的放线菌数量逐渐减少，根部组织内生放线菌多样性最丰富，呈现从根到茎到叶逐渐减少的规律；比较了可培养和高通量测序方法内生放线菌的分布特征和差异，可培养法下共分离到6个放线菌属，优势菌属为链霉菌（49.5%）和小单孢菌（32.4%），而高通量测序检测到组织样品中40个放线菌属的分布，优势菌属为链孢子囊菌属、小单孢菌、链霉菌和分枝杆菌属等，多样性更丰富，而疣孢菌属、小双孢菌和野野村菌属放线菌未能在高通量测序中检测到，只有将两种方法有机结合起来才能更全面了解植物内生放线菌的多样性。通过抗菌活性筛选获得大量具有抑制病原细菌和植物病原真菌的内生放线菌，从4株活性菌株中共分离到靛红类、Fluostains类、烟曲霉酸类代谢产物39个，其中4个为新化合物，一些化合物具有较好的水稻细菌性条斑病菌、金黄色葡萄球菌和酪氨酸酶抑制活性。随后对2株活性菌株进行全基因组测序，获得相关化合物生物合成基因簇并分析其合成途径。基于固氮、溶磷、产IAA、分泌铁载体等促生活性分析，选择了一些具有促生潜能的内生细菌定殖水稻种子，开展栽培稻盆栽实验，检测栽培稻的生长指标，利用转录组分析初步验证其促生机制。本研究不仅能系统揭示东野内生放线菌结构动态规律和多样性，丰富我国放线菌资源，筛选到良好生物活性的内生微生物，而且可为发掘新型农用生物活性化合物、阐明内生放线菌与宿主间的相互作用奠定基础。