竹种子内生真菌多样性及其抑菌活性的研究

Bamboos (Bambusoideae, Poaceae) have been used broadly for traditional medicine, food material, building element and paper pulp. However, there is a considerable ecological problem in most of bamboo speices, where sexual propagation plays a vital part in the sustainable production of bamboos, but the seeds are uncommon and have a low germination. ..The seed-associated fungal endophytes were usually implicated in assisting seeds in germination of seed pods and some fungal endophytes had serious negative effects on the seed survival in tissue culture. These seeds colonized by field and storage fungi could be a source of potential pathogens, which might pose problems in nurseries. Furthermore, many endophytic fungi are capable of synthesizing bioactive compounds which can be used by the plants for defense against pathogenic fungi and bacteria, and producing a wide range of novel metabolites of pharmaceutical and agricultural importance...Our project is for the first time to investigate the diversity of endophytic fungi associated with bamboo seeds comprehensively, and screen the antimicrobial activity of culturable strains. . In the 3-year plan, amount of fungal endophytes would be isolated from three types of bamboo (Phyllostachys edulis, Dendrocalamus latiflorus Munro, and Dendrocalamopsis vario-striata (W. T. Lin) Keng f.) seeds, at different locations (Guangdong, Guangxi, Guizhou, Hainan). These strains would be categorized into different morphotypes according to culturable characteristics and their taxonomic position would be validated by molecular identification. The diversities of the fungal community structures would be compared among three species of bamboos at the same location, or among different locations by the same species. Furthermore, inhibitory effects against clinical pathogens and phytopathogens would be screened globally and the candidate isolates should display high and broad-spectrum bioactivities by the agar diffusion method. The effective agents extracted from these represented strains under submerged fermentation, would be analyzed preliminarily and checked again for the antimicrobiol activities. .After the future project, a good deal of fungal strains would be collected from bamboo seeds, which could improve to understand the interaction between bamboo seeds and endophytic fungi, and be anticipated as a potential source of novel plant defense activators and bioactive compounds.

竹子属于禾本科竹亚科(Bambusoideae)植物，被广泛的应用于医药、食品、建材、造纸等行业，其稀有的种子资源对竹林的可持续发展和避免生态灾难具有重要的意义。种子内生真菌与种子的萌发和幼苗的生长密切相关，并在防治病害、提高种子资源利用效率等方面发挥了重要的作用，但竹种子内生真菌的相关研究却少有人涉及。鉴于此，项目申请者在前期工作的基础上较系统的开展竹种子内生真菌的研究工作，从不同地区（广东、广西、贵州、海南）不同竹种（毛竹、麻竹、吊丝单）的种子中，分离培养内生真菌；通过形态观察和分子鉴定确定内生真菌的种类组成，利用真菌群落的结构变化反映内生真菌与生态环境、寄主种类的相关性；采用抑菌实验大规模快速筛选高生物活性的菌株，初步分析相关的有效成份。通过本项目的研究，收集大量的菌株，为进一步探讨竹种子与内生真菌的相互作用奠定基础，也为下一步研发新生防制剂和生物活性物质提供重要的资源。

内生真菌广泛存在于植物体内，是重要的生物资源。竹子兼具食用、观赏、药用等多种功效，其种子更是稀有的研究材料。本研究以毛竹、翠竹、方竹、黄竹、筇竹以及黄条金刚竹的种子为研究材料，采用形态结合分子系统发育的方法鉴定竹种子的内生真菌，并进一步分析其种类组成、分布、多样性及其与寄主关系。从1700个种子组织块中共分离出1553株内生真菌菌株，通过形态结合分子系统的方法鉴定，将其归类为2门，5纲，13目，22科，28属。分析竹种子内生真菌的菌群组成和分布规律，不同的竹种子其真菌群落具有不同的多样性：Shannon-Wiener多样性指数（H）的顺序为：毛竹（2.97）>筇竹（2.43）>黄竹（2.31）>翠竹（2.25）>方竹（2.21）>黄条金刚竹（0.85）；Margalef丰富度指数（R）的排序为：毛竹（2.77）>筇竹(1.8)>翠竹(1.63)>方竹(1.36)>黄竹(1.26)>黄条金刚（0.89）；根据Pielou’s evenness均匀度指数（J），黄竹、翠竹、毛竹、方竹和筇竹没有显著差异（均在1.0-1.1之间），但黄条金刚竹仅为0.2。采用乙酸乙酯萃取发酵液和菌丝体活性物质，以病原菌为指示菌进行抗性检测，HZP74（Pestalotiopsis sp.）和菌株MZP227（Penicillium sp.）的提取物对五种指示菌具有抑菌作用，其中菌株HZP74提取物对枯草芽孢杆菌和白色念珠菌的抑制作用极其明显。本研究首次对竹种子内生真菌进行了大规模的分离培养和鉴定，获得了大量竹种子内生真菌资源，并进一步对其组成、分布及多样性进行了全面系统的分析。研究结果丰富了对竹种子内生真菌这一特殊的真菌类群的认识，并为竹子病害防治和内生真菌资源进一步开发利用提供了理论依据，尤其是具有明显生物活性的相关菌株有望成为提供天然产物和药物前体的资源宝库。