花榈木根瘤菌多样性及幼苗根瘤形成调控因素研究

Considered the status of wild seedling regeneration difficult of rare and endangered species Ormosia henryi and low nodulation rate of artificial planting seedlings, widely collected nodules and investigate habitat condition of Ormosia henryi, and sampling analysis in their distribution areas. The rhizobium will be isolated and purified, be verified by using microscope.The genetic diversity, DNA sequencing and phenotypic traits of rhizobium strains of Ormosia henryi will be studied.The study will reveal the phenotype and gentic diversity of Ormosia henryi rhizobia and clear the taxonomic status of them,will clarify the coupling relationship between diversity of rhizobia in nature condition and their environment.By the factorial test that artificially simulate different inoculation conditions and vaccination materials, measure the nodule status, nitrogenase activity, anatomical features of root,growth and physiological indices of Ormosia henryi seedling.The internal and external effect factors on nodule formation of Ormosia henryi will be clarified , the key factors for restricting nodule formation in wild habitats and under artificial cultivation condition will be explored. We will filter out fine strains,and will establish optimized symbiotic nitrogen fixation system of Ormosia henryi. The study will provid a theoretical basis and technical guidance for the protection of wild populations of Ormosia henryi and rhizobia inoculation for nurturing seedlings of Ormosia henryi.

针对珍稀濒危物种花榈木野生幼苗更新困难，人工播种幼苗结瘤率低的现状，通过在花榈木分布区广泛采集花榈木根瘤，并进行野生生境调查及取土样分析；对花榈木根瘤菌进行分离纯化和镜检验证，进行根瘤菌遗传多样性分析、根瘤菌株DNA测序和表型性状分析，揭示花榈木根瘤菌的表型多样性和遗传多样性，明确花榈木根瘤菌的分类地位，阐明花榈木野生状态根瘤菌多样性与环境的耦合关系；通过人工模拟不同接种条件和接种材料的析因试验，测定不同处理的结瘤状况、固氮酶活性、根系解剖特征、幼苗生长和生理生化指标,阐明影响花榈木根瘤形成的内外因素，揭示花榈木野生生境和人工育苗中制约根瘤形成的关键因素，筛选出优良菌株，建立优化的花榈木根瘤菌共生固氮体系，为花榈木野生种群的保护和苗木培育中接种花榈木根瘤菌提供理论依据和技术指导。

针对花榈木野生幼苗更新困难，人工播种幼苗结瘤率低的现状，开展了花榈木根瘤菌生物生态学特性与幼苗根瘤形成调控内外因素研究。结果表明：1）花榈木根瘤因生态环境的不同，其形状、大小、颜色、以及重量均呈现出丰富的多样性，花榈木根瘤属于无限根瘤类型。花榈木根瘤主要集中分布在0-30cm的土层内，其中以10-20cm土层最多最大。2）花榈木根瘤具有丰富的表型多样性和遗传多样性。按照表型可分为5个表观类群；分离出的57株菌株遗传多样性丰富，分别属于Rhizobium、Azorhizobium、Mesorhizobium、Bradyrhizobium、Sinorhizobium、Burkholderia 6个属中。其中根瘤菌属（Rhizobium）最多，占42.1%。固氮根瘤菌属Azorhizobium）次之，占的17.5%。3）花榈木从接种开始15d开始显瘤，30-40d发育完成，60d稳定；根瘤菌侵染花榈木的部位是侧根的根毛。幼龄根瘤仅有一团分生组织，而后分化形成根瘤原基，再发育为具有表皮、皮层、维管束和含菌细胞区完整的根瘤结构。随着根瘤的发育，根瘤颜色的变化从最初的白色逐渐变成浅黄色到黄褐色，时间再延长，变成红褐色到深褐色；根瘤的形状从不明显的小突起到球形、棒状以及不规则形；根瘤表面从光滑变得粗糙。4）花榈木根瘤接种的适宜方式是幼苗长出真叶时用菌液浇灌根部，幼苗结瘤株数、结瘤率最高，结瘤质量好，幼苗生长、生理状况和固氮效果均最好。5）促进花榈木结瘤适宜的氮浓度为26mol/L～78mol/L，最佳基质酸碱度为pH6.0，基质相对含水量控制在60%左右，温度为25℃，透光度75%。该条件与花榈木本身对光照、水分、温度等的生态适应性相一致。结合对花榈木生存的环境和人工播种环境分析，认为氮浓度是影响根瘤形成的关键因素，其次是光照和温度。6）筛选出促生抗旱的菌株6株，分别是10 号、11 号、9 号、16 号、7 号、13 号菌株。