大豆根内特异微生物及疫霉病菌侵染后根招募微生物代谢活性化合物研究

Microbes have been screened from rhizosphere and roots of the disease- and insect pest- caused plants as well as the adjacent normal plants for many years in our lab. 44 new genera and species of actinomycetes, over 200 active compounds with novel structures and three commercialized strains have been obtained. For exploring the specific microbes within roots and those recruited by disease and insect pest infestation with the remarkable abilities of synthesizing active compounds, the 16S rRNA metagenomes of microbiota residing in the soil, soybean (normal and Phytophthora-infected) rhizosphere and roots will be sequenced to define the composition and abundance of different genera, therefore, leading to their identification of these specific microbes. According to the distribution of different genera colonizing the roots, special media and separation methods are employed for isolating specific strains. The isolated specific strains will be screened by PCR for genes associated with secondary-metabolite biosynthesis. Three primer sets were used to specifically target adenylation domains associated with nonribosomal peptide synthetases (NRPSs) and ketosynthase (KS) domains associated with type I modular, iterative, hybrid, and enediyne polyketide synthases (PKSs). Subsequently, sequence clustering patterns were conducted to provide an estimate of PKS pathway diversity and to evaluate the biosynthetic richness of individual strains. Furthermore, novel purified compounds will be identified and their activities will be assessed. This study exploits the potential of antibiotic biosynthesis of specific microbes within roots and those recruited by infestation comprehensively and deeply, thus contributing great scientific significance and applicable value to the discovery of novel agroantibiotics.

本实验室从病虫害侵染植物及相邻正常植物的根际、根内筛选微生物，已高效发现44个放线菌新种、属，200多个新结构活性化合物，3个产业化菌株。植物根内是否存在一类特异微生物，和植物被病虫害侵染后根能招募微生物，且它们能代谢农用活性化合物？为探明这一科学假设，拟采用16S rRNA宏基因组测序，明确土壤、大豆及被疫霉病菌侵染大豆根际、根内微生物群落组成及丰度，确定大豆根内存在的特异微生物和侵染后招募的微生物，选择专用、特殊培养基及方法分离这类菌株；然后以参与主要抗生素生物合成的非核糖体多肽合成酶腺苷酰作用模块、PKS I型酮基合成酶模块、烯二炔聚酮合成酶的3个基因，进行基因探针扫描菌株基因组，聚类分析生物合成化合物结构的多样性，纯化、表征新型化合物并评估活性。研究将全面、深入探明大豆根内特异微生物和被疫霉病菌侵染后根招募的微生物代谢活性化合物的能力，对引领发现新型农用抗生素有重大科学和应用价值。

为了探明植物被病虫害侵染后根能招募微生物，且能代谢农用活性化合物，采用16S rRNA宏基因组测序，明确土壤、大豆及被疫霉病菌侵染大豆根际、根内微生物群落组成及丰度，确定了大豆侵染后招募微生物，并分离招募菌株，获得了抗病和代谢的活性化合物。研究结果为高效发现新型农用抗生素提供了新方法。.疫霉病菌侵染大豆12天时，根内样品中的放线菌门和放线菌门内的链霉菌科、小单孢菌科、原小单孢菌科、类诺卡氏菌科、束丝放线菌科在侵染大豆中的增加幅度远高于正常大豆的，这些物种在侵染大豆根内得到富集。纯培养招募微生物并活性测定，大豆根内招募微生物有强的抗病害能力。. 筛选发现抗多种植物病害、解磷能力强的小孢根霉NEAU-8和黑曲霉NEAU-L7，是国际上首次发现小孢根霉菌属具有相关农业用途功能。小孢根霉NEAU-8和黑曲霉NEAU-L7对番茄、黄瓜、辣椒、玉米、大豆、水稻、小麦等作物的不同真菌病害都有较好的防效。同时，对磷矿粉、磷酸钙、磷酸锌有强的解磷能力。.从新放线菌Streptomyces sp. NEAU6中分离新型植物生长调节剂---五谷丰素。五谷丰素化学结构是在腺嘌呤N9位上结合经修饰稀有阿洛酮糖C2′位和C5′位的新型核苷类化合物，与细胞分裂素作用于植物的表型明显不同，在使用时期上也有别于细胞分裂素、赤霉素、油菜素内酯等植物激素。五谷丰素具有优良特性：（1）用量低，增产明显，提高品质。水稻亩有效用量0.2g、玉米亩有效用量0.08g、蔬菜等亩有效用量0. 2g-1g，大田增产10%-39%。（2）毒性低。大鼠口经急毒LD50 >5000mg/kg，毒性低于食盐。（3）浸种处理水稻、玉米、小麦等，作物整个生长周期株高、茎粗及产量都能直观看见，且为最经济、最安全、最简单的施药方式。（4）低用量、低毒，早期浸种处理，作物无残留，环境无影响。（5）五谷丰素水溶性好，提取和剂型加工工艺安全、简单，生产成本低，浸种水溶性粉剂不用加任何助剂。（6）具有生长素、细胞分裂素促进植物根、芽生长的双重特性，且诱导植物抗病，是极少有既能促进植物生长，又能增强植物抗病的新型化合物。