棉花根际微生物与内生菌对黄萎病的共调控效应及防病机理

Verticillium wilt has seriously restricted the sustainable yield of cotton in Xinjiang, China. Rhizosphere microorganisms and endophytic bacteria in cotton (Gossypium hirsutum L.) have great potential for biological control of cotton verticillium wilt. There are the close relation to cotton verticillium wilt and rhizosphere and endophytic microorganisms, but no study has investigated the co-regulation effect of the rhizosphere microorganisms and endophytes on verticillium wilt and their mechanism to control disease. This project intends to collect plant and root soil samples at cotton fields of various degree of cotton verticillium wilt, and to study the relativity between the diversity of endophytes and rhizosphere microorganisms of cotton and the verticillium wilt incidence degree using high-throughput sequencing and RT-QPCR technology combined with the traditional separation method and to screen dominant species of endogenous and rhizosphere biocontrol microorganisms. With seed, soil culture and suspension culture, combined with the instrument and technology of laser confocal microscope, fungi dyeing, RT-QPCR, liquid chromatography, mass spectrometry and infrared spectrum, coordination control effect of dominant species of endogenous and rhizosphere biocontrol microorganisms on cotton verticillium wilt was to be considered from the perspective of antimicrobial effects and detoxification. The interaction inducing effects of dominant species of endogenous and rhizosphere biocontrol microorganisms was to be explored using microbial and plant cell culture method. Pathogenic mechanisms of rhizosphere microorganisms and endophytic bacteria in cotton were to be elucidated from the angle of siderophores production, resistance-related hydrolase and oxidase production, induced resistance and so on. The study is so as to provide new ideas and theoretical basis for the biological control of cotton Verticillium Wilt.

棉花黄萎病严重制约新疆棉花持续高产和稳产，根际微生物及内生菌在棉花黄萎病生物防治中潜力巨大。棉花黄萎病发生与根际微生物及内生菌有密切关系，但根际微生物及内生菌对黄萎病的共调控效应及防病机理研究尚无报道。本项目拟采集新疆黄萎病发生程度不同棉田棉花样品，采用高通量测序和RT-QPCR技术结合传统分离方法，研究不同发病程度棉田棉花根际微生物及内生菌多样性与黄萎病发病程度的相关性，筛选优势内生及根际生防菌。采用组培、土培及悬浮培养，结合激光共聚焦显微镜、真菌染色、RT-QPCR、液相色谱、质谱及红外光谱等仪器技术，从抑菌及解毒角度，考察生防菌对棉花黄萎病的协同调控效应。应用微生物与植物细胞共培养方法，探讨生防菌对棉花黄萎病的交互诱导效应。从优势内生及根际生防菌铁载体的产生、抗病性相关水解酶和氧化酶的产生、诱导抗性等方面，阐明棉花根际微生物与内生菌防病机理，为防治棉花黄萎病提供新思路和理论依据。

棉花黄萎病严重制约新疆棉花持续高产和稳产，根际微生物及内生菌在棉花黄萎病生物防治中潜力巨大。棉花黄萎病发生与根际微生物及内生菌有密切关系，但根际微生物及内生菌对黄萎病的共调控效应及防病机理研究尚无报道。.本项目研究表明，棉花根际与内生细菌、古菌和真菌具有丰富的多样性。棉花根际细菌中，变形菌门、酸杆菌门、芽单胞菌门为三大优势菌群，其中变形菌门丰度最高；根际古菌中含量最高的是泉古菌类，其次是广古菌门；土壤真菌中含量最高的是Sodariomycetes，其次是座囊菌纲。棉花内生细菌中，主要有变形菌门、放线菌门、厚壁菌门的细菌，变形菌门含量较高，为优势菌群；内生古菌含量最高的是泉古菌门，为优势菌群，其次是未分类古菌，Thermoplasmata；内生真菌中含量最高的是座囊菌纲，其次是粪壳菌纲。.棉花根际与体内黄萎病菌、细菌、古菌和真菌数量在南疆、北疆、东疆呈一定规律。发病与健康棉花根际与内生细菌、古菌、真菌组间有一定差异。不同地点、不同生育期棉花根际和内生菌与黄萎病病原菌有相关性。.对新疆棉区8个采样点的320份样品进行了分离，筛选到4株棉花黄萎病高效拮抗细菌，通过形态特征、生理生化特征和16S rDNA序列分析将拮抗菌株BHZ-29、SHT-15、SHZ-24和SMT-24分别鉴定为贝莱斯芽孢杆菌、枯草芽孢杆菌、萎缩芽孢杆菌和香草芽孢杆菌。.棉花根际与内生拮抗细菌主要通过减少黄萎病菌数量，破坏黄萎病菌细胞形态，导致黄萎病菌细胞膜破裂，降低黄萎病菌毒蛋白浓度来协同调控棉花黄萎病。盆栽试验表明，拮抗细菌能够提高棉株生物量，增加棉花根际细菌数量降低真菌数量，提高棉花体内POD酶和SOD酶活性，降低棉花黄萎病病情指数，对棉花黄萎病具有良好的交互诱导效应。.棉花根际与内生拮抗细菌的防病机理主要在于拮抗菌产生几丁质酶、葡聚糖酶、纤维素酶、蛋白酶、儿茶酚型铁载体，产铁载体能力SHZ-24强，SHT-15与SMT-24一般，BHZ-29弱，低铁环境对BHZ-29、SHZ-24、SMT-24抑菌有影响，对SHT-15抑菌无影响，同时拮抗细菌诱导棉苗体内SOD、POD、PPO、CAT、PAL和MDA变化，增加棉苗株高和根毛数量，在棉苗土壤中呈先降后增的趋势，刺激棉花使其增强抗病性。