棉花内生蜡状芽孢杆菌YUPP-10对棉花黄萎病的防治作用及机制

【目的】棉花黄萎病(Verticillium wilt)是世界性难以防治的病害,筛选有效的生防微生物资源成为解决棉花黄萎病的重要途径之一,本研究旨在分离一株高效拮抗细菌,并明确其防治机理,为生物防治棉花黄萎病提供技术支持。【方法】用葡甘聚糖作为碳源筛选一株能够降解β-1,4糖苷键的棉花内生细菌YUPP-10,通过平板对峙培养、平板对扣培养和悬滴法等测定YUPP-10对大丽轮枝菌(Verticillium dahliae)菌丝生长和孢子萌发的抑制效果;用YUPP-10无菌滤液培养大丽轮枝菌微菌核,检测其对微菌核萌发的影响;利用基质接种法进行盆栽试验探究其对棉花黄萎病的防治效果;通过胚根、叶片接种病原菌,木质化和活性氧爆发研究YUPP-10诱导抗病能力;采用实时荧光定量PCR(qRT-PCR)方法检测防御相关基因的表达。【结果】成功获取一株蜡状芽孢杆菌(Bacillus cereus)YUPP-10,其整体代谢产物在7 d时对大丽轮枝菌的抑菌带宽为0.73 cm,挥发性代谢产物在10 d时对大丽轮枝菌菌落的抑制率为77.03%;YUPP-10无菌培养液对大丽轮枝菌孢子和菌核萌发的抑制率分别为17.22%—71.25%和10.69%—26.62%,且抑制率与无菌滤液的浓度呈正相关。在用YUPP-10玉米蛭石培养物拌土处理的盆栽试验中,其对棉花黄萎病的最高防效达到80.60%。诱导抗性试验发现YUPP-10诱导了胚根和叶片抗大丽轮枝菌的侵染,诱导了细胞木质化和活性氧的爆发。qRT-PCR检测结果显示YUPP-10成功激活了苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)、多酚氧化酶(PPO)、几丁质酶(CHI)和病程相关基因(PR10)的表达,对大丽轮枝菌在棉花体内的扩散和生长起到了抑制作用。【结论】YUPP-10通过直接抑制病原菌的生长,同时诱导植物防御反应来抵抗病原菌的侵染和扩展,其具有良好的应用前景。

Biocontrol Effect and Mechanism of Cotton Endophytic Bacterium Bacillus cereus YUPP-10 Against Verticillium Wilt in Gossypium hirsutum

【Objective】Control of Verticillium wilt is a worldwide problem in agriculture and horticulture, screening of effective biocontrol microbial resources has become one of the important ways to control Verticillium wilt. The use of microbial antagonists to control these pathologies fits modern sustainable agriculture criteria. The objective of this study is to screen an efficient antagonistic bacteria, and characterize the biocontrol mechanism of bacteria against Verticillium wilt, thus providing a technical basis for control of Verticillium wilt of cotton with biocontrol bacteria. 【Method】An endophytic bacterium, which can hydrolyze polysaccharides with β-1,4 linkage, was isolated by enrichment medium, which was made of konjac glucomannan as carbon source. The inhibition and prevention of YUPP-10 against V. dahliae and Verticillium wilt were tested. The inhibition rate of YUPP-10 against V. dahliae was assessed using the confront culture method, enclosed chamber test and hanging drop method, cultured microsclerotia with aseptic culture filtrate, and the control effect of cottonon Verticillium wilt was detected by substrate inoculation method in a pot experiment. Induced disease resistance of YUPP-10 in cotton were analysed by embryo and leaves inoculated with V. dahliae, sedimentation of lignin and reactive oxygen species (ROS). In addition, the expression level of defense genes in G. hirsutum leaves were detected by real-time quantitative PCR (qRT-PCR). 【Result】A Bacillus cereus isolate YUPP-10 was screened. The results showed that YUPP-10 and its volatile organic compounds significantly inhibited the colony growth of V. dahliae, with the width of the inhibition zone of YUPP-10 was 0.73 cm after 7 days, and the inhibition rate of volatile organic compounds of YUPP-10 was 77.03% after 10 days. B. cereus YUPP-10 culture filtrate (CF) suppressed V. dahliae spore and microsclerotia germination in a dose-dependent manner. The inhibition rate of CF on V. dahliae spore and microsclerotia germination ranged from 17.22% to 71.25% and 10.69% to 26.62%, respectively. The inhibition rate was 80.60%, when cotton seedlings were pro-inoculated with substrate containing YUPP-10 in a pot experiment. YUPP-10 induced disease resistance, including immature embryo and leaves against V. dahliae, ROS, sedimentation of lignin and some defense genes, such as PAL, POD, PPO, CHI and PR10.【Conclusion】It was concluded that YUPP-10 is an efficient biocontrol agent that protects cotton plant from V. dahliae infection. Our data demonstrate that V. dahliae growth is restricted and the additional signals from YUPP-10 must participate in the regulation of the immune response against V. dahliae. Therefore, the YUPP-10 has a great potential in controlling Verticillium wilt.