棉花内生细菌YUPP-10及其分泌蛋白CGTase对棉花枯萎病的防治作用及机理  

Biocontrol Effect and Mechanism of Cotton Endophytic Bacterium YUPP-10 and Its Secretory Protein CGTase Against Fusarium Wilt in Cotton

作　　者：周京龙[1,2] 冯自力 魏锋 赵丽红 张亚林 周燚 冯鸿杰[2] 朱荷琴[2] ZHOU JingLong;FENG ZiLi;WEI Feng;ZHAO LiHong;ZHANG YaLin;ZHOU Yi;FENG HongJie;ZHU HeQin(College of Agriculture,Yangtze University,Jingzhou 434025,Hubei;Institute of Cotton Research,Chinese Academy of Agricultural Sciences/State Key Laboratory of Cotton Biology,Anyang 455000,Henan)

机构地区：[1]长江大学农学院,湖北荆州434025 [2]中国农业科学院棉花研究所/棉花生物学国家重点实验室,河南安阳455000

出　　处：《中国农业科学》2021年第17期3691-3701,共11页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31901938);中央级公益性科研院所基本科研业务费专项(1610162021031);中国农业科学院科技创新工程。

摘　　要：【目的】棉花枯萎病(Fusarium wilt)是棉花种植中较为严重的土传病害之一,主要采用化学方法进行防治,但对环境和人畜安全造成一定影响。生物防治因其专一性强、安全性高的特点,成为防治棉花枯萎病的重要途径。本研究旨在获得一株高效拮抗细菌,并明确其防治机理,为棉花枯萎病生物防治提供技术支持。【方法】前期获得一株能够破坏β-1,4糖苷键的棉花内生蜡状芽孢杆菌(Bacillus cereus)YUPP-10,通过平板对峙培养、共培养法和悬滴法分别测试其对棉花枯萎病菌(尖镰孢,Fusarium oxysporum)菌丝生长、产孢和孢子萌发的影响;利用YUPP-10菌液浸种处理,研究其对棉花生物量的影响;以尖镰孢菌土为基质种植棉花,生长1周后,用LB液体培养基培养的YUPP-10制成不同浓度(分别为1×10^(8)、1×10^(7)和1×10^(6) cfu/mL)的生防菌剂灌根处理棉苗,于温室中进行棉花枯萎病的防治效果研究;通过Fosmid文库筛选到了YUPP-10关键抑菌物质为环糊精糖基转移酶(CGTase,EC 2.4.1.19),研究其对尖镰孢菌丝生长、产孢和孢子萌发的影响;利用拟南芥花序侵染法将其转化拟南芥,获得能够稳定表达CGTase的转基因株系,研究转基因拟南芥对枯萎病的抗性,以及在病原胁迫下部分防御基因的转录。【结果】YUPP-10菌株发酵液对尖镰孢菌丝生长、产孢和孢子萌发具有显著的抑制作用,随着发酵液浓度的升高,对尖镰孢产孢量和孢子萌发的抑制率越高,最高抑制率分别为98.41%和51.65%。低浓度的YUPP-10菌液能促进棉花种子发芽、出苗和地上部分的生长。YUPP-10发酵液灌根处理后,棉苗的病情指数和病株率显著降低,其中,1×10^(7) cfu/mL的YUPP-10发酵液对棉花枯萎病的防治效果最高,达到45.11%。YUPP-10菌株的关键抑菌物质CGTase能够水解羧甲基纤维素和葡甘聚糖,表明其具有破坏β-1,4糖苷键的能力,进而检测了其对尖镰孢的抑制效果,�【Objective】Fusarium wilt is one of the most important soil-borne diseases in cotton planting.Chemical methods are mainly used to control this disease,however,it has certain impact on the environment,human and animal safety.Biological control has become an important way to control cotton Fusarium wilt because of its high specificity and safety.The objective of this study is to screen an efficient antagonistic bacteria,and characterize the biocontrol mechanism of bacteria against Fusarium wilt,thus providing a technical basis for cotton Fusarium wilt control with biocontrol bacteria.【Method】In a previous study,an endophytic bacterium Bacillus cereus YUPP-10 was isolated from vascular of cotton,which can hydrolyze polysaccharides withβ-1,4 linkage.The effects of YUPP-10 on hyphal growth,sporulation,and spore germination of F.oxysporum were tested using the confront culture method,enclosed chamber test and hanging drop method,respectively.The seeds were soaked by YUPP-10,and then,the seeding germination and the biomass of cotton were detected.The cotton were cultivated in substrate with F.oxysporum,and after a week of growth,YUPP-10 cultured with LB liquid medium was treated at different concentrations(1×10^(8),1×10^(7)and 1×10^(6) cfu/mL,respectively),and the control efficacy against Fusarium wilt was studied in the greenhouse.The key antibacterial substances of YUPP-10 were obtained by Fosmid library,and the direct effects of recombinant cyclodextrin glycosyltransferase(CGTase)on F.oxysporum hyphal growth,sporulation,and spore germination were studied.The overexpression vector was transformed into Arabidopsis thaliana Col-0 via the floral dip method.The resistance of CGTase-overexpressing transgenic plants against Fusarium wilt was assessed with an in vitro technique.The transcriptional levels of some defense genes were analyzed under pathogen challenge.【Result】YUPP-10 significantly inhibited the hyphal growth,sporulation,and spore germination of F.oxysporum,the most inhibition rate of spore yield a

关 键 词：蜡状芽孢杆菌 棉花枯萎病 尖镰孢萎蔫专化型病菌 环糊精糖基转移酶 防治效果 

分 类 号：S435.621[农业科学—农业昆虫与害虫防治]