生防菌NCD-2菌株定量检测体系的建立及其在棉花根际定植检测中的应用  

Development of quantitative detection system for biocontrol strain NCD-2 and its application in rhizosphere colonization of cotton

作　　者：苏星 苏振贺 宣立锋[3] 李社增[2] 王培培[2] 郭庆港[2] 马平[2] Su Xing;Su Zhenhe;Xuan Lifeng;Li Shezeng;Wang Peipei;Guo Qinggang;Ma Ping(College of Plant Protection,Agricultural University of Hebei,Baoding,Hebei 071000,China;Institute of Plant Protection,Hebei Academy of Agriculture and Forestry Sciences/Integrated Pest Management Center of Hebei Province/Key Laboratory of IPM on Crops in Northern Region of North China,Ministry of Agriculture and Rural Affairs,Baoding,Hebei 071000,China;Shijiazhuang Institute ofFruit Trees,Hebei Academy of Agriculture and Forestry Sciences,Shijiazh uang 050061,China)

机构地区：[1]河北农业大学植物保护学院,河北保定071000 [2]河北省农林科学院植物保护研究所/河北省农业有害生物综合治理中心/农业农村部华北北部作物有害生物综合治理重点实验室,河北保定071000 [3]河北省农林科学院石家庄果树研究所,石家庄050061

出　　处：《棉花学报》2022年第2期162-172,共11页Cotton Science

基　　金：国家现代农业产业技术体系——棉花产业技术体系(CARS-15-19);河北省重点研发计划(19226510D);国家自然科学基金(32172487);河北省农林科学院现代农业科技创新工程(2022KJCXZX-ZBS-1)。

摘　　要：【目的】建立生防枯草芽孢杆菌NCD-2菌株的定量检测体系,明确生防菌在棉花根际土壤中的定植情况。【方法】根据NCD-2菌株全基因组序列设计特异性引物;利用聚合酶链式反应(polymerase chain reaction,PCR)技术,构建NCD-2菌株的实时PCR检测体系,并检测NCD-2菌株在棉花根际的定植情况。【结果】所构建的NCD-2菌株实时PCR检测体系能够特异性地定量检测土壤中NCD-2菌株的数量。用有效活菌数10^(9)mL^(-1)的NCD-2菌液处理棉种,在灭菌土中播种后8 d和16 d,用实时PCR检测其在棉花根际土壤中的定植数量分别为1.14×10^(5) g^(-1)和9.5 ×10^(4) g^(-1),与传统计数法检测的结果(2.15 ×10^(5) g^(-1)和2.45 ×10^(5 )g^(-1))高度相关,2种方法结果的相关系数在播种后8 d时为0.99,在播种后16 d时为0.95。而将NCD-2菌株处理后的棉种播种于含有立枯丝核菌的土壤中后16 d,用实时PCR检测其在根际土壤中的定植数量为7.6×10^(5) g^(-1),此时NCD-2菌株对棉花立枯病防治效果达到67.9%。【结论】所构建的实时PCR检测体系能够准确检测NCD-2菌株在根际土壤中的定植情况,为高效使用NCD-2菌株防控病害奠定了基础。[Objective] A quantitative detection technique for biocontrol Bacillus subtilis NCD-2 was developed and used to evaluate the colonization of strain NCD-2 in cotton rhizosphere. [Method] Specific primers for strain NCD-2 were designed based on the whole genome sequences of strain NCD-2. The colonization dynamics of strain NCD-2 in cotton rhizosphere soil were quantified by real-time polymerase chain reaction (PCR). [Result] The real-time PCR detection technique for strain NCD-2was successfully developed. Real-time PCR detection result showed that the population contents of strain NCD-2 in cotton rhizosphere soil were 1.14×10^(5) g^(-1)and 9.5×10^(4) g^(-1)on the 8;day and 16;day after sowing, respectively. Comparatively, 2.15×10^(5) g^(-1)and 2.45×10^(5) g^(-1)of strain NCD-2 in soil at the 8;day and the 16;day after sowing were obtained by traditional colony counting method. The correlation coefficients between the two methods were 0.99 at the 8;day and 0.95 at the 16;day. The control effect of NCD-2 strains against cotton rhizoctonia damping-off was 67.9% at 16 days after seed treatment. At that time the population content of strain NCD-2 was 7.6×10^(5) g^(-1)in soil with real-time PCR detection. [Conclusion] The established real-time PCR detection technique in this study can accurately reflect the colonization of NCD-2 strain in cotton rhizosphere, and lays a foundation for using the strain to effectively control diseases.

关 键 词：枯草芽孢杆菌 定量检测 根际 实时聚合酶链式反应 棉花 

分 类 号：S435.621.2[农业科学—农业昆虫与害虫防治] S476.1[农业科学—植物保护]