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棉花学报 ›› 2022, Vol. 34 ›› Issue (2): 162-172.doi: 10.11963/cs20210062

• 研究与进展 • 上一篇    

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

苏星1,2(),苏振贺2,宣立锋3,李社增2,王培培2,郭庆港2,马平2,*()   

  1. 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
  • 收稿日期:2021-10-28 出版日期:2022-03-15 发布日期:2022-05-30
  • 通讯作者: 马平 E-mail:pingma88@126.com
  • 作者简介:苏星(1999―),女,硕士, suxing66882021@163.com
  • 基金资助:
    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)

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

Su Xing1, 2(), Su Zhenhe2, Xuan Lifeng3, Li Shezeng2, Wang Peipei2, Guo Qinggang2, Ma Ping2, *()   

  1. 1. College of Plant Protection, Agricultural University of Hebei, Baoding, Hebei 071000, China
    2. 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
    3. Shijiazhuang Institute of Fruit Trees, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang 050061, China
  • Received:2021-10-28 Online:2022-03-15 Published:2022-05-30
  • Contact: Ma Ping E-mail:pingma88@126.com

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109 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1.14×105 g-1和9.5×104 g-1,与传统计数法检测的结果(2.15×105 g-1和2.45×105 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7.6×105 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。

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

Abstract:

[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-2 was successfully developed. Real-time PCR detection result showed that the population contents of strain NCD-2 in cotton rhizosphere soil were 1.14×105 g-1 and 9.5×104 g-1 on the 8th day and 16th day after sowing, respectively. Comparatively, 2.15×105 g-1 and 2.45×105 g-1 of strain NCD-2 in soil at the 8th day and the 16th day after sowing were obtained by traditional colony counting method. The correlation coefficients between the two methods were 0.99 at the 8th day and 0.95 at the 16th 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 7.6×105 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.

Key words: Bacillus subtilis; quantification; rhizosphere; real-time polymerase chain reaction; cotton