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草业学报 ›› 2013, Vol. 22 ›› Issue (5): 136-144.DOI: 10.11686/cyxb20130516

• 研究论文 • 上一篇    下一篇

发生马铃薯立枯病土壤中立枯丝核菌的荧光定量PCR快速检测

李瑞琴1,2,刘星1,3,4,邱慧珍1,3,4*,张文明1,3,4,张春红1,3,4,王蒂3,4,5*,张俊莲3,4,5,沈其荣6   

  1. 1.甘肃农业大学资源与环境学院,甘肃 兰州 730070;
    2.甘肃省农业科学院农业质量标准与检测技术研究所,甘肃 兰州 730070;
    3.甘肃省
    干旱生境作物学重点实验室,甘肃 兰州 730070;
    4.甘肃省作物遗传改良与种质创新重点实验室,甘肃 兰州 730070;
    5.甘肃农业大学农学院,甘肃 兰州 730070;
    6.南京农业大学资源与环境学院,江苏 南京 210095
  • 出版日期:2013-10-20 发布日期:2013-10-20
  • 通讯作者: E-mail:hzqiu@gsau.edu.cn,wangd@gsau.edu.cn
  • 作者简介:李瑞琴(1969-),女,甘肃庆阳人,在读博士。
  • 基金资助:
    农业部公益性行业(农业)科研专项(201103004),甘肃省科技重大专项(1102NKDM025)和国家科技支撑计划(2012BAD06B03)资助。

Rapid detection of Rhizoctonia in rhizosphere soil of potato using real-time quantitative PCR

LI Rui-qin1,2, LIU Xing1,3,4, QIU Hui-zhen1,3,4, ZHANG Wen-ming1,3,4, ZHANG Chun-hong1,3,4, WANG Di3,4,5, ZHANG Jun-lian3,4,5, SHEN Qi-rong6   

  1. 1.College of Resources and Environmental Sciences, Gansu Agricultural University, Lanzhou 730070, China;
    2.Gansu Academy of Agricultural Sciences, Institute of Agricultural Quality Standard and Testing Technology, Lanzhou 730070, China;
    3.Gansu Province Key Laboratory of Arid-land Crop Science, Lanzhou 730070, China;
    4.Gansu Province Key Laboratory of Crop Genetic & Germplasm Enhancement, Lanzhou 730070, China;
    5.College of Agronomy, Gansu Agricultural University, Lanzhou 730070, China;
    6.College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, China
  • Online:2013-10-20 Published:2013-10-20

摘要: 连作马铃薯根际土壤中立枯丝核菌的大量累积可能是导致马铃薯连作障碍发生的主要原因之一。为了解根际土壤中土传病害病原菌的积累与连作障碍之间的关系,进一步寻求缓解和克服马铃薯连作障碍土传病害的有效途径,本研究建立优化了荧光定量PCR(real-time PCR)方法,对引起马铃薯立枯病的病原菌立枯丝核菌进行了快速监测和绝对定量,对马铃薯连作1~5年根际土壤立枯丝核菌的动态变化趋势进行了检测分析。结果显示,研究建立优化的荧光定量PCR检测方法,可直接应用土壤DNA进行病原菌的定量检测,能检测到土壤中浓度为1×102个拷贝/g土的马铃薯立枯丝核菌,扩增效率为1.04,具有检出限低、扩增效率高的特点,实现了不通过病土分离培养方法,就可掌握病原菌在根际土壤中的累积状况;在马铃薯立枯病发病严重的连作根际土壤中,立枯丝核菌的累积数量随连作年限的递增呈上升趋势;病原菌的累积随生育进程的推进呈下降趋势;病原菌累积量最大的是连作5年的播前土壤,每g土壤达3.75×107个拷贝数,由此可见,连作导致了土壤微生物种群结构发生改变,土壤致病真菌数量增加,相应地也增加了马铃薯立枯病的初侵染机率。

Abstract: A leading obstacle to continuous cropping of potatoes is Rhizoctonia solani in the rhizosphere. An understanding of the relationship between potato soil-borne pathogen accumulation and continuous cropping was sought to overcome the obstacles to continuous cropping of soil borne diseases. Synergy Brands (SYBR) Green I Dye (SGI) real time fluorescent quantitative Polymerase Chain Reaction (Rt-PCR) was optimised to perform quick checks and quantification of R. solani in soils with potato damping off disease. The dynamic changes of R. solani in the rhizosphere soil of continuously cropped potato was investigated. Results show that, the methods can be directly applied to soil pathogens with quantitative detection of DNA, which can be detected to a minimum detection level of 1×102 copies/g in potato soil concentrations of R. solani. Amplification efficiency is high (1.04) with a low detection limit. The cumulative status of pathogenic bacteria in the rhizosphere soil can be readily assessed without isolation and culturing. With increasing years of continuous potato cropping, the numbers of R. solani in rhizosphere soil had an upward trend with the increasing year of potato’s continuous cropping, by the growth process, the numbers of rhizoctonia solani in rhizosphere soil were downward trend. The largest amount of pathogens accumulated is the pre-seeding period of five years, that is 3.75×107 copies/g. This shows that continuous cropping led to a soil microbial population structure that increased the amount of change of soil pathogenic fungi. The infection probability of Rhizoctonia solani in early potato blight increased.

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