沙棘枝枯病的定量检测体系构建和应用  

Construction and application of a quantitative detection system for sea buckthorn stem wilt

作　　者：陈悦 孙思雨 胡建忠[2] 郝凯强 于曼[1] 吴元华[1] 夏博[1] CHEN Yue;SUN Siyu;HU Jianzhong;HAO Kaiqiang;YU Man;WU Yuanhua;XIA Bo(Shenyang Agricultural University,Shenyang 110866,Liaoning,China;Seabuckthorn Development and Management Center,Ministry of Water Resources,Beijing 100038,China)

机构地区：[1]沈阳农业大学,辽宁沈阳110866 [2]水利部沙棘开发管理中心,北京100038

出　　处：《经济林研究》2022年第1期258-264,273,共8页Non-wood Forest Research

基　　金：水利部沙棘开发管理中心沙棘病虫害研究专项(2019-zk-kj-012)。

摘　　要：【目的】找到一种灵敏度较高的定量检测沙棘枝干以及土壤中病原菌的方法,为沙棘枝枯病的早期诊断和防控提供参考。【方法】以17份沙棘枝条样本、4份沙棘根际土壤样本为材料,提取拟枝孢镰刀菌DNA,基于翻译延伸因子(EF-1α)设计特异性扩增拟枝孢镰刀菌的引物,检测PCR引物的特异性,检测拟枝孢镰刀菌实时荧光定量PCR的灵敏性,并采用实时荧光定量PCR技术检测沙棘枝条和根际土壤微生物DNA样品。【结果】建立了拟枝孢镰刀菌质粒检测标准曲线(R²=0.9849),使用该曲线得到的每微升DNA样本的检测拷贝数是10^(3)~10^(8)。拟枝孢镰刀菌实时荧光定量PCR检测结果表明,在17份枝条样品和4份土壤微生物DNA样品中,有7份样品出现荧光信号。【结论】与普通PCR扩增方法相比,建立的拟枝孢镰刀菌实时荧光定量PCR检测体系的检测结果更精确,其具有更强的实用性和可操作性,既可以用于定性检测病原菌,又可以用于定量检测沙棘枝干和土壤中病原菌密度。实时荧光定量PCR技术凭借其优异的特异性和灵敏性可以被广泛地应用于多种植物、空气和土壤中病原菌含量的测定。【Objective】Aiming to find a new method to specifically detect and quantitatively measure the pathogen in sea buckthorn branches and soil,which will be useful for the prevention and control of sea buckthorn stem wilt in earlier period.【Method】Using 17 branch samples and 4 rhizosphere soil samples in sea buckthorn as materials,a real-time fluorescence quantitative PCR technology was used to design specific amplification primers based on translation extension factor(EF-1α),and compared with traditional PCR methods,to optimize and establish a detection system for accurate detection of sea buckthorn stem wilt by specific primers design.【Result】The established standard curve for the detection of Fusarium sporotrichioides plasmids had a detection range of 10^(3)-10^(8) copies/μL.Using this method,21 branch samples and soil samples were tested,and fluorescence signals were found in 7 samples.【Conclusion】The real-time fluorescence quantitative PCR detection system established in this study is more accurate,practical and maneuverable than the ordinary PCR amplification technology.It has achieved a breakthrough in both the qualitative identification of pathogenic bacteria and the quantitative detection of pathogenic bacteria density in the sea buckthorn branches and soil,laying a foundation for the early diagnosis,prevention and control of sea buckthorn stem wilt.With its excellent specificity and sensitivity,real-time quantitative PCR can be widely used in the determination of pathogens in various plants,air and soil.

关 键 词：沙棘枝枯病 拟枝孢镰刀菌 实时荧光定量PCR 分子检测 

分 类 号：S665.9[农业科学—果树学]