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大豆根腐病致病镰孢菌的多重PCR检测技术

引用本文：何宛芹,付瑶,鲁雯璐,常小丽,杨文钰.大豆根腐病致病镰孢菌的多重PCR检测技术.植物保护学报,2017,44(4):609-616

DOI：10.13802/j.cnki.zwbhxb.2017.2016234

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作者	单位	E-mail
何宛芹	四川农业大学农学院, 四川省作物带状复合种植工程技术研究中心, 成都 611130
付瑶	四川农业大学农学院, 四川省作物带状复合种植工程技术研究中心, 成都 611130
鲁雯璐	四川农业大学农学院, 四川省作物带状复合种植工程技术研究中心, 成都 611130
常小丽	四川农业大学农学院, 四川省作物带状复合种植工程技术研究中心, 成都 611130	xl_changkit@126.com
杨文钰	四川农业大学农学院, 四川省作物带状复合种植工程技术研究中心, 成都 611130	mssiyangwy@sicau.edu.cn

中文摘要:为建立大豆根腐病镰孢菌的多重PCR检测方法，以四川大豆根腐病致病菌包括尖孢镰孢菌、腐皮镰孢菌、禾谷镰刀菌和木贼镰孢菌为对象，设计镰孢菌翻译延伸因子基因EF-1α的种特异引物，建立多重PCR扩增体系，并进行优化与验证。结果表明：25 μL体系为最优镰孢菌多重PCR扩增体系，4种镰孢菌等体积混合DNA 4.0 μL，各镰孢菌特异正向引物1.0 μL，共用反向引物4.0 μL，最佳退火温度为54℃，当循环30次时，能清晰地扩增出各镰孢菌EF-1α条带，对4种镰孢菌混合DNA的检测灵敏度可达0.1 ng/μL。室内环境样本验证结果表明，依据EF-1α扩增片段大小，该体系能够特异地检测出大豆黄化苗与致病镰孢菌混合样本中的镰孢菌，但无法从其它真菌的DNA中扩增获得目的片段。表明基于EF-1α基因特异引物建立的镰孢菌多重PCR检测技术可快速、特异地检测大豆根腐病镰孢菌。

中文关键词:大豆根腐病镰孢菌多重 PCR 翻译延伸因子基因病原菌鉴定

A multiplex PCR detection technique for the pathogenic Fusarium species causing soybean root rot

Author Name	Affiliation	E-mail
He Wanqin	Sichuan Engineering Research Center for Crop Strip Intercropping System, College of Agriculture, Sichuan Agricultural University, Chengdu 611130, Sichuan Province, China
Fu Yao	Sichuan Engineering Research Center for Crop Strip Intercropping System, College of Agriculture, Sichuan Agricultural University, Chengdu 611130, Sichuan Province, China
Lu Wenlu	Sichuan Engineering Research Center for Crop Strip Intercropping System, College of Agriculture, Sichuan Agricultural University, Chengdu 611130, Sichuan Province, China
Chang Xiaoli	Sichuan Engineering Research Center for Crop Strip Intercropping System, College of Agriculture, Sichuan Agricultural University, Chengdu 611130, Sichuan Province, China	xl_changkit@126.com
Yang Wenyu	Sichuan Engineering Research Center for Crop Strip Intercropping System, College of Agriculture, Sichuan Agricultural University, Chengdu 611130, Sichuan Province, China	mssiyangwy@sicau.edu.cn

Abstract:To establish a multiplex PCR detection technique for Fusarium species causing soybean root rot, the four major pathogenic Fusarium species, Fusarium oxysporum, F. solani, F. equiseti and F. graminearum, in southwest China were selected. Based on the sequence of the transcription elongation factor gene EF-1α, the Fusarium species-specific primers were designed, and the multiplex PCR were established. The reaction and amplification parameters were optimized, and the sensitivity and specificity were verified. The results showed that all specific EF-1α gene fragments of four Fusarium species were amplified by the multiplex PCR in a total volume of 25 μL with 4.0 μL mixed DNA, 1.0 μL specific forward primer for each Fusarium species, and 4.0 μL common reverse primer at an annealing temperature of 54℃ and 30 cycles. The multiplex PCR system was capable of detecting the mixed DNA with a concentration 0.1 ng/μL. The pathogenic Fusarium species was successfully tested from the mixed samples of soybean etiolated seedlings with Fusarium species by using the optimized multiple PCR according to fragment size of EF-1α gene, whereas this gene was not amplified from DNA samples of other tested fungi. The study indicated that the multiplex PCR technique based on specific EF-1α gene primer pairs of four species of Fusarium could be used for the rapid and precise detection of the pathogenic Fusarium species causing soybean root rot.

keywords:soybean root rot Fusarium species multiplex PCR EF-1α pathogen identification

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