Sensitive and Direct Detection of Heterodera filipjevi in Soil and Wheat Roots by Species-Specific SCAR-PCR Assays.
Sensitive and Direct Detection of Heterodera filipjevi in Soil and Wheat Roots by Species-Specific SCAR-PCR Assays.
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DOI：
10.1094/pdis-02-13-0132-re
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发表时间：
2013-09
期刊：
影响因子：
4.5
通讯作者：
Huan Peng;Xiaoli Qi;D. Peng;H. Long;Xu-feng He;Wen-Kun Huang;Wen-ting He
中科院分区：
文献类型：
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作者：
Huan Peng;Xiaoli Qi;D. Peng;H. Long;Xu-feng He;Wen-Kun Huang;Wen-ting He
谷物囊线虫是世界小麦产区谷物作物上最重要的植物寄生线虫。2010年，filipjevi在中国首次被报道。在本研究中，建立了一种基于种特异性序列特征的扩增区域聚合酶链反应（SCAR-PCR）检测和鉴定小麦根和土壤中filipjevi的方法。根据随机引物OPK16扩增的RAPD标记设计物种特异性引物。生成了一个646 bp的特异性片段，该片段表征了H. filipjevi的扩增区域。PCR检测限低至0.125 μl二期幼鱼（J2）裂解液、3.9 × 10-3 μl成年雌虫裂解液和10-3 μl包囊裂解液。该方法可在0.5 g土壤中检测到不同阶段（J2、J3、J4和雌虫）和单一线虫。该方法在6个野外样本中检测到2个菲利普耶夫氏蜱，其中1个样本中有菲利普耶夫氏蜱和维纳氏蜱的混合种群。本研究首次为小麦根系和土壤中的H. filipjevi提供了明确的诊断分析。
Cereal cyst nematodes are the most important plant-parasitic nematodes on cereal crops in wheat producing areas of the world. Heterodera filipjevi was first reported in China in 2010. In this study, species-specific sequence characterized amplified region-polymerase chain reaction (SCAR-PCR) assays for detection and identification of H. filipjevi from infected wheat roots and soil were developed. The species-specific primers were designed according to the randomly amplified polymorphic DNA (RAPD) markers amplified with random primer OPK16. A 646-bp specific fragment of sequence was generated, which characterized amplified regions in H. filipjevi. The detection limitation of the PCR assay was as low as 0.125 μl second-stage juvenile (J2) lysate, 3.9 × 10-3 μl adult female lysate, and 10-3 μl cyst lysate. The method was able to detect the various stages (J2, J3, J4, and female) of H. filipjevi, and a single of nematode in 0.5 g of soil. H. filipjevi was detected by the method in two of six field samples, and one of those samples contained a mixed population of H. filipjevi and H. avenae. This study is the first to provide a definitive diagnostic assay for H. filipjevi in wheat roots and soil.