用SSR标记和巢式PCR快速检测大豆疫霉菌

Rapid Detection of Phytophthora sojae Using SSR Marker and Nested PCR

【目的】建立快速、准确的大豆疫霉菌检测和病害早期诊断分子技术。【方法】基于大豆疫霉菌SSR标记Psc239设计两对引物Psc239EF/Psc239ER和Psc239F/Psc239R,建立特异性检测大豆疫霉菌的巢式PCR方法。【结果】用36个大豆疫霉菌分离物和25个其他卵菌和真菌分离物基因组DNA评价引物及巢式PCR的专化性,引物对Psc239EF/Psc239ER、Psc239F/Psc239R和巢式PCR反应只在大豆疫霉菌中分别扩增出519、242和242bp的特异片段,在其它卵菌和病原真菌中不扩增片段;用两对引物进行常规PCR扩增,检测大豆疫霉菌DNA的灵敏度均为50pg·μl-1,而巢式PCR的灵敏度为50fg·μl-1;巢式PCR检测土壤中卵孢子的灵敏度为每克土壤0.4个卵孢子;巢式PCR能够特异地从大豆病株和土壤中检测到大豆疫霉菌。【结论】基于SSR标记建立的巢式PCR方法能够用于大豆疫霉菌的快速检测和病害诊断。

[Objective] This study was aimed at developing a rapid and accurate molecular technique for detection of Phytophthora sojae and diagnosis of soybean Phytophthora root rot at early stage. [Method] Based on the SSR marker Psc239 of P. sojae, two primer pairs, Psc239EF/Psc239ER and Psc239F/Psc239R, were designed specifically to amplify DNA from P. sojae by nested PCR. [Result] The primer pairs and nested PCR assay were assessed for specificity using DNA from 36 isolates ofP. sojae and 25 isolates of other oomycetes and fungi, Psc239EF/Psc239ER, Psc239F/Psc239R and nested PCR could amplify 519 bp, 242 bp and 242 bp fragments only from P. sojae, respectively, no amplification product was observed from other oomycetes and fungi. In conventional PCR, the sensitivity of detection was of 50 pg·μl-^1 P. sojae DNA for both primer pairs, in the nested PCR the detection limit was 50 f g·μl-^1. The detection limit for oospores of P. sojae in soil was of 0.4 oospores in 1 g soils by the nested PCR. The nested PCR assay was also used to detect DNA extracted from infected soybean tissues and soil samples. [ Conclusion ] The nested PCR assay based on SSR marker Psc239 could be used to rapidly detect P. sojae in soil and diagnose Phytophthora root rot on soybean.