PCR-based assays to detect and quantify Phomopsis sclerotioides in plants and soil
PCR-based assays to detect and quantify Phomopsis sclerotioides in plants and soil
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DOI：
10.1007/s10327-009-0209-6
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发表时间：
2010-02
影响因子：
1.2
通讯作者：
M. Shishido;Kyoko Sato;Nanako Yoshida;Rie Tsukui;T. Usami
中科院分区：
文献类型：
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作者：
M. Shishido;Kyoko Sato;Nanako Yoshida;Rie Tsukui;T. Usami
我们开发了聚合酶链反应 (PCR) 检测方法来检测和定量拟核茎点菌（葫芦黑根腐病的致病因子）。我们使用来自代表性分离株的核糖体 DNA (rDNA) 的内转录间隔区 1 和 2 来搜索目标序列。在对包括 13Ph 在内的 40 种真菌分离株进行测试后选择了引物对。菌核分离株，除Ph.以外的9种拟茎点茎分离株。菌核，以及 18 种对葫芦科植物致病或非致病的土传真菌。使用 CPs-1（正向）和 CPs-2（反向）引物对的常规 PCR 测定可产生来自 allPh 的目标 DNA 扩增子。菌核分离株，但没有测试其他分离株。从自然感染黄瓜和甜瓜黑根腐病的田地采集的土壤和根部样本中，CPs-1/CPs-2 引物对在常规 PCR 检测中成功扩增了目标 DNA 片段。此外，我们将 CPs-1/CPs-2 引物对应用于 SYBR Green I 的实时 PCR 测定，并通过参考添加已知量的目标 DNA 生成的标准曲线成功地对 PCR 扩增产物进行定量。目标Ph.在人工接种的黄瓜、甜瓜、南瓜和西瓜的根中也同样检测到了菌核 DNA 片段，但数量较多。寄主下胚轴中的菌核DNA变化如下：甜瓜≥黄瓜≥西瓜>南瓜。这些结果表明Ph。菌核感染不具有物种特异性，但不同宿主物种的感染率可能不同。
We developed polymerase chain reaction (PCR) assays to detect and quantifyPhomopsis sclerotioides, the causal agent of black root rot of cucurbits. We used internal transcribed spacers 1 and 2 of the ribosomal DNA (rDNA) from representative isolates to search for target sequences. Primer pairs were selected after testing against 40 fungal isolates including 13Ph. sclerotioidesisolates, 9Phomopsisisolates other thanPh. sclerotioides,and 18 soilborne fungi that were either pathogenic or nonpathogenic to cucurbits. Conventional PCR assays with the primer pair of CPs-1 (forward) and CPs-2 (reverse) produced target DNA amplicons from allPh. sclerotioidesisolates but none of the other isolates tested. From soil and root samples collected from fields naturally infested with black root rot of cucumber and melon, the CPs-1/CPs-2 primer pair successfully amplified target DNA fragments in conventional PCR assays. Moreover, we applied the CPs-1/CPs-2 primer pair in a real-time PCR assay with SYBR Green I, and PCR-amplified products were successfully quantified by reference to a standard curve generated by adding known amounts of target DNA. TargetPh. sclerotioidesDNA fragments were similarly detected in artificially inoculated roots of cucumber, melon, pumpkin, and watermelon, but quantities ofPh. sclerotioidesDNA in their hypocotyls of the hosts varied as follows: melon ≥ cucumber ≥ watermelon > pumpkin. These results suggest thatPh. sclerotioidesinfection is not species-specific but the rate of infection may differ among host species.