Kontz Brian, Adhikari Sajag, Subramanian Senthil, Mathew Febina M
Department of Plant Science, South Dakota State University, Brookings 57007.
Plant Dis. 2016 Aug;100(8):1669-1676. doi: 10.1094/PDIS-10-15-1204-RE. Epub 2016 May 9.
Diaporthe caulivora and D. longicolla are the causal agents of stem canker of soybean (Glycine max L.). Accurate identification of stem canker pathogens upon isolation from infected soybean plants is difficult and unreliable based on morphology. In this study, two TaqMan probe-based quantitative polymerase chain reaction (qPCR) assays were optimized for detection of D. caulivora and D. longicolla in soybean plants. The assays used previously reported D. caulivora-specific (DPC-3) and D. longicolla-specific (PL-3) probe/primer sets. The sensitivity limit of the two assays was determined to be over a range of 100 pg to 10 fg of pure D. caulivora and D. longicolla genomic DNA. The qPCR assays were validated with plant samples collected from commercial soybean fields. The PL-3 set detected D. longicolla in soybean plants collected from the fields (quantification cycle value <35), which was confirmed by isolation on potato dextrose agar (PDA). D. caulivora was detected only in low levels (quantification cycle value <40) by DPC-3 set in a few of the symptomatic field samples, although the pathogen was not isolated on PDA. The qPCR assays were also useful in quantitatively phenotyping soybean plants for resistance to D. caulivora and D. longicolla under greenhouse conditions.
菜豆间座壳菌和长柄间座壳菌是大豆（Glycine max L.）茎溃疡病的病原体。从受感染的大豆植株中分离出茎溃疡病病原体后，基于形态学进行准确鉴定既困难又不可靠。在本研究中，优化了两种基于TaqMan探针的定量聚合酶链反应（qPCR）检测方法，用于检测大豆植株中的菜豆间座壳菌和长柄间座壳菌。这些检测方法使用了先前报道的菜豆间座壳菌特异性（DPC-3）和长柄间座壳菌特异性（PL-3）探针/引物组。确定这两种检测方法的灵敏度极限为纯菜豆间座壳菌和长柄间座壳菌基因组DNA的100 pg至10 fg范围。qPCR检测方法通过从商业大豆田采集的植物样本进行了验证。PL-3引物组在从田间采集的大豆植株中检测到了长柄间座壳菌（定量循环值<35），通过在马铃薯葡萄糖琼脂（PDA）上分离得到了证实。在少数有症状的田间样本中，DPC-3引物组仅检测到低水平的菜豆间座壳菌（定量循环值<40），尽管该病原体未在PDA上分离出来。qPCR检测方法在温室条件下对大豆植株抗菜豆间座壳菌和长柄间座壳菌的定量表型分析中也很有用。
