Natesan Karthi, Park Byeong-Yong, Ko Hyoung-Rai, Kim Eunhwa, Park Sohee, Park Sekeun
Crop Protection Division, National Institute of Agricultural Sciences, Rural Development Administration, Wanju 55365, Korea.
Plant Pathol J. 2025 Jun;41(3):330-340. doi: 10.5423/PPJ.OA.11.2024.0181. Epub 2025 Jun 1.
Pratylenchus penetrans, an important soil pathogen, has been reported on various crops in the temperate regions of South Korea. In concern, there is an urgent need for a precise, species-specific quantitative polymerase chain reaction (qPCR) kit to detect and quantify root lesion nematodes for early pest management and to controls yield losses. The present study focuses on D2-D3 region, a known marker for molecular profiling of Pratylenchus sp. A primer set mined from the highly conserved D2-D3 region of P. penetrans was used in a SYBR green based qPCR assay. Initial examination identified P. penetrans from infested soil samples using morphological and phylogenetic analyses. The DPp7F12R primer set demonstrated significant specificity in identifying P. penetrants by both conventional polymerase chain reaction (PCR) and qPCR assays. Linear regression of serially diluted DNA from nematode and nematode inoculated soil revealed a limit of quantification of 2 picograms (r² = 0.984), while also highlighting the impact of soil inhibitors. The qPCR using the DNA from varying densities of P. penetrans inoculated in soil demonstrated a robust correlation (r² = 0.98), indicating the limit of detection down to single nematode. Primer specificity evaluation with field soil sample precisely detected only P. penetrants. Species-specific DPp7F12R facilitate the direct detection of P. penetrants from soil DNA in very shorter time. Reliability of PCR was confirmed using BLAST algorithm, which identified partial sequence of PCR amplicon (300 bp) as P. penetrants. Finally, PCR assay using DPp7F12R is crucial for early detection of P. penetrans infestations, helping improve the plant health.
穿刺短体线虫是一种重要的土壤病原菌，在韩国温带地区的各种作物上均有报道。因此，迫切需要一种精确的、针对特定物种的定量聚合酶链反应（qPCR）试剂盒，用于检测和定量根腐线虫，以便进行早期害虫管理并控制产量损失。本研究聚焦于D2-D3区域，这是短体线虫属分子谱分析的已知标记。从穿刺短体线虫高度保守的D2-D3区域挖掘的一组引物用于基于SYBR Green的qPCR分析。通过形态学和系统发育分析，初步从受侵染的土壤样本中鉴定出穿刺短体线虫。DPp7F12R引物组在通过常规聚合酶链反应（PCR）和qPCR分析鉴定穿刺短体线虫时显示出显著的特异性。对来自线虫和接种线虫的土壤的系列稀释DNA进行线性回归分析，得出定量限为2皮克（r² = 0.984），同时也突出了土壤抑制剂的影响。使用接种在土壤中不同密度的穿刺短体线虫的DNA进行qPCR分析显示出很强的相关性（r² = 0.98），表明检测限低至单个线虫。用田间土壤样本进行引物特异性评估，仅精确检测到穿刺短体线虫。物种特异性的DPp7F12R有助于在非常短的时间内直接从土壤DNA中检测穿刺短体线虫。使用BLAST算法确认了PCR的可靠性，该算法将PCR扩增子的部分序列（300 bp）鉴定为穿刺短体线虫。最后，使用DPp7F12R的PCR分析对于早期检测穿刺短体线虫侵染至关重要，有助于改善植物健康状况。
