ABSTRACT
Evaluation of multiple PCR techniques for rapid detection of Meloidogyne spp. directly from soil

Yupapon Boonrat1, Pornsuk Chaisuk1, On-uma Ruangwong1, Nuttapon Khongdee2, 3, Milan Chameera Samarakoon1*, and Nilita Mukjang3, 4, 5*
 
Meloidogyne spp. (root-knot nematodes; RKNs) are parasitic nematodes that cause significant damage to diverse economically important plants such as various herbaceous species. However, research focusing on direct soil-based detection remains limited research updates, as identifying these pathogens directly from complex soil matrices presents significant technical challenges. This study developed and optimized rapid, specific protocols for end-point polymerase chain reaction (PCR), multiplex PCR, and real-time PCR (qPCR) for the direct detection of M. incognita and M. javanica in soil. Nematodes from infected roots were identified morphologically, then spiked into soil for DNA extraction. Species-specific primers (Inc-K14-F/Inc-K14-R for M. incognita and Fjav/Rjav for M. javanica) were used for target detection, with PCR annealing optimized at 60-64 °C (30 s). In end-point PCR, the minimum of detection for M. incognita was 6 adult females g-1 soil and 8 second-stage juveniles (J2) g-1 soil. For M. javanica, the sensitivity was 4 adult females and 4 J2 g-1 soil. Furthermore, the multiplex PCR protocol developed in this study successfully identified both species in a single reaction, with a minimum of detection of 6 adult females individuals and 20 J2 g-1 soil for each species. Additionally, a qPCR assay was successfully developed using SYBR Green qPCR and conditions adapted from the multiplex PCR for M. javanica, achieving a minimum of detection of 4 adult females and 8 J2 g-1 soil. Overall, these molecular protocols provide rapid and accurate tools for nematode identification, helping to reduce crop yield losses.
Key words: Detection sensitivity, plant disease detection, plant protection, root-knot disease, soil-borne pathogen.
1Chiang Mai University, Faculty of Agriculture, Department of Entomology and Plant Pathology, Chiang Mai 50200, Thailand.
2Chiang Mai University, Faculty of Agriculture, Department of Highland Agriculture and Natural Resources, Chiang Mai 50200, Thailand.
3Chiang Mai University, Agrobiodiversity in Highland and Sustainable Utilization Research Group, Chiang Mai 50200, Thailand.
4Chiang Mai University, Faculty of Science, Department of Biology, Chiang Mai 50200, Thailand.
5Chiang Mai University, Faculty of Science, Center of Excellence in Microbial Diversity and Sustainable Utilization, Chiang Mai 50200, Thailand.
*Corresponding authors (nilita.m@cmu.ac.th; milanchameerasamar.s@cmu.ac.th)
Received: 22 May 2026; Accepted: 14 September 2026, Available online: 29 October 2026.