Multiplex Real-Time qPCR for the Detection of Heterodera schachtii and Heterodera trifolii in Kimchi cabbage Fields

Cyst nematodes, such as Heterodera schachtii (HS) and H. trifolii (HT), are major pathogens affecting kimchi cabbage production in Korea. Accurate species identification is essential for effective management, as these nematodes exhibit similar morphological features but differ in host range and pathogenicity. In this study, we developed a multiplex real-time TaqMan qPCR assay for the simultaneous detection of HS and HT. Species-specific primers and fluorescent probes targeting the coxI gene (HS) and a hypothetical protein gene (HT) were designed using MIG-seq-based polymorphism data. The assay demonstrated high amplification efficiency (HS: 89.3%, HT: 121.7%) and strong linearity (R² > 0.98) across serial DNA dilutions (1 ng to 100 fg). Specificity testing against 8 non-target plant-parasitic nematodes, including H. glycines, H. sojae, Meloidogyne incognita, and Pratylenchus penetrans, confirmed that no cross-reactivity occurred. Notably, the assay permits direct use of crushed cyst sap as a template, eliminating DNA purification. Detection was consistent from a single cyst, and reproducible across mixtures with varying ratios of HS and HT. The multiplex format reliably identified both species in co-infected samples without interference. This assay provides a rapid, sensitive, and field-adaptable molecular tool for the diagnosis of HS and HT, contributing to early detection and improved management of cyst nematodes in cabbage-growing regions.