Plant-parasitic nematode diversity in Spanish pea fields with species-specific qPCR-based identification of <i>Pratylenchus thornei</i> and <i>Pratylenchus mediterraneus</i>

Pea (Pisum sativum L.) is a cool-season annual legume crop cultivated globally. It is increasingly integrated into cereal-based rotations across Mediterranean agroecosystems, raising concerns about plant-parasitic nematodes (PPNs) that affect both legumes and subsequent crops. This study establishes the prevalence, diversity, and abundance of PPNs in a novel cropping system in southern Spain, where pea is introduced into cereal rotations. Nematode community structure in the surveyed fields is strongly influenced by intensive tillage practices and the desiccation tolerance of certain species, which facilitates their persistence through the dry summer season. The dominant PPNs affecting pea crops are Pratylenchus spp., with P. mediterraneus frequently occurring in mixed infections alongside closely related species such as P. thornei, P. neglectus, and P. crenatus. Given the absence of molecular tools specifically designed for P. mediterraneus, we developed a species-specific qPCR assay targeting P. mediterraneus and its close relative P. thornei. The newly developed qPCR assay was validated for specificity and sensitivity, and successfully applied to nematode communities extracted from pea root tissues. This molecular tool enables precise detection and quantification of P. mediterraneus and P. thornei, even in complex mixtures, offering a robust resource for nematode diagnostics and integrated management in legume-based cropping systems.