Development of the duplex droplet digital PCR for quantitative monitoring of mixed infections of Meloidogyne incognita and M. enterolobii

Abstract BACKGROUND Root‐knot nematodes (RKN, Meloidogyne spp. ) are economically significant pests that cause immense damage to a wide range of crops. Among them, M. incognita and M. enterolobii are of particular concern, as their high virulence and broad host range. RKN are challenging for detection due to their subterranean lifestyle underground. Also, the mixed infection of nematodes in field crops complicates the need for more accurate diagnostic and quantification technologies. RESULTS To address this challenge, we developed and optimized a novel duplex droplet digital PCR (ddPCR) method, using primer/probe sets targeting M. incognita and M. enterolobii , to simultaneously identify and quantify both species within a single assay. The innovative ddPCR diagnostic demonstrated excellent performance in terms of sensitivity, precision and reproductivity when quantifying the eggs and soil samples containing juveniles of both species. Moreover, the application of the duplex ddPCR method enables the monitoring of population dynamics of M. incognita and M. enterolobii under competitive environmental conditions. Our results indicated that the reproduction factor of M. incognita possibly inhibited when in mixed populations of M. enterolobii . Conclusion In this study, we first applied duplex ddPCR technique for differentiating mixed infections of M. incognita and M. enterolobii , offering a valuable tool for species detection and quantification. It enables the monitoring of population dynamics for both species, which is crucial for providing theoretical guidance for the implementation of timely and effective control measures. © 2024 Society of Chemical Industry.