Development Of PCR Assays For Rapid Detection And Identification Of Three Major Root-knot Nematodes, Meloidogyne Incognita, M.Javanica And M.arenaria

Root-knot nematodes (Meloidogyne spp.) constitute a major group of pathogens of agricultural crops and have been listed as quarantine pests in many countries. Among the approximate 80 species of Meloidogyne described, M. incognita, M. javanica and M arenaria, which reproduce by mitotic parthenogenesis, are considered major species duo to their wide distribution and host range. To develop a rapid and sensitive method for the detection and identification of the three root-knot nematodes, random amplified polymophic DNA (RAPD) was employed to analyze the genomic differences among populations of M incognita, M. javanica, M. arenaria, M. hapla and M. enterolobii. Four RAPD fragments specific for M. incognita (OPF-01600, OPF-01800, OPG-11740 and OP26-011200), and three RAPD fragments specific for M. javanica (OPF-12600, OPG-18850 and OP26-1269o) were identified. These RAPD markers were subsequently cloned and sequenced. Based on the sequences, various sequence characterized amplified region (SCAR) primers were designed and tested for their specificity against 21 populations of M incognita, nine populations of M. javanica, eight populations of M arenaria, four populations of M. hapla and one population of M. enterolobii. Three primer pairs showed potential for routine rapid and accurate detection and identification of M. incognita, M. javanica and M arenaria in root and soil samples. Using primer set MI-779-F/MI-955-R, a fragment of 955 bp was specifically amplified from the M incognita populations; using primer set MJ-517-F/R, a product of 517 bp was specifically amplified from the M javanica populations; and with primer set MI-779-F/R a product of 799 bp was obtained specifically from the populations of M incognita, M. javanica and M. arenaria. The three primer sets can be combined in a multiplex PCR assay to amplify the species-specific markers from ca. 1 ng of nematode DNA, but not from a single juvenile. However, when the primer sets were used separately in standard PCR assays, the species-specific markers can be reliably produced from one third to one portion of a single juvenile, male and female. Successful development of SCAR-based PCR assays that can sensitively diagnose a single nematode of M. incognita, M. javanica and M. arenaria is reported for the first time. It isexpected that application of the assays will contribute to the management of root-knot nematode diseases in China through the proper use of crop resistance and the effective implementation of plant quarantine measures.