Identification of Meloidogyne chitwoodi, M. fallax and M. hapla based on SCAR-PCR: a powerful way of enabling reliable identification of populations or individuals that share common traits

Abstract

This study describes the development of species-specific pairs of PCR primers for the root-knot nematodes Meloidogyne chitwoodi, M. fallax and M. hapla that amplify species-specific RAPD fragments. After sequencing the fragments, longer primers were designed to complement the terminal sequences of the polymorphic DNA fragments. The resulting pairs of primers were used to generate the sequence-characterized amplified regions (SCARs). Using the developed pairs of SCAR primers, SCAR fragments of M. chitwoodi, M. fallax or M. hapla were easily amplified from DNA extracts from juveniles, egg masses, females of the particular nematode species investigated, either present alone, in a mixture with other nematode species or in infested plant material. A specially designed multiplex assay using three pairs of SCAR primers enabled the identification of multiple species in a mixture in a single PCR step. Single juveniles were easily identified by applying this multiplex assay followed by a subsequent multiplex PCR using three pairs of nested primers. The SCAR-PCR-based assays described have potential to be optimized for routine practical diagnostic tests. The usefulness of converting RAPD markers into SCAR markers is discussed.