Specific probes efficiently distinguish root-knot nematode species using signature sequences in the ribosomal intergenic spacer

Molecular probes were designed to identify Meloidogyne species by hybridizing to unique signature sequences located within variable regions of the ribosomal intergenic spacer (IGS). IGS nucleotide sequences were obtained by sequencing the corresponding PCR-amplified DNA. Sequence alignments of the IGS from M. chitwoodi and M. fallax revealed several areas of localized dissimilarity to which species-specific PCR primers were synthesized. When used in combination with nonspecific (conserved) primers, these primer pairs produced species-specific PCR amplification products as revealed by agarose gel electrophoresis. Size separation of amplified products from a single PCR reaction utilizing a combination of five specific and non-specific primers was demonstrated to provide an accurate, single-test assay, without the need for restriction digests. Multiplex-PCR amplification of DNA from single juveniles or a small number of eggs efficiently distinguished M. chitwoodi and M. fallax from M. hapla, M. incognita, M. javanica, M. arenaria, and M. mayaguensis.