Diagnostics of the Peach Root-Knot Nematode, Meloidogyne Floridensis Using Multiplex Real-Time PCR

The peach root-knot nematode, Meloidogyne floridensis is an emerging pest of peach and other crops that is currently known to occur only in California, Florida, Georgia and South Carolina, USA. Morphological and morphometric differentiation of this species from other related species is difficult and requires taxonomic expertise. Molecular methods can provide rapid and reliable diagnostics of M. floridensis. Currently, molecular identification of M. floridensis is based on the analysis of the intergenic mitochondrial COII-16S region and the nad5 gene sequence polymorphism using PCR-RFLP and sequencing. However, these techniques are time-consuming and not appropriate for the analysis of large numbers of samples. In this study, RAPD and whole genome sequence datasets were used to reveal species-specific DNA fragments for M. floridensis to develop a new diagnostic method. A rapid method of M. floridensis detection using multiplex Real-time PCR with TaqMan probes of three different fluorescent reporter dyes was developed in this study. Multiplex Real-time PCR simultaneously observed amplifications of three fragments with the following primer/probe sets: set 1 – species-specific for M. floridensis/ M. hispanica; set 2- specific for the tropical group of the root-knot nematodes and set 3 - specific for the tropical group of the root-knot nematodes excluding M. floridensis. Positive signals from primer/probe sets 1 and 2 and a negative signal from primer/probe set 3 indicated the presence of M. floridensis in a sample. These results showed that Real-time PCR with specific primers and probes, was sensitive enough to detect the M. floridensis DNA extracted from a single second-stage juvenile specimen per a reaction tube alone or in mixture with other nematodes. Our results also showed that widely used diagnostics for the southern root-knot nematode, M. incognita based on PCR with species-specific primers can generate amplicons of similar lengths with M. floridensis samples and therefore, compromise these methods that were specifically developed for the detection of M. incognita. The Seville root-knot nematode, M. hispanica was also detected in Florida and Mexico for the first time as a results of our field sample testing.