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A molecular protocol is presented for distinguishing seven of the most common and economically important Meloidogyne spp. DNA was extracted from individual second-stage juvenile (J2) nematodes of Meloidogyne spp. and amplified by PCR (polymerase chain reaction). Fifteen PCRs including amplification of rDNA, specific SCAR (sequence characterized amplified region) and RAPD (random amplified polymorphic DNA) fragments were possible from the extracted DNA. This enabled a molecular diagnostic key for M. incognita, M. javanica, M. arenaria, M. mayaguensis, M. hapla, M. chitwoodi and M. fallax to be designed. The key unifies published methods into a single logical schematic using primer combinations that were previously validated and shown to work reliably and specifically. The protocol can be used with single juvenile or adult nematodes and the schematic can readily be expanded to accommodate more species. The use of RAPD amplification to assist with identification of samples which do not yield diagnostic amplification products after the first three steps of the molecular key is also described. [ABSTRACT FROM AUTHOR]

Several Paratrichodorus and Trichodorus (trichodorid) nematode species are natural vectors of Tobacco rattle virus (TRV) and cause economically important diseases, especially in potato and ornamental bulbous crops. Identification of trichodorid species based on morphological characters is laborious, time-consuming, and requires the services of highly trained personnel. Molecular diagnostics for trichodorid nematodes, using the ribosomal DNA repeat unit, were successfully developed to distinguish two Paratrichodorus and two Trichodorus species. The complete sequences of the 18S genes and the ITS-1 regions for these species were obtained and species-specific primers successfully designed for them. An RT-PCR assay was developed utilizing isolate-specific primers that amplify serologically distinguishable strains of TRV in individual trichodorid nematodes. The primers were based on the highly conserved RNA-1 segment of the bipartite genome and also on different parts of the RNA-2 segment of the virus genome. [ABSTRACT FROM AUTHOR]