Molecular characterisation and diagnostics of the red ring nematode, Bursaphelenchus cocophilus, from Mexico

The red ring nematode, Bursaphelenchus cocophilus, vectored by the South American palm weevil, Rhynchophorus palmarum, is the causal agent of red ring disease in coconut and other palms in countries of Central and South America. The populations of B. cocophilus collected in the states of Guerrero and Tabasco, Mexico, were molecularly characterised using the D2-D3 expansion segments of 28S rRNA, ITS rRNA and COI gene sequences. Mexican B. cocophilus populations were molecularly different from other Central and South American populations. Comparative analysis of available rRNA sequences obtained from several countries showed that B. cocophilus consists of molecularly different populations and its species structure is likely congruent with that of the beetle vector. Conventional PCR, real-time PCR and recombinase polymerase amplification (RPA) with lateral flow dipstick (LF) assays have been developed for the identification of the red ring nematode in this study. The specificity of the ITS rRNA primers in the assays were examined using B. cocophilus and other 15 species of family Aphelenchoididae. Detection sensitivity levels, determined by using a dilution series of B. cocophilus extracts, was 0.13 nematode per reaction tube for real-time PCR and 0.25 nematode per reaction tube for conventional PCR and LF-RPA assays. The application of the LF-RPA assay has great potential for diagnosing infestation of this species with a minimal laboratory infrastructure.