Molecular diagnosis of trichodorid species from Portugal

Abstract

A PCR-RFLP assay was developed for the identification of trichodorids belonging to Nanidorus, Paratrichodorus and Trichodorus genera. Exploring the variability of the 18S SSU rDNA gene this method provides for the first time a molecular diagnosis tool alternative to the difficult and time consuming phenotyping especially of quite similar species, enabling selectivity in mixed samples with non-trichodorid species, and also the differentiation of juveniles. Based on the alignment of previously obtained 18S rDNA nucleotide sequences of trichodorids from Portugal, a pair of selective primers was designed in conserved regions to allow the amplification in all known Portuguese species of a variable region located at the 3’ end of the gene. The PCR product, 615bp in length, exhibits nucleotide variability to generate restriction fragment patterns which were consistent among populations of the same species and allowed to discriminate trichodorids at the species level. The proposed protocol was tested and proved effective with twelve trichodorid species from Portugal (N. minor, P. allius, P. anemones, P. divergens, P. hispanus, P. pachydermus, P. porosus, T. beirensis, T. lusitanicus, T. primitivus and two other Trichodorus species, A and B) and six non-indigenous trichodorid populations (N. minor, P. allius, P. anemones, P. pachydermus, P. porosus, T. primitivus).