TaqMan Real-Time PCR Assay for Specific Detection and Differentiation of Xanthomonas translucens pv. undulosa from other pathovars targeting a recombination mediator gene, recF.

Abstract

Bacterial leaf streak and black chaff diseases of wheat caused by Xanthomonas translucens pv. undulosa is becoming a major constraint to growers and trade since it is seed-borne. Molecular tools for specific detection/differentiation of pv. undulosa are lacking. We report the development of a TaqMan real-time PCR for specific detection/identification of pv. undulosa targeting the recombination mediator gene (recF). Analysis of the complete recF (1,117 bp) sequences identified the gene as a reliable phylogenetic marker for identification of pv. undulosa, differentiating it from the other pathovars. recF-based sequence homology values among the eleven pathovars correlated well with genome-based DNA-DNA hybridization values. The discriminatory power of recF to differentiate pv. undulosa from the other pathovars is due to nucleotide polymorphic positions. We used these nucleotide polymorphisms to develop a TaqMan PCR for specific detection of pv. undulosa/secalis. The specificity of the assay was validated using sixty-seven bacterial and fungal/Oomycete strains. The selected primers and the double-quenched FAM-labeled TaqMan probe were specific for the detection of eleven pv. undulosa/secalis strains. The 56 strains of other X. translcens pathovars (n = 39) and non-Xanthomonas spp. (n = 17) did not exhibit any detectable fluorescence. Also, greenhouse-inoculated and naturally-infected wheat leaf samples showed positive reactions for the presence of pv. undulosa DNA but not healthy control plants. The TaqMan assay reliably detected as low as 1-pg DNA amount and 10 colony forming units of the target pathogen per reaction. This TaqMan assay could be useful to regulatory agencies with economic benefits to wheat growers.