engleski

Epidemiology of grapevine phytoplasma pathosystems: multilocus seqeunce typing approach

Phytoplasmas (genus 'Candidatus Phytoplasma') are uncultivable wall-less bacteria with reduced genomes that lack many basic metabolic pathways. These pathogenic bacteria inhabit plant phloem and insects and need both hosts for their survival and dispersal in nature. The most important etiological agents of grapevine yellows (GY) diseases in Europe are associated with Flavescence Dorée (FD) and Bois Noir (BN) phytoplasmas, belonging to the ribosomal groups 16SrV and 16SrXII-A, respectively. Surveys of GY in Croatia are being continuously conducted since 1997, with BN phytoplasmas being widespread, and recently discovered FD phytoplasmas being restricted to the areas of the country bordering Slovenia and Hungary. The aim of this study was to investigate a variability of phytoplasmas involved in grapevine phytoplasma pathosystems from several locations. Grapevine, weed and insect (Hyalesthes obsoletus Signoret as BN phytoplasmas vector and Scaphoideus titanus Ball as FD phytoplasma vector) samples were collected. Total nucleic acids were extracted from all samples and PCR/RFLP analysis of phytoplasma 16S rDNA was performed together with triplex real-time PCR assay, in order to simultaneously detect the presence of both BN and FD phytoplasmas. Samples that tested positive for phytoplasma presence were subjected to the multilocus sequence typing (MLST) analysis, in order to further elucidate epidemiology of the pathosystems studied. Following genes were amplified and analyzed: housekeeping genes tufB and secY, together with stamp and vmp1 genes that are BN-specific and potentially involved in phytoplasma-insect interactions (for BN phytoplasma positive samples) and secY, map, uvrB-degV and vmpA (for FD phytoplasma positive samples). MLST analyses of the amplified phytoplasma sequences, including RFLP and phylogenetic analyses, have revealed a strong relatedness between phytoplasmas detected in grapevine and insect samples, showing their affiliation to the same pathosystem at the same location. However, variability was found in some of the analyzed gene regions. BN phytoplasma found in weed Convolvulus arvensis has shown a considerable genetic variability in all of the analyzed genes. The latter result, together with the finding of FD phytoplasmas at the same location in both grapevine and insect samples, suggests a co-existence of different phytoplasma pathosystems.

BN; Croatia; FD; insect vector; MLST; phytoplasma; weed

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