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A case study of Bois noir and Flavescence dorée phytoplasma pathosystems: multilocus sequence typing approach

Grapevine yellows (GY) diseases have been continuously monitored and studied in Croatia since 1997. Although main etiological agents are still associated with Bois noir (BN) phytoplasmas belonging to ribosomal subgroup 16SrXII-A (stolbur) which are widespread, Flavescence dorée (FD) phytoplasmas (16SrV-C) have also been detected in several areas of the country close to borders with Slovenia and Hungary. Three locations have been chosen for a case study of grapevine phytoplasma pathosystems: Vukanovec, as a BN focus, and Brckovština and Jagnjić dol, representing FD phytoplasma foci. Grapevine and weed samples were collected together with samples of BN and FD phytoplasma insect vectors Hyalesthes obsoletus Signoret (Vukanovec) and Scaphoideus titanus Ball (Brckovština and Jagnjić dol). Total nucleic acids were extracted from all samples and triplex real-time PCR assay was performed in order to simultaneously detect the presence of both BN and FD phytoplasmas. Additionally, PCR/RFLP analysis of phytoplasma 16S rDNA was also performed. At location Vukanovec, the presence of BN phytoplasmas was confirmed in grapevine samples as well as in insect vector H. obsoletus, while no phytoplasmas were detected in weed samples. At location Brckovština, both FD and BN phytoplasmas were detected in grapevine samples, while in insect vector S. titanus samples, FD phytoplasmas presence was confirmed. One weed sample Convolvulus arvensis L. from the same location tested positive for BN phytoplasmas. FD phytoplasmas were also detected in grapevine as well as in S. titanus samples from location Jagnjić dol. In order to study the variability of detected phytoplasmas in more detail and further elucidate epidemiology of the pathosystems studied, multilocus sequence typing (MLST) analysis approach was used. Following genes were amplified from BN phytoplasma positive samples: housekeeping genes tufB and secY, together with stamp and vmp1 genes that are BN-specific and potentially involved in phytoplasma-insect interactions. In FD phytoplasma positive samples, secY, map, uvrB-degV and vmpA genes were analyzed. MLST analyses, 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. However, variability was found in some of the analyzed gene regions. BN phytoplasma found in weed C. arvensis from location Brckovština has shown a considerable genetic variability in all of the analyzed genes. The latter result, together with the finding of FD phytoplasmas in both grapevine and insect samples, suggests a co-existence of different phytoplasma pathosystems at the same location.

epidemiology; genotyping; MLST; phytoplasma

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