engleski

Molecular epidemiology of grapevine phytoplasmoses in Croatia: multigene sequence analysis approach

Phytoplasmas (genus 'Candidatus Phytoplasma') are wall-less bacteria belonging to the class Mollicutes, along with mycoplasmas, spiroplasmas and acholeplasmas which are all believed to share a common Bacillus/Clostridium-like ancestor. Their genomes are relatively small and reduced with many basic metabolic pathways missing, yet many predicted genes are present in multiple copies. Phytoplasma axenic cultivation is still challenging and not common. However, four phytoplasma genomes have been completely sequenced and annotated so far which started a new era in functional and comparative genomics research. These endocellular pathogens have a unique life-style as they have hosts from two kingdoms – Plantae (plants) and Animalia (insects), and need both for their survival and dispersal in nature. Numerous plant species worldwide are affected by phytoplasmoses resulting in serious yield losses, including a range of economically important crops such as wheat, maize, rice, potato, sugarcane, oilseed rape, coconut and grapevine as well as many fruit trees. The most important etiological agents of grapevine yellows (GY) diseases in Europe are also associated with phytoplasma species: ‘Ca. P. solani’ and ‘Ca. P. vitis’, also referred to as Bois Noir (BN ; ribosomal group 16SrXII-A) and Flavescence Dorée (FD ; ribosomal group 16SrV), respectively. In Croatia, surveys of GY molecular epidemiology have been continuously conducted since 1997, with recently discovered FD phytoplasmas being restricted to the areas of the country bordering Slovenia and Hungary and BN phytoplasmas being widespread and diverse. 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 and Scaphoideus titanus Ball. Triplex real-time PCR assay was performed in order to simultaneously detect the presence of both BN and FD phytoplasmas, as well as the PCR/RFLP analysis of phytoplasma 16S rDNA. Samples that tested positive for phytoplasma presence were subjected to the multigene sequence analysis in order to further elucidate epidemiology of the pathosystems studied. Following genes were amplified and analyzed: 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).SecY, map, uvrB-degV and vmpA genes were analyzed for FD phytoplasma positive samples. Multigene sequence analysis has 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.

Epidemiology; MLST; MLSA; phytoplasmas; pathosystem

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