engleski

CSI Vineyard: Trac(k)ing of Grapevine Yellows Phytoplasmas Emergence and Spread by MLST

Insect-transmitted plant pathogens from the genus ‘Candidatus Phytoplasma’ are associated with numerous diseases affecting plant species worldwide. Since their axenic cultivation in a pure culture is still challenging, phytoplasma detection, diagnosis and classification are still based primarily on the 16S rRNA gene analysis. However, when a distinction between closely related strains or the elucidation of a complex pathosystem is needed, information obtained solely on the basis of highly conserved 16S rDNA is not adequate. Thus, the genotyping of additional housekeeping genes together with specific variable genes is performed. Multi-locus sequence typing (MLST) is commonly used tool in population, evolutionary and epidemiological studies of various bacterial pathogens and is recognized as a reproducible approach. In Euro-Mediterranean region, economically important grapevine yellows (GY) diseases are mainly associated with phytoplasmas belonging to the ribosomal groups 16SrV-C and -D (flavescence dorée ; FD) and 16SrXII-A (stolbur ; bois noir ; BN, ‘Ca. Phytoplasma solani’) causing similar symptoms but differing in epidemiology. The main objective of this work was to demonstrate tracking and tracing of both BN and FD phytoplasmas emergence and spread by MLST approach. In the period 2012-2016, over 500 samples of grapevine, insect vectors and wild plants were taken from different viticultural regions of Croatia. Total nucleic acids were extracted and triplex real-time PCR assay was performed, together with PCR/RFLP. Subsequently, distinctive sets of gene fragments differing in conservation and evolution were amplified and sequenced and the phylogenetic analysis was performed. Real-time PCR revealed the continuous emergence of new locations and areas affected by FD over the years and a clear trend of the disease spread with Istria as a new hotspot heavily affected since 2014. For FD phytoplasma, MLST demonstrated the existence of 2 map, 4 uvrB-degV and 5 secY genotypes with a strong relatedness between strains, showing their affiliation to the same pathosystem. MLST analysis of BN isolates revealed the presence of at least 17 different comprehensive genotypes encompassing 3 tuf, 5 secY, 7 vmp1 and 10 stamp genotypes and the prevalent ones were identified. Finding of distinct BN and FD clusters and different distribution of genotypes based on MLST suggests separate routes of the disease introduction in Croatia and spread of the disease.

Bois noir; Flavescence dorée; Hyalesthes obsoletus; Scaphoideus titanus; weeds; map; uvrB-degV; secY; tuf; vmp1; 16S rRNA gene

Istraživanje je ostvareno u okviru HRZZ uspostavnog istraživačkog projekta "Komparativn i funkcionalna genomika fitoplazmi-emergentnih biljnih patogena u Hrvatskoj" (voditeljica Martina Šeruga Musić)