engleski

PLANT PATHOGEN FORENSICS: A DETECTIVE STORY OF TRAC(K)ING PHYTOPLASMA EMERGENCE AND SPREAD BY MLST

Uncultivable and insect-transmitted bacteria from the genus ‘Candidatus Phytoplasma’ are associated with diseases affecting various plant species worldwide, including many economically important crops. Classification and detection of phytoplasmas are based mainly on the 16S rRNA gene analysis. However, for the elucidation of a complex pathosystem or a distinction between strains, genotyping of additional housekeeping and specific variable genes is applied. Multi-locus sequence typing (MLST) is widely used in population, evolutionary and epidemiological studies of diverse bacterial pathogens. Yet unassigned to a species taxon within the ‘Ca. Phytoplasma’ genus, Flavescence dorée (FD) phytoplasma is one of the main agents associated with grapevine yellows diseases in Europe. It is transmitted by insect vector Scaphoideus titanus Ball. and possesses a high epidemic potential. First report of FD phytoplasma in Croatia was given in 2009 for restricted continental areas of the country. The main objective of this study was to demonstrate tracking and tracing of FD phytoplasmas emergence and spread by MLST approach. In the 4-year period, over 500 samples of grapevine, S. titanus and wild plants from different regions of Croatia were analyzed. Triplex real- time PCR was performed for detection together with PCR/RFLP of 16S rDNA. Subsequently, secY, map and uvrB-degV genes were amplified and sequenced and the phylogenetic analysis was performed. Continuous emergence of new locations affected by FD over the years and a clear trend of the disease spread with Istrian peninsula heavily affected since 2014 was revealed. FD-related phytoplasmas were found for the first time in alder and invasive tree species Ailanthus altissima Mill. Phylogenetic analyses of map gene revealed the existence of 2 distinct FD clusters: map-FD2 and map-FD3, with the latter being prevalent. Different genotypes within both FD clusters could be distinguished based on additional SNPs. Furthermore, MLST revealed a strong relatedness between strains detected in grapevines and insects, showing their affiliation to the same pathosystem. Finding of distinct FD clusters and different distribution of genotypes based on MLST suggests separate routes of the disease introduction in Croatia and spread of the disease.

phytoplasma; genotyping; infection; plants; insects; secY; imp; phylogenetic analysis

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