Variability of rplF and tufB genes in flavescence dorée phytoplasma isolates

Fitoplazme (rod 'Candidatus Phytoplasma') su bakterije bez staniĉne stijenke izrazito reduciranih genoma koje parazitiraju u floemu biljaka i stanicama kukaca, a nije ih moguće uzgajati u ĉistoj kulturi in vitro. Uzrokuju velik broj ekonomski znaĉajnih bolesti biljaka u cijelom svijetu meĊu kojima su i ţutice vinove loze. Svrstane su u razred Mollicutes unutar kojeg tvore veliku monofiletsku grupu. Prenose se kukcima-vektorima iz reda Hemiptera koji se hrane floemom te zaraţenim sadnim materijalom. Postoje dvije fitoplazmatske vrste koje su većinski uzroĉnici ţutica vinove loze u Europi: flavescence dorée (FD) i bois noir (BN; Candidatus 'Phytoplasma solani'). Cilj ovog diplomskog rada bio je tipizacija izolata fitoplazme FD analizom konstitutivnih gena rplF (gen za 50S ribosomski protein L6) i tufB (gen za elongacijski faktor EF-Tu) koji do sada nisu korišteni kao molekularni markeri u multigenskoj tipizaciji ove fitoplazmatske vrste. U analizu su ukljuĉeni uzorci vinove loze i biljaka iz okoline vinograda, kao i kukaca-vektora, prethodno prikupljeni na lokacijama na podruĉju ĉitave kontinentalne Hrvatske i Istarske ţupanije. Fragmenti gena rplF i tufB umnoţeni su lanĉanom reakcijom polimerazom te sekvencirani. Sravnjivanjem i filogenetskom analizom dobivenih nukleotidnih sljedova gena rplF i tufB utvrĊeno je postojanje 5, odnosno 6 razliĉitih genotipova meĊu analiziranim uzorcima. Gen rplF se pokazao konzerviranijim od gena tufB, ali su se zbog svoje varijabilnosti oba pokazala kao potencijalno dobri kandidati za dodatne molekularne markere u tipizaciji fitoplazme FD.Phytoplasmas (genus 'Candidatus Phytoplasma') are non-cultivable wall-less bacteria from the class Mollicutes, with extremely reduced genomes, found in plant phloem and insect vector cells. They cause many economically important diseases worldwide including Grapevine yellows (GY) diseases. Diseases are transmitted by infected planting material and phloem-feeding insects – vectors of the order Hemiptera. Two phytoplasma species are major causeing agents of GY: flavescence dorée (FD) and bois noir (BN; Candidatus 'Phytoplasma solani'). The aim of this thesis was to perform typing of phytoplasma FD isolates by analysis of housekeeping genes rplF (encoding 50S ribosomal protein L6) and tufB (encoding the elongation factor EF-Tu). These genes have not been commonly used as molecular markers in multilocus sequence typing (MLST) of phytoplasma FD so far. The analysis included samples of grapevine, wild plants from the vicinity of vineyards and insect-vectors previously collected all over continental Croatia and in Istrian county. Fragments of rplF and tufB genes were amplified by PCR and sequenced. Alignment and phylogenetic analysis of rplF and tufB gene fragments revealed the presence of 5 and 6 genotypes, respectively. Gene rplF showed to be more conserved than the tufB gene, but both proved as good candidates as potential additional molecular markers for FD phytoplasma genotyping due to their variability