Molecular characterization of 'Candidatus Phytoplasma solani' isolates from periwinkle (Catharanthus roseus (L.) G. Don) of the Zagreb area

Fitoplazme (rod 'Candidatus Phytoplasma’) su bakterije bez stanične stijenke iz razreda Mollicutes koje imaju domaćine u dva carstva jer ih nalazimo u floemu biljaka kao i stanicama kukaca. Nije ih moguće uzgojiti u čistoj kulturi in vitro jer se radi o unutarstaničnim organizmima kod kojih je došlo do redukcije genoma i gubitka mnogih metabolički važnih gena. Uzročnici su ekonomski značajnih bolesti biljaka u čitavom svijetu. Fitoplazmatska vrsta 'Ca. Phytoplasma solani' jedna je od fitoplazmi s najširim krugom prirodnih domaćinskih vrsta, a karakteristična je za europski kontinent. Cilj ovog diplomskog rada bio je karakterizirati izolate bakterije 'Ca. Phytoplasma solani' iz madagaskarskog zimzelena (Catharanthus roseus (L.) G. Don) prikupljenih na području užeg centra grada Zagreba. U multigensku tipizaciju izolata bili su uključeni sljedeći fitoplazmatski geni: 16S rRNA, secY, tufB, vmp1 i stamp. Fragmenti gena za 16S rRNA, tufB i vmp1 umnoženi su lančanom reakcijom polimerazom korištenjem specifičnih početnica te nakon toga analizirani metodom RFLP (polimorfizam duljine restrikcijskih fragmenata). Specifično umnoženi fragmenti gena secY i stamp sekvencirani su te analizirani bioinformatičkim metodama. Filogenetske analize dobivenih nukleotidnih sljedova gena secY i stamp kao i rezultati analize RFLP ostalih gena, potvrdile su pripadnost izolata fitoplazmatskoj vrsti 'Ca. Phytoplasma solani' te njihovu varijabilnost unutar vrste. Analize gena secY i tufB pokazale su očuvanost ovih gena dok je kod gena stamp i vmp1, specifičnih za vrstu, uočena veća raznolikost genotipova.Phytoplasmas (genus "Candidatus Phytoplasma") are bacteria from the class Mollicutes without cell wall found in hosts from two kingdoms, in plant phloem and insect hemolymph. They are intracellular organisms that have undergone reduction of genome with a loss of many metabolically important genes. Therefore they cannot be cultured in vitro in cell-free media. Phytoplasma infect plants all over the world causing significant economic damage. Species 'Ca. Phytoplasma solani' is phytoplasma with widest natural host species range which is characteristic for the European continent. The aim of this thesis was to characterize 'Ca. Phytoplasma solani' isolates from periwinkle (Catharanthus roseus (L.) G. Don) collected at the Zagreb downtown area. In multigene typization of isolates following genes were analyzed: 16S rRNA, secY, tufB, vmp1 and stamp. Fragments of 16S rRNA, tufB and vmp1 genes were amplified by polymerase chain reaction using phytoplasma specific primers and further analyzed by RFLP (restriction fragment length polymorphism) analyses. Amplified fragments of genes secY and stamp were sequenced and analyzed by using bioinformatics tools. Phylogenetic analysis of secY and stamp nucleotide sequences together with RFLP analysis of other genes confirmed the affiliation of all isolates to the 'Ca. Phytoplasma solani' species and their intraspecies variability. Analyses of housekeeping genes secY and tufB showed that these sequences were highly conserved, while the analyses of stamp i vmp1 species specific genes, revealed more genotype variability