Rasprostranjenost PPV-D i PPV-Rec sojeva virusa šarke šljive (Plum pox virus) u Srbiji i dinamika njihovog širenja u zasadu šljive

Abstract

Virus šarke šljive (Plum pox virus-PPV), prouzrokovač bolesti šarke šljive, je prisutan u Srbiji skoro 80 godina i smatra se ekonomski najznačajnijim virusom koštičavih voćaka. Od 7 opisanih sojeva PPV, u Srbiji je potvrđeno prisustvo tri soja: PPV-M, PPV-D i PPV-Rec. U cilju utvrđivanja raširenosti sojeva šarke u zasadima šljive i kajsije u Srbiji, u periodu 2008‒2010. godine, standardizovanom procedurom je sakupljeno ukupno 283 uzoraka lišća šljive i kajsije iz preko 90 zasada. Tipiziranje detektovanih izolata sprovedeno je IC-RT-PCR metodom kroz 4 odvojene PCR reakcije sa PPV-M i PPV-D specifičnim prajmerima sa ciljnim sekvencama u dva regiona genoma. Za filogenetsku analizu i utvrđivanje stope diverziteta odabran je 41 izolat za sekvencioniranje fragmenata iz dva regiona genoma (C-ter NIb‒N-ter CP i C-ter P3‒6K1‒N-ter CI). U cilju ispitivanja dinamike širenja PPV-D i PPV-Rec sojeva u direktnoj kompeticiji, tokom 2008. godine podignut je eksperimentalni zasad sa 400 stabala šljive sorte Čačanska lepotica. Osam stabala, koja su locirana u sredini zasada, su inokulisana sa potpuno okarakterisanim PPV-D i PPV-Rec izolatima (4 stabla po izolatu-interni izvor infekcije). Sva stabla Prunus vrsta u neposrednoj okolini zasada su markirana, uzorkovana i testirana na prisustvo PPV (eksterni izvor infekcije). Pozitivni uzorci su sekvencionirani u C-ter NIb‒N-ter CP regionu genoma za dalju uporednu analizu sa sekvencama inokulisanih i novoinficiranih stabala u zasadu. Svake godine, od 2008‒2011., sva stabla u eksperimentalnom zasadu su vizuelno pregledana i ispitivana ELISA testom na prisustvo PPV. Pozitivni uzorci su tipizirani IC-RT-PCR metodom i sekvencionirani u C-ter NIb‒Nter CP regionu genoma. Tokom poslednje tri godine istraživanja vršeno je praćenje prisustva i dinamike brojnosti lisnih vaši u eksperimentalnom zasadu...

Plum pox virus (PPV), the agent of Sharka disease, is present in Serbia almost 80 years and it is considered as economically the most important virus of stone fruits. Three strains, out of 7 described, are present in Serbia: PPV-M, PPV-D and PPV-Rec. In order to determine the prevalence of strains in plum and apricot orchards in Serbia during the three-year period (2008-2010), a total of 283 plum and apricot leaf samples from over 90 orchards were collected by standardized procedure. Strain-typing of isolates was done by IC-RT-PCR in 4 separate PCR reactions with PPV-M and PPV-D specific primers with target sequences in two different genomic regions. For phylogenetic analysis and determination of diversity rate, 41 isolates were selected for sequencing fragments from two genomic regions (C-ter NIb‒N-ter CP and C-ter P3‒6K1‒N-ter CI). In order to study the dynamics of spread of PPV-D and PPV-Rec strains in direct competition, an experimental plum orchard of 400 trees of Čačanska lepotica variety was built in 2008. Eight trees, which are located in the middle of the orchard, were inoculated with a fully characterized PPV-D and PPV-Rec isolates (4 trees per isolate-internal source of infection). All Prunus trees in the vicinity of the experimental orchard were marked, sampled and tested for the presence of PPV (external source of infection). Positive samples were sequenced in C-ter NIb‒N-ter CP genomic region for further analysis with sequences of inoculated and newly infected trees in the orchard. Every year, from 2008‒2011, all trees from the orchard were visually inspected and tested by ELISA for the presence of PPV. ELISA positive samples were typed by IC-RT-PCR and sequenced in the C-ter NIb‒N-ter CP genomic region. During the last three years of research, the presence and the number of aphids in the experimental orchard were monitored...