Genetička struktura populacije virusa bronzavosti paradajza (Tomato spotted wilt tospovirus) poreklom iz različitih domaćina u Srbiji

Abstract

Analiza genetičke strukture populacije virusa bronzavosti paradajza (Tomato spotted wilt tospovirus, TSWV) u Srbiji, obuhvatila je molekularnu karakterizaciju 41 izolata poreklom iz različitih biljaka domaćina sakupljenih sa različitih lokaliteta u različitom vremenskom periodu. Filogenetske analize i mreža haplotipova na osnovu delimičnih sekvenci pet gena (N, NSs, NSm, Gn- Gc i RdRp), kao i na osnovu sekvenci dela S i M segmenta dobijenih spajanjem odgovarajućih sekvenci, ukazale su na postojanje genetičkog diverziteta u populaciji TSWV u Srbiji. Filogenetske analize su pokazale da je većina srpskih izolata TSWV evropskog porekla, međutim neki izolati su pokazali veću bliskost sa izolatima iz severnoameričkog klastera, ukazujući da su srpski izolati TSWV uneti kroz više nezavisnih introdukcija. Osim toga, filogenetsko stablo na osnovu sekvenci M i L segmenta ukazalo je i na postojanje rekombinantnih, odnosno pseudorekombinantnih izolata. Analiza sekvenci M segmenta primenom RDP softvera potvrdila je da je izolat 224-16 poreklom iz Cinia sp. prirodni rekombinant dva srpska izolata. Analiza prirodne selekcije pokazala je da na sve testirane genske regione i parove sekvenci TSWV deluje negativna selekcija, dok je pozitivna selekcija ustanovljena samo između nekoliko parova sekvenci NSm, odnosno Gn-Gc gena. Osim toga, poređenje aminokiselinske sekvence NSm proteina ukazalo je na mutacije koje su izazvale dve supstitucije, ili C118Y ili T120N, kod srpskih RB (resistance-breaking) izolata poreklom iz otpornog hibrida paradajza ‘Wrestler F1’, gajenih na dva lokaliteta, ukazujući na njihov RB fenotip i nezavisnu evoluciju. Biološka karakterizacija je potvrdila da su srpski RB izolati sposobni da prevaziđu otpornost paradajza sa Sw-5b, ali ne i paprike sa Tsw genom. Rezultati ovih istraživanja ukazuju da je populacija TSWV u Srbiji heterogena usled više nezavisnih introdukcija virusa, ali i pojave genetičke i fenotipske varijabilnosti izazvane mutacijama, rekombinacijama i pseudorekombinacijama što je doprinelo raznolikosti virusa u našoj zemlji.

The study aimed at defining population structure of Serbian Tomato spotted wilt tospovirus (TSWV) isolates is based on molecular characterization of 41 selected isolates from different host plants collected from different locations over different period of time. Phylogenetic analyses and the median-joining haplotype network of the partial sequences of each of five genes (N, NSs, NSm, Gn- Gc and RdRp), as well as concatenated sequences of segments S and M obtained by merging the corresponding sequences, have shown genetic diversity of TSWV in Serbia. The phylogenetic analyses showed that most Serbian TSWV isolates originated from Europe, but several isolates were more closely related to isolates from North America clade, indicating that Serbian TSWV isolates were introduced through several independent introductions. In addition, the phylogenetic tree based on segments M and L revealed the presence of recombination and reassortment, respectively. A sequence analysis of segment M using RDP software confirmed that isolate 224-16 originating from Cinia sp. represents a natural recombinant between two Serbian isolates. Purifying selection was determined as the major natural force influencing all five genes and pairs of sequences, while positive selection was found only between a few pairs of sequences of NSm and Gn-Gc genes. Moreover, the NSm amino acid sequence comparison revealed the presence of two amino acid substitutions (C118Y or T120N) in Serbian RB (resistance-breaking) isolates originating from the resistant tomato cultivar ‘Wrestler F1’ grown at two locations, indicating their RB phenotype and independent evolutionary events. A bioassay confirmed that Serbian RB isolates had the ability to break the Sw-5b-mediated resistance in the tomato, but were unable to overcome the Tsw resistance gene in the pepper. The results of this research indicate that the population of TSWV in Serbia is heterogeneous due to several independent introductions of the virus, but also due to the occurrence of mutations, recombination and reassortment, which have all contributed to the molecular diversity of the virus in our country.