Diversity and characterization of pepper viruses in Serbia

Dvogodišnjim proučavanjima pojave i rasprostranjenosti virusa u usevu paprike u Srbiji utvrđeno je da se virusi javljaju svake godine kako u proizvodnji rasada, tako i u usevu paprike bilo da je gajen u zaštićenom prostoru ili na otvorenom polju. Serološkim analizama prikupljenih uzoraka paprike primenom DAS–ELISA testa, detektovano je prisustvo pet virusa: virus mozaika krastavca (Cucumber mosaic virus, CMV), virus crtičastog mozaika krompira (Potato virus Y, PVY), virus bronzavosti paradajza (Tomato spotted wilt virus, TSWV), virus mozaika lucerke (Alfalfa mosaic virus, AMV) i virus blagog šarenila paprika (Pepper mild mottle virus, PMMoV), koji su se javili u pojedinačnim ili kompleksnim infekcijama. U obe godine istraživanja, utvrđeno je dominantno prisustvo CMV i/ili PVY u proizvodnji rasada, kao i kod oba načina gajenja paprika. Pregledom useva paprike u različitim lokalitetima gajenja zabeležena je pojava niza simptoma na lišću, stablu, cvetovima i plodovima, koji su upućivali na virusnu zarazu, ali nije bilo moguće utvrditi njihovu povezanost sa detektovanim virusom. Za molekularnu detekciju i identifikaciju kao i za potvrdu rezultata dobijenih na osnovu antigenih osobina, korišćeni su odabrani izolati virusa paprike koji su detektovani u usevu paprike serološkim analizama. Amplifikacija različitih regiona genoma virusa obavljena je primenom RT–PCR metode korišćenjem odgovarajućih specifičnih prajmera. Nakon sekvencioniranja i višestrukog uparivanja sekvenci izolata sa sekvencama drugih virusa dostupnih u GenBank bazi podataka i proračunom genetičke sličnosti obavljena je molekularna identifikacija odabranih izolata virusa paprike poreklom iz naše zemlje. Determinisane su nukleotidne sekvence CP gena tri izolata CMV, AMV i PMMoV, sekvence gena za P1 protein tri izolata PVY, sekvence NC gena tri izolata TSWV, kao i nukleotidne sekvence RdRp gena tri izolata PMMoV. Filogenetska analiza na osnovu sekvenci gena za P1 protein, pokazala je grupisanje izolata PVY iz Srbije u klaster sa ostalim evropskim izolatima nekrotičnih sojeva, zatim na osnovu sekvenci CP gena izolata CMV, utvrđena je pripadnost ispitivanih izolata iz paprike podgrupi IA gde je svrstana većina sojeva iz drugih delova sveta. Filogenetska analiza izolata TSWV pokazuje grupisanje ispitivanih izolata u klaster koji čini većina izolata iz Evrope, dok je na osnovu sekvence CP gena izolata AMV, pokazano grupisanje izolata u četiri genetička soja, gde se većina izolata, uključujući i ispitivane, izdvojila u grupu I...A two-year investigation of the presence and distribution of pepper viruses in Serbia revealed that viruses occur each year of transplant, and also in greenhouse and in open field production. Serological analyses of collected pepper samples using DAS-ELISA test detected the presence of five viruses: Cucumber mosaic virus (CMV), Potato virus Y (PVY), Tomato spotted wilt virus (TSWV), Alfalfa mosaic virus (AMV) and Pepper mild mottle virus (PMMoV), which occurred in single or complex infections. The prevalence of CMV and/or PVY was found in both years of investigation in transplant production and in both ways pepper is grown in Serbia. Survey of pepper crops in different growing regions indicated the occurrence of a number of symptoms on leaves, stem, flowers and fruit, which resembled those of virus infection, but it was not possible to determine their association with detected virus. Selected pepper virus isolates were used for molecular detection and identification as well as for confirmation of the results obtained on the basis of antigenic properties, which have been detected in a crop of pepper by serological analysis. The amplification of different genome regions was performed by RT-PCR method, using specific corresponding primers of the respective viruses. After the sequencing, sequences of Serbian virus isolates were compared with the respective virus sequences available in the GenBank and calculation of genetic homology was performed for molecular identification of selected isolates of the pepper viruses originating from our country. Nucleotide sequences of CP gene of three isolates of CMV, AMV and PMMoV, the P1 protein gene sequence of three PVY isolates, sequences of NC gene of three TSWV isolates and nucleotide sequences of RdRp gene of three PMMoV isolates were determined. Phylogenetic analysis based on gene sequences for P1 protein showed a grouping of PVY isolates from Serbia in a cluster with other European isolates of necrotic strains. On the basis of CP gene phylogenetic analysis, it was shown that tested pepper CMV isolates from Serbia belong to subgroup IA, where most strains from the other parts of the world are classified. Phylogenetic analysis of TSWV isolates showed grouping of Serbian isolates into cluster with most of the isolates from Europe, while, based on the CP gene sequences of AMV isolates, showed grouping of isolates into four genetic strains, where most of the isolates, including tested, allocated in group I..