Presence and molecular characterization of alfalfa mosaic virus on tobacco in Serbia

Abstract

Three-year investigation of the presence and distribution of tobacco viruses in Serbia revealed that Alfalfa mosaic virus (AMV) appeared every year with different frequency in tobacco crops. During 2008, the presence of AMV was detected in most of the tested samples (58.82%) and it was the second most common compared to all other viruses which presence was confirmed in Serbia. In 2006 and 2007, AMV was detected in a significantly lower percentage (2.80% and 13.64%, respectively). This study showed that Alfalfa mosaic virus was more commonly found in multiple infections with two, three or even four detected viruses. Single infections were detected only in 2006, in one tobacco field in the locality of Futog. During this investigation, a rapid and simple protocol was optimized and developed for molecular detection of AMV in tobacco leaves, using primers CPAMV1/CPAMV2 and commercially available kits for total RNA extraction as well as for RT-PCR (reverse transcription - polymerase chain reaction). Using RT-PCR and these primers that flank the AMV coat protein gene, a DNA fragment of 751 bp was amplified, sequenced, and compared with the sequences available in GenBank database. The sequence of isolate 196-08 (GenBank Acc. No. FJ527749) proved to be identical at the nucleotide level of 99 to 93% with those from other parts of the world. Phylogenetic analysis of 27 isolates based on 528 bp sequences of the coat protein gene did not show correlation of the isolates with their geographic origin or plant host and showed that these isolates fall into four molecular groups of strains. Serbian AMV isolate from tobacco belongs to group IV, the group that includes most of the isolates selected for phylogenetic analysis.

Trogodišnjim proučavanjima pojave i rasprostranjenosti virusa u usevu duvana u Srbiji utvrđeno je da se virus mozaika lucerke (Alfalfa mosaic virus, AMV) javlja svake godine sa različitom učestalošću. Prisustvo AMV ustanovljeno je u najvećem broju testiranih uzoraka (58,82%) tokom 2008. godine kada je bio drugi po zastupljenosti u odnosu na sve ostale viruse čije je prisustvo utvrđeno u Srbiji. AMV je detektovan u znatno nižem procentu 2006. (2,80%) i 2007. godine (13,64%). Tokom ovih istraživanja virus mozaika lucerke češće je bio prisutan u mešanim infekcijama sa dva, tri ili čak četiri detektovana virusa. Prisustvo pojedinačne zaraze utvrđeno je samo 2006. godine u jednom usevu duvana na lokalitetu Futog. U toku ovih istraživanja razvijen je i optimiziran brz i precizan protokol za molekularnu detekciju AMV u lišću duvana primenom specifičnih prajmera CPAMV1/CPAMV2 i komercijalnih kitova za ekstrakciju ukupnih RNK i RT-PCR (reverzna transkripcija praćena lančanom reakcijom polimeraze). Korišćenjem ovih prajmera, koji omogućavaju umnožavanje gena za protein omotača, umnožen je fragment dužine 751 bp, sekvencioniran i upoređen sa sekvencama dostupnim u GenBank bazi podataka. Dobijena sekvenca izolata 196-08 (GenBank Acc. No. FJ527749) pokazala je 99-93% nukleotidne identičnosti sa izolatima AMV iz drugih delova sveta. Filogenetske analize 27 izolata, na osnovu 528 bp sekvence gena za protein omotača, nisu ukazale na korelaciju sa geografskim poreklom izolata ili biljkom domaćinom, a pokazale su grupisanje izolata u četiri molekularne grupe sojeva. Izolat AMV iz duvana iz Srbije pripada grupi IV, koja obuhvata većinu izolata odabranih za filogenetsku analizu.