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