Short communication: Pepino mosaic virus, a new threat for Serbia’s tomatoes

Aim of study: To report the occurrence of Pepino mosaic virus (PepMV) on tomato in Serbia and to genetically characterize Serbian PepMV isolates.Area of study: Tomato samples showing virus-like symptoms were collected in the Bogojevce locality (Jablanica District, Serbia).Material and methods: Collected tomato samples were assayed by DAS-ELISA using antisera against eight economically important or quarantine tomato viruses. Three selected isolates of naturally infected tomato plants were mechanically transmitted to tomato ‘Novosadski jabučar’ seedlings. For confirmation of PepMV infection, RT-PCR was performed using specific primers PepMV TGB F/PepMV UTR R. Maximum-likelihood phylogenetic tree was constructed with 47 complete CP gene sequences of PepMV to determine the genetic relationship of Serbian PepMV isolates with those from other parts of the world.Main results: The results of DAS-ELISA indicated the presence of PepMV in all tested samples. Mechanically inoculated ‘Novosadski jabučar’ seedlings expressed yellow spots and light and dark green patches, bubbling, and curled leaves. All tested tomato plants were RT-PCR positive for the presence of PepMV. The CP sequence analysis revealed that the Serbian PepMV isolates were completely identical among themselves and shared the highest nucleotide identity of 95.1% (99.2% aa identity) with isolate from Spain (FJ263341). Phylogenetic analysis showed clustering of the Serbian PepMV isolates into CH2 strain, but they formed separate subgroup within CH2 strain.Research highlights: This is the first data of the presence of PepMV in protected tomato production in Serbia. Considering increased incidence and rapid spread in Europe, the presence of PepMV on tomato could therefore represent serious threat to this valuable crop in Serbia

Učestalost pojave i molekularna karakterizacija virusa mozaika lubenice poreklom iz Srbije

Watermelon mosaic virus (WMV) is widespread in cucurbit crops, most commonly occuring in temperate and Mediterranean regions. In Serbia WMV has been detected in single and mixed infections with Zucchini yellow mosaic virus and Cucumber mosaic virus in field-grown pumpkin and squash crops. Among pumpkin-affecting viruses WMV is the most frequent one, both by the number of localities and its incidence at each location. During the growing season of 2009, samples from 583 plants of Cucurbita pepo cvs. Olinka, Belgrade zucchini and Tosca (Zucchini group), as well as from C. maxima and C. moschata showing symptoms of virus infection were collected from 12 commercial fields at eight localities and analyzed by DAS-ELISA using polyclonal antisera specific to six most important cucurbit viruses. Interestingly, WMV was detected at fewer sites and had lower incidence rate than in two previous years. In single infections, WMV was found in 11% of tested plants in three fields; in mixed infections with ZYMV, it was recorded in 9.9% of plants in five fields and with CMV in only 0.2% in one field. The partial coat protein gene and 3’ non-translated region from two representative isolates of WMV originating from different localities and host plant species were amplified by RT-PCR, sequenced, and compared with the sequences available in GenBank database. The PCR-amplified fragment of predicted size of approximately 1017 bp was obtained. The sequences of isolates 137-08 (Acc. No. GQ259958) and 159-08 (GU144020) proved to be 94-99% identical at the nucleotide level with those from other parts of the world. The sequences of these two isolates differed from each other only at two nucleotide positions, without any amino acid substitution. Phylogenetic analysis of 57 isolates based on 750 bp sequences of the coat protein gene showed no correlation between isolates and their geographic origin, and it also indicated that these isolates fell into three molecular groups of strains. Serbian WMV isolates belong to group I, the most widespread group of isolates of this virus in the world.Virus mozaika lubenice (Watermelon mosaic virus, WMV) široko je rasprostranjen u usevima vrežastih kultura, mada se najčešće javlja na Mediteranu i regionima sa umerenom klimom. U Srbiji, WMV je detektovan u pojedinačnim i mešanim infekcijama sa virusom žutog mozaika cukinija (Zucchini yellow mosaic virus, ZYMV) i virusom mozaika krastavca (Cucumber mosaic virus, CMV) u usevima tikava i bundeva gajenim na otvorenom polju. Od virusa tikava, WMV je često prevalentan virus, kako po broju lokaliteta u kojima je dokazan, tako i po učestalosti u samom usevu. Iz ukupno 12 komercijalnih useva na osam lokaliteta, 2009. godine sakupljeno je 583 biljaka Cucurbita pepo cvs. Olinka, Beogradska tikvica i Tosca (cukini forma), kao i C. maxima i C. moschata, sa simptomima virusnih zaraza, koje su analizirane na prisustvo šest najznačajnijih virusa tikava primenom DAS-ELISA metode. WMV je detektovan na manjem broju lokaliteta i u manjoj učestalosti u poređenju sa prethodne dve vegetacije. U pojedinačnim infekcijama, WMV je detektovan u 11% testiranih biljaka u tri useva, a u mešanim sa ZYMV u 9,9% testiranih biljaka u pet useva i sa CMV u 0,2% testiranih biljaka u jednom usevu. Deo gena za protein omotača virusa i deo 3’ neprepisujućeg regiona dva odabrana izolata WMV poreklom iz različitih lokaliteta i biljaka domaćina umnožen je primenom RT-PCR metode, sekvencioniran i upoređen sa sekvencama dostupnim u GenBank bazi podataka. Dobijen je PCR produkt očekivane veličine od 1017 bp. Sekvence izolata 137-08 (Acc. No. GQ259958) i 159-08 (GU144020) pokazale su 94-99% nukleotidne identičnosti sa izolatima WMV iz drugih delova sveta. Sekvence ova dva izolata razlikovala su se jedna od druge samo u dva nukleotidna mesta što nije imalo za posledicu aminokiselinske supstitucije. Filogenetske analize 57 izolata, na osnovu 750 bp sekvence gena za protein omotača, nisu ukazale na korelaciju sa geografskim poreklom izolata, a pokazala su grupisanje izolata u tri molekularne grupe sojeva. Izolati WMV iz Srbije pripadaju grupi I, grupi koja obuhvata najrasprostranjenije izolate ovog virusa u svetu

Biološka varijabilnost virusa žutog mozaika cukinija u Srbiji

Zucchini yellow mosaic virus (ZYMV) has become an important pathogen of cucurbits over the past 20 years. In our country, this virus also causes significant losses, especially in oil pumpkin production. Although ZYMV variability has been extensively studied worldwide by biological and molecular characterization, such information is still limited for the European region. Herein, we present the first analysis of phenotypic variations of ZYMV isolates sampled from oil pumpkin cv. Olinka from different locations in Serbia during 2008. Twelve isolates were obtained by mechanical inoculation on the C. pepo hybrid Ezra F1, and four of them were chosen for further detailed biological study. These investigated isolates exhibited different phenotypical features and thus represented different variants, concerning their host range, and type and severity of symptoms. A previously developed primer pair, ZYMV CPfwd/CPrev, was tested to determine its suitability for detection of Serbian ZYMV isolates. Amplification of Serbian ZYMV isolates of different origin, in single and mixed infection, resulted in one distinctive band. The used primer pair proved to be useful not only for routine molecular detection, but also for further molecular characterization of Serbian ZYMV isolates because it amplifies the hypervariable genomic region of ZYMV. The result obtained in this study is a contribution to investigation of genetic diversity of Serbian ZYMV isolates and their molecular relationship with ZYMV isolates from other parts of the world.Poslednjih 20 godina virus žutog mozaika cukinija (Zucchini yellow mosaic virus, ZYMV) se javlja kao značajan patogen biljaka iz familije tikava. Kod nas ovaj virus, takođe, izaziva značajne gubitke, naročito u proizvodnji uljane tikve. Iako je biološka i molekularna varijabilnost ZYMV dosta ispitivana, još uvek nema dovoljno informacija o varijabilnosti evropskih izolata ovog virusa. Ovaj rad predstavlja prvu analizu fenotipske varijabilnosti izolata ZYMV poreklom iz Srbije, koji su tokom 2008. godine sakupljeni iz različitih lokaliteta gajenja uljane tikve sorte Olinka. Mehaničkim inokulacijama C. pepo hibrid Ezra F1, dobijeno je 12 izolata, od kojih su četiri odabrana za dalju biološku karakterizaciju. Na osnovu različitog kruga domaćina, tipa i jačine simptoma na test-biljkama, utvrđena je fenotipska varijabilnost ispitivanih izolata. Par prajmera ZYMV CPfwd/Cprev testiran je radi utvrđivanja pogodnosti za rutinsku detekciju izolata ZYMV poreklom iz Srbije. Na osnovu pojave traka očekivane veličine u gelu, ZYMV je detektovan i u pojedinačnim i mešanim infekcijama u biljkama sa različitih lokaliteta, što dokazuje pogodnost ovih prajmera. Kako ovi prajmeri amplifikuju hipervarijabilni deo genoma virusa, pogodni su ne samo za rutinsku molekularnu detekciju, već i za molekularnu karakterizaciju izolata ZYMV iz Srbije. Rezultati dobijeni u ovom radu omogućiće dalja proučavanja genetičke varijabilnosti izolata ZYMV iz Srbije, kao i njihove evolutivne povezanosti sa izolatima iz drugih delova sveta

Novi pravci primene otpornosti tikava u kontroli virusnih oboljenja

As there is a growing frequency of viral plant diseases in epidemic proportions, the possibilities for successful control are constantly being explored. Despite the fact that integral and simultaneous employment of numerous control measures may contribute to the decreasing amount of yield losses, especially concerning non-persistently aphid-transmitted viruses, these measures are often not efficient enough. Research into the basis of resistance to viral infection and principles of its inheritance, introduction of sources of resistance in susceptible genotypes, by conventional or genetic manipulations, are very intensive for cucurbit crops, especially pumpkins. Pumpkin crops are being endangered by a great number of different viruses, among which the Zucchini yellow mosaic virus, (ZYMV), Watermelon mosaic virus (WMV) and Cucumber mosaic virus (CMV) are present every year in Serbia, frequently causing epidemics. The majority of pumpkin cultivars are not resistant or tolerant to viral infections, but sources of resistance have been identified in various related species. So far, the identified sources of resistance to the ZYMV are found in Cucurbita moschata and Citrullus lanatus var. lanatus genotypes and consist of one or several major dominant genes of resistance. It is a similar case with WMV, although the sources of dominant major genes are identified in C. lanatus and C. colocynthis. The sources of resistance to CMV in the form of one dominant gene have been identified in the genotype C. moschata, although the introduction of this gene by conventional means proved to be very difficult. Besides the aforementioned, substantial efforts are being made in developing genotypes with multiple resistance against several viruses and even other pathogens, as well as genotypes with resistance to the most significant plant aphid species, through mechanisms of antixenosis or antibiosis. The other way of obtaining resistant genotypes includes genetic manipulation. Genetically modified resistant pumpkins have been among the first successfully developed crops. Genotypes with pathogen derived resistance can already be found in commercially grown pumpkins in some parts of the world, and they have been developed by introducing the coat protein gene of one, two or all three viruses which are the most frequent, ZYMV, WMV and CMV. Yet, this approach to the control of pumpkin viral diseases is related to possible negative consequences, mostly through the already detected gene transfer to wild plants and development of resistant transgenic weeds of unpredictable impact on the environment. Improved host plant genetic resistance to viral infections or biological vectors, developed by conventional or genetic engineering methods, represents the most dynamic and prominent field of research. It is economically and ecologically the most justified approach to the control of pumpkin and other plant diseases caused by viruses non-persistently transmitted by aphids.Kako je pojava brojnih viroza gajenih biljaka u epidemijskim razmerama sve češća, mogućnosti za uspešnu kontrolu stalno se ispituju. Integralna i istovremena primena brojnih pojedinačnih mera kontrole, naročito u slučaju virusa koji se prenose biljnim vašima na neperzistentan način, mogu da doprinesu smanjenju šteta, ali često nisu dovoljno efikasne. Proučavanja osnova otpornosti i načina nasleđivanja, unošenje izvora otpornosti u osetljive genotipove, konvencionalnim ili genetičkim manipulacijama, vrlo su intenzivna kod vrežastih kultura, posebno tikve. Tikve ugrožava veliki broj raznorodnih virusa od kojih se u Srbiji svake godine javljaju virus žutog mozaika cukinija (Zucchini yellow mosaic virus, ZYMV), virus mozaika lubenice (Watermelon mosaic virus, WMV) i virus mozaika krastavca (Cucumber mosaic virus, CMV), a pojedinih godina izazivaju epidemije. Većina sorti gajenih tikava ne poseduje otpornost ili tolerantnost na virusne zaraze, ali izvori otpornosti identifikovani su u različitim srodnim vrstama. Do sada identifikovani izvori otpornosti na ZYMV nalaze se u genotipovima Cucurbita moschata i Citrullus lanatus var. lanatus i obuhvataju jedan ili nekoliko major gena rezistentnosti koji se nasleđuju dominantno. Slična situacija je i sa WMV, mada su izvori dominantnih major gena identifikovani u C. lanatus i C. colocynthis. Izvor otpornosti na CMV u vidu jednog dominantnog gena identifikovan je u genotipu C. moschata, mada je unošenje ovog gena konvencionalnim putem bilo veoma teško. Pored toga, veliki napori ulažu se u dobijenje genotipova sa istovremenom otpornošću na više virusa čak i drugih patogena, kao i genotipova koji ispoljavaju otpornost na najznačajnije vrste biljnih vaši, kroz mehanizme antiksenoze ili antibioze. Drugi pravac dobijanja otpornih genotipova podrazumeva genetičke manipulacije. Genetski modifikovane otporne tikve su među prvima, od svih gajenih biljaka, uspešno razvijene. Genotipovi sa PD (pathogen derived) otpornošću već se nalaze u komercijalnoj proizvodnji tikava u nekim delovima sveta i dobijeni su unošenjem gena za CP (coat protein) jednog, dva ili sva tri u svetu najraširenija virusa, ZYMV, WMW i CMV. Ipak, ovaj prilaz kontroli virusnih oboljenja tikava vezan je za uočene moguće negativne posledice, pre svega kroz već registrovan transfer gena u biljke spontane flore i nastanak otpornih transgenih korova nepredvidljivog ponašanja i značaja u prirodi. Poboljšana genetička otpornost biljke domaćina bilo na infekciju virusima, bilo na biološke vektore, dobijena konvencionalnim ili metodama genetičkog inženjeringa predstavlja najdinamičnije i najperspektivnije polje istraživanja kao ekonomski i ekološki najopravdaniji pristup kontroli oboljenja tikava i drugih biljaka koje izazivaju virusi koji se neperzistetno prenose biljnim vašima

Prisustvo i rasprostranjenost virusa uljane tikve i molekularna detekcija virusa žutog mozaika cukinija

Over the past decade, intensive spread of virus infections of oilseed pumpkin has resulted in significant economic losses in pumpkin crop production, which is currently expanding in our country. In 2007 and 2008, a survey for the presence and distribution of oilseed pumpkin viruses was carried out in order to identify viruses responsible for epidemics and incidences of very destructive symptoms on cucurbit leaves and fruits. Monitoring and collecting samples of oil pumpkin, as well as other species such as winter and butternut squash and buffalo and bottle gourd with viral infection symptoms, was conducted in several localities of Vojvodina Province. The collected plant samples were tested by DAS-ELISA using polyclonal antisera specific for the detection of six most economically harmful pumpkin viruses: Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMW), Squash mosaic virus (SqMV), Papaya ringspot virus (PRSV) and Tobacco ringspot virus (TRSV) that are included in A1 quarantine list of harmful organisms in Serbia. Identification of viruses in the collected samples indicated the presence of three viruses, ZYMV, WMV and CMV, in individual and mixed infections. Frequency of the identified viruses varied depending on locality and year of investigations. In 2007, WMV was the most frequent virus (94.2%), while ZYMV was prevalent (98.04%) in 2008. High frequency of ZYMV determined in both years of investigation indicated the need for its rapid and reliable molecular detection. During this investigation, a protocol for ZYMV detection was developed and optimized using specific primers CPfwd/Cprev and commercial kits for total RNA extraction, as well as for RT-PCR. In RT-PCR reaction using these primers, a DNA fragment of approximately 1100 bp, which included coat protein gene, was amplified in the samples of infected pumkin leaves. Although serological methods are still useful for large-scale testing of a great number of samples, this protocol, due to its high sensitivity and specificity, is an important improvement in rapid diagnosis of diseases caused by this virus. In addition, the protocol provides a basis for further characterization of ZYMV isolates originating from Serbia.Intenzivno širenje virusa infektivnih za uljanu tikvu (Cucurbita pepo), poslednjih deset godina imalo je za posledicu značajne ekonomske gubitke u proizvodnji ove kulture koja se gaji na sve većim površinama u našoj zemlji. Kako bi se identifikovali virusi, odgovorni za epidemijsku pojavu i ispoljavanje veoma destruktivnih simptoma na lišću i plodovima uljane tikve, tokom 2007. i 2008. sprovedeno je ispitivanje njihove pojave i rasprostranjenosti. Pregled i sakupljanje uzoraka uljane tikve, kao i nekih drugih vrsta tikava, kao što su bundeva, muskatna i bizonska tikva i vrg sa simptomima virusnih zaraza, obavljeno je na više različitih lokaliteta gajenja uljane tikve u Vojvodini. Sakupljeni uzorci testirani su DAS-ELISA metodom primenom poliklonalnih antiseruma specifičnih za detekciju u svetu šest ekonomski najznačajnijih virusa tikava: virusa mozaika krastavca (Cucumber mosaic virus, CMV), virusa žutog mozaika cukinija (Zucchini yellow mosaic virus, ZYMV), virusa mozaika lubenice (Watermelon mosaic virus, WMW), virusa mozaika bundeve (Squash mosaic virus, SqMV), virusa prstenaste pegavosti papaje (Papaya ringspot virus, PRSV) i virusa prstenaste pegavosti duvana (Tobacco ringspot virus, TRSV), koji se nalazi na A1 karantinskoj listi štetnih organizama u Srbiji. Identifikacija virusa u sakupljenim uzorcima ukazala je na prisustvo tri virusa, ZYMV, WMV i CMV, koji su se javili u pojedinačnim ili mešanim infekcijama. Njihova učestalost je po pojedinim godinama i lokalitetima bila različita. Tokom 2007. najčešće je dokazan WMV (94,2%), dok je 2008. godine prevalentan virus bio ZYMV (98,04%). Velika učestalost ZYMV u obe godine ispitivanja ukazala je na potrebu za brzom i pouzdanom molekularnom detekcijom ovog virusa. U toku ovih istraživanja razvijen je i optimizovan protokol za detekciju ZYMV primenom specifičnih prajmera CPfwd/CPrev i komercijalnih kitova za ekstrakciju ukupne RNA i RT-PCR. Korišćenjem ovih prajmera, kojim se amplifikuje deo genoma ZYMV kojim je obuhvaćen i gen za proteinski omotač, umnožen je DNK fragment dužine oko 1100 bp iz lišća zaraženih biljaka. Mada serološke metode i dalje imaju veliku prednost u primeni za masovna testiranja velikog broja uzoraka, razvijeni protokol molekularne detekcije, zbog visoke osetljivosti i specifičnosti predstavlja značajno poboljšanje brze dijagnoze oboljenja koja ovaj virus izaziva. Osim toga, ovaj protokol pruža osnovu za dalju karakterizaciju ZYMV izolata poreklom iz Srbije

Karakterizacija virusa mozaika krastavca poreklom iz tikava u Srbiji

Cucumber mosaic virus (CMV) is considered one of the most economically important plant viruses and has a worldwide distribution and a very wide host range including plants from family Cucurbitaceae. In Serbia, on cucurbits CMV was detected in single and mixed infections with Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus (WMV). Viruses, including CMV, are constantly present in cucurbit crops, but their frequency changes by year and locality. Surveys and sample collections were conducted in cucurbit crops in the period from 2008 to 2009 at 15 localities in Vojvodina province, and sample testing was carried out using the DAS-ELISA method and commercially available antisera for six economically most important cucurbit viruses. In 2008, a total of 51 samples were collected from 13 cucurbit crops of oilseed pumpkin Olinka variety, squash, and bottle gourd and CMV was detected in a total of 55% of tested samples with symptoms of viral infection. The most common infectious type was mixed infection with ZYMV and WMV (35.3%), and then mixed infection with ZYMV (17.7%) and WMV (2%). A total of 599 symptomatic samples of oilseed pumpkin Olinka variety, zucchini squash varieties Beogradska and Tosca, squash, and winter squash were collected in 15 cucurbits crops in 2009. CMV was present in 4.4% of total collected samples, in single infections in 1.3%, and in mixed with WMV or ZYMV in 1.3%, and 1.8%. Five CMV isolates were obtained by mechanical inoculations of N. glutinosa and one of them was selected for further biological characterization. Test plants which were described to be hosts of CMV expressed symptoms characteristic for those caused by CMV after inoculations by isolate 115-08. CMV specific primers Au1u/Au2d were used to amplify an 850 bp fragment using RT-PCR method. Amplified fragment encodes the entire viral coat protein (CP) gene and partial 5’ and 3’ UTRs of two selected CMV isolates. Amplified fragments were sequenced and deposited in the NCBI, where they were assigned accession numbers, HM065510 (115-08) and HM065509 (151-08). The sequences of CMV isolates from Serbia shared the highest nucleotide and amino acid identity with isolates from subgroup IA, from 99.5 to 97.4% and 99.1 to 97.4%, and the lowest identities were with the subgroup II isolates from 66.9 to 64, 5%, from 75.8 to 74.1%.Virus mozaika krastavca (Cucumber mosaic virus, CMV) je veoma široko rasprostranjen i jedan je od ekonomski najznačajnijih virusa za poljoprivrednu proizvodnju, sa veoma širokim krugom domaćina u koji spada i veliki broj gajenih biljaka, među kojima i predstavnici familije Cucurbitaceae. Epidemiologija ovog virusa je veoma složena, zbog čega je i njegova kontrola otežana. Na tikvama u Srbiji CMV je detektovan u pojedinačnim i mešanim infekcijama sa virusom žutog mozaika cukinija (Zucchini yellow mosaic virus, ZYMV) i virusom mozaika lubenice (Watermelon mosaic virus, WMV). Virusi, pa tako i CMV, su stalno prisutni u usevima tikava, ali se njihova učetalost menja po godinama i lokalitetima. Pregledi terena i sakupljanje uzoraka tikava obavljeni su u periodu 2008-2009. godine na 15 lokaliteta gajenja tikava u Vojvodini, a testiranje sakupljenih uzoraka izvršeno je primenom DAS-ELISA metode i komercijalno dostupnih antiseruma za šest ekonomski najznačajnih virusa tikava u svetu. Tokom 2008. godine u 13 useva gajenih tikava sakupljen je ukupno 51 uzorak uljane tikve sorte Olinka, bundeve i vrga, a CMV je detektovan u ukupno 55% testiranih uzoraka sa simptomima virusnih zaraza. CMV je najčešće bio detektovan u mešanoj infekciji sa ZYMV i WMV (35,3%), a zatim u mešanoj infekciji sa ZYMV (17,7%) i sa WMV (2%). Ukupno 599 simptomatičnih uzoraka uljane tikve sorte Olinka, tikvica sorti Beogradska i Toska, bundeve i muskatne tikve sakupljeno je u 15 useva tokom 2009. godine. U sakupljenim uzorcima CMV je bio prisutan u ukupno 4,4% testiranih uzoraka. U pojedinačnim infekcijama u 1,3%, a u mešanim sa WMV ili ZYMV u 1,3%, odnosno 1,8% testiranih uzoraka. Mehaničkim inokulacijama N. glutinosa dobijeno je pet izolata CMV od kojih je jedan izabran za dalju biološku karakterizaciju. Odabrani izolat (115-08) izazvao je na test biljkama koje su opisane kao domaćini CMV pojavu karakterističnih simptoma. Specifičnim prajmerima CMV Au1u/Au2d uz upotrebu RT-PCR metode umnožen je deo genoma od oko 850 bp koji kodira ceo protein omotača virusa i delove 5’ i 3’ neprepisujućih regiona dva odabrana izolata CMV. Amplifikovani fragmenti su sekvencionirani i prijavljeni u NCBI, gde su im dodeljeni pristupni brojevi, HM065510 (115-08) i HM065509 (151-08). Sekvence CMV izolata iz Srbije dele najveću nukleotidnu i aminokiselinsku sličnost sa izolatima iz podgrupe IA, od 99,5 do 97,4%, odnosno 99,1 do 97,4%, a najmanju sa izolatima II podgrupe od 66,9 do 64,5%, odnosno od 75,8 do 74,1%

Prisustvo i molekularna karakterizacija virusa mozaika lucerke u usevu duvana u Srbiji

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