Diverzitet, biološka i molekularna karakterizacija virusa tikava i epidemologija oboljenja u Srbiji

In the period from 2007 to 2011 constant high levels of viral infections were detected in 61 cucurbit growing localities in Serbia. The frequency of viral infection fluctuated over the years, ranging from 30-50% in 2007, to the extremely high 80% in the epidemic year of 2008, while it was constant and around 40% in the next two years. In the last year of investigation, the frequency reached a very high level of 60%. Serological testing of 1100 collected samples proved the presence of three viruses: Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV) and Cucumber mosaic virus (CMV). Additional testing of symptomatic samples revealed the presence of Cucurbit aphid-borne yellows virus (CABYV), which represents the first detection of CABYV in Serbia, and Tomato spotted wilt virus (TSWV), which is the first report of TSWV on cucurbits in Serbia and the first report of Cucurbita maxima as natural host plant worldwide. ZYMV was the most prevalent virus during this five year survey. Mixed infections were the most frequent infection type in the first two years, while single infection prevailed in the last three years of investigation. During this investigation, transmission of ZYMV by seeds of C. pepo var. styriaca, C. maxima and C. moschata was proved. Both annual and perennial weeds, which are present during the establishment of cucurbit crops, and in the beginning stages of vegetation, were infected with ZYMV, WMV, CMV and TSWV. The most prevalent was TSWV which was detected in eight different weed species, the second most prevalent virus in weeds was CMV, detected in seven different weed species, while the prevalence of ZYMV and WMV was similar and they were detected in five and four weed species, respectively. A high diversity of aphids which are visiting cucurbit crops was detected through the determination of 57 aphid taxons. The most common aphid species were Aphis fabae, Anoecia corni, Aphis gossypii, Myzus persicae and Acyrthosiphon pisum. The greatest diversity and the largest number of aphids were detected in the first few weeks after plant sprouting. The detection and identification of the species level for all five cucurbit viruses in Serbia were confirmed by RT-PCR method and suitable specific primers. Bioassay on 45 test plants was used to determine phenotypic variability and host range for ZYMV, WMV and CMV...U periodu od 2007. do 2011. godine na 61 lokalitetu gajenja tikava u Srbiji utvrđeno je stalno prisustvo virusa sa visokim intenzitetom zaraze. Učestalost virusne zaraze se po godinama mijenjala i kretala od 30-50% u 2007. godini, preko izuzetno visokih 80% u epidemijskoj 2008. godini, da bi tokom naredne dvije godine bila konstantna i kretala se oko 40%. U posljednjoj godini ispitivanja zaraza je ponovo dostigla čak 60%. Serološkim testiranjem 1100 uzoraka sakupljenih sa 19 vrsta, sorti i varijeteta tikava detektovano je prisustvo tri virusa: virusa žutog mozaika cukinija (Zucchini yellow mosaic virus, ZYMV), virusa mozaika lubenice (Watermelon mosaic virus, WMV) i virusa mozaika krastavca (Cucumber mosaic virus, CMV). Dodatnim testiranjem simptomatičnih uzoraka detektovano je prisustvo virusa žutila tikava perzistentno prenosivog vašima (Cucurbit aphid-borne yellows virus, CABYV), što predstavlja prvi nalaz ovog virusa u Srbiji i virusa bronzavosti paradajza (Tomato spotted wilt virus, TSWV), što je prvi nalaz ovog virusa na tikvama u Srbiji, ali i prvi nalaz u prirodnoj infekciji na Cucurbita maxima u svijetu. Najzastupljeniji virus tokom pet godina ispitivanja bio je ZYMV. Tokom prve dvije godine najčešće su bile detektovane miješane infekcije, a u poslednje tri godine pojedinačne. U okviru ovih istraživanja dokazano je prenošenje ZYMV sjemenom Cucurbita pepo var. styriaca, C. maxima i C. moschata. Utvrđena je zaraženost jednogodišnjih i višegodišnjih korovskih biljaka, koje su prisutne u vrijeme zasnivanja usjeva tikava i u početnim fazama vegetacije, sa ZYMV, WMV, CMV i TSWV. Najzastupljeniji je bio TSWV, a njegovo prisustvo je dokazano u osam različitih korovskih vrsta, sljedeći po zastupljenosti je bio CMV, dokazan u sedam vrsta korova, dok su ZYMV i WMV bili slični po zastupljenosti, a dokazani su u pet, odnosno četiri korovske vrste. Pokazan je visok diverzitet biljnih vaši koje posjećuju usjeve tikava kroz determinaciju 57 taksona. Najzastupljenije vrste vaši bile su Aphis fabae, Anoecia corni, Aphis gossypii, Myzus persicae i Acyrthosiphon pisum. Najveći diverzitet i najveća brojnost vrsta vaši bila je u prvim nedjeljama po nicanju biljaka. Detekcija i identifikacija do nivoa vrste svih prouzrokovača virusnih oboljenja na tikvama u Srbiji potvrđena je primjenom RT-PCR metode i odgovarajućih specifičnih prajmera i sekvencioniranjem. Fenotipska varijabilnost i krug domaćina za ZYMV, WMV i CMV ispitivani su primjenom biotesta na ukupno 45 test biljaka..

Prisustvo i rasprostranjenost virusa žute prugavosti praziluka u usevima različitih vrsta lukova u Srbiji

Leek yellow stripe virus (LYSV) is one of the most frequent and important viruses in leek and garlic crops worldwide. In Serbia this virus is found both in leek and garlic, and often at high percentages. During two consecutive years, 2012 and 2013, a total 92 samples were collected from 11 inspected leek-, garlic- and onion-growing locations and they were analyzed for the presence of LYSV using DAS-ELISA. LYSV was detected in 31.5% of the tested samples. In 2012, the presence of LYSV was only detected in leek plants, and in 55.6% of the tested samples. During 2013, LYSV was detected in 85% of leek and 58.3% of garlic samples. In total, LYSV was detected in 56.4% of leek samples and 17.1% garlic samples. LYSV incidence was confirmed using RT-PCR with LYSV specific primers amplifying 1020 bp fragment representing coat protein and part of nuclear inclusion B genes. Molecular identification was confirmed by sequencing of two selected isolates, 181-13 (MG242625) from garlic and 298-13 (MG242624) from leek, and comparing them to the GenBank sequences of LYSV. Phylogenetic analysis of 55 sequences of LYSV from all over the world showed some correlation between host plant and geographical origin of the isolates, forming five separate clades. Two Serbian LYSV isolates fell into distant clades. The Serbian leek isolate 298-13 of LYSV belongs to clade B, while isolate 181-13 originating from garlic belongs in clade E.Virus žute prugavosti praziluka (Leek yellow stripe virus, LYSV) je jedan od najčešćih i najznačajnijih virusa na praziluku i belom luku. U Srbiji je virus detektovan na obe kulture, i praziluku i belom luku, a često se javlja u visokim procentima. Tokom dve uzastopne godine (2013. i 2014.) pregledano je 11 lokaliteta gajenja praziluka, belog i crnog luka i sakupljena su 92 uzorka koja su DAS-ELISA metodom testirana na prisustvo LYSV. Prisustvo LYSV je dokazano u 31,5% testiranih uzoraka. Tokom 2012., prisustvo LYSV dokazano je samo u praziluku, u 55,6% testiranih uzoraka. Tokom 2013., LYSV je dokazan u 85% uzoraka praziluka i 58,3% uzoraka belog luka. Ukupno, prisustvo LYSV je dokazano u 56,4% uzoraka praziluka i 17,1% uzoraka belog luka. Prisustvo LYSV u testiranim uzorcima potvrđeno je primenom RT-PCR metode i specifičnih prajmera za LYSV koji umnožavaju fragment od 1020 bp koji obuhvata gen za proteinski omotač i deo gena za nuklearne inkluzije B. Molekularna identifikacija LYSV obavljena je sekvenciranjem dva odabrana izolata iz belog luka 181-13 (MG242625) i praziluka 298-13 (MG242624) i poređenjem dobijenih sekvenci sa sekvencama LYSV iz GenBank baze podataka. Filogenetske analize 55 sekvenci izolata LYSV iz različitih delova sveta ukazale su na delimičnu korelaciju između biljke domaćina i geografskog porekla izolata, formiranjem pet odvojenih grupa izolata u stablu. Dva izolata iz Srbije grupisala su se u udaljene grupe. Izolat iz Srbije iz praziluka 298-13 grupisao se u grupu B, dok se izolat iz belog luka 181-13 grupisao u grupu E

Diversity, biological and molecular characterization of cucurbit viruses and disease epidemiology in Serbia

U periodu od 2007. do 2011. godine na 61 lokalitetu gajenja tikava u Srbiji utvrđeno je stalno prisustvo virusa sa visokim intenzitetom zaraze. Učestalost virusne zaraze se po godinama mijenjala i kretala od 30-50% u 2007. godini, preko izuzetno visokih 80% u epidemijskoj 2008. godini, da bi tokom naredne dvije godine bila konstantna i kretala se oko 40%. U posljednjoj godini ispitivanja zaraza je ponovo dostigla čak 60%. Serološkim testiranjem 1100 uzoraka sakupljenih sa 19 vrsta, sorti i varijeteta tikava detektovano je prisustvo tri virusa: virusa žutog mozaika cukinija (Zucchini yellow mosaic virus, ZYMV), virusa mozaika lubenice (Watermelon mosaic virus, WMV) i virusa mozaika krastavca (Cucumber mosaic virus, CMV). Dodatnim testiranjem simptomatičnih uzoraka detektovano je prisustvo virusa žutila tikava perzistentno prenosivog vašima (Cucurbit aphid-borne yellows virus, CABYV), što predstavlja prvi nalaz ovog virusa u Srbiji i virusa bronzavosti paradajza (Tomato spotted wilt virus, TSWV), što je prvi nalaz ovog virusa na tikvama u Srbiji, ali i prvi nalaz u prirodnoj infekciji na Cucurbita maxima u svijetu. Najzastupljeniji virus tokom pet godina ispitivanja bio je ZYMV. Tokom prve dvije godine najčešće su bile detektovane miješane infekcije, a u poslednje tri godine pojedinačne. U okviru ovih istraživanja dokazano je prenošenje ZYMV sjemenom Cucurbita pepo var. styriaca, C. maxima i C. moschata. Utvrđena je zaraženost jednogodišnjih i višegodišnjih korovskih biljaka, koje su prisutne u vrijeme zasnivanja usjeva tikava i u početnim fazama vegetacije, sa ZYMV, WMV, CMV i TSWV. Najzastupljeniji je bio TSWV, a njegovo prisustvo je dokazano u osam različitih korovskih vrsta, sljedeći po zastupljenosti je bio CMV, dokazan u sedam vrsta korova, dok su ZYMV i WMV bili slični po zastupljenosti, a dokazani su u pet, odnosno četiri korovske vrste. Pokazan je visok diverzitet biljnih vaši koje posjećuju usjeve tikava kroz determinaciju 57 taksona. Najzastupljenije vrste vaši bile su Aphis fabae, Anoecia corni, Aphis gossypii, Myzus persicae i Acyrthosiphon pisum. Najveći diverzitet i najveća brojnost vrsta vaši bila je u prvim nedjeljama po nicanju biljaka. Detekcija i identifikacija do nivoa vrste svih prouzrokovača virusnih oboljenja na tikvama u Srbiji potvrđena je primjenom RT-PCR metode i odgovarajućih specifičnih prajmera i sekvencioniranjem. Fenotipska varijabilnost i krug domaćina za ZYMV, WMV i CMV ispitivani su primjenom biotesta na ukupno 45 test biljaka...In the period from 2007 to 2011 constant high levels of viral infections were detected in 61 cucurbit growing localities in Serbia. The frequency of viral infection fluctuated over the years, ranging from 30-50% in 2007, to the extremely high 80% in the epidemic year of 2008, while it was constant and around 40% in the next two years. In the last year of investigation, the frequency reached a very high level of 60%. Serological testing of 1100 collected samples proved the presence of three viruses: Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV) and Cucumber mosaic virus (CMV). Additional testing of symptomatic samples revealed the presence of Cucurbit aphid-borne yellows virus (CABYV), which represents the first detection of CABYV in Serbia, and Tomato spotted wilt virus (TSWV), which is the first report of TSWV on cucurbits in Serbia and the first report of Cucurbita maxima as natural host plant worldwide. ZYMV was the most prevalent virus during this five year survey. Mixed infections were the most frequent infection type in the first two years, while single infection prevailed in the last three years of investigation. During this investigation, transmission of ZYMV by seeds of C. pepo var. styriaca, C. maxima and C. moschata was proved. Both annual and perennial weeds, which are present during the establishment of cucurbit crops, and in the beginning stages of vegetation, were infected with ZYMV, WMV, CMV and TSWV. The most prevalent was TSWV which was detected in eight different weed species, the second most prevalent virus in weeds was CMV, detected in seven different weed species, while the prevalence of ZYMV and WMV was similar and they were detected in five and four weed species, respectively. A high diversity of aphids which are visiting cucurbit crops was detected through the determination of 57 aphid taxons. The most common aphid species were Aphis fabae, Anoecia corni, Aphis gossypii, Myzus persicae and Acyrthosiphon pisum. The greatest diversity and the largest number of aphids were detected in the first few weeks after plant sprouting. The detection and identification of the species level for all five cucurbit viruses in Serbia were confirmed by RT-PCR method and suitable specific primers. Bioassay on 45 test plants was used to determine phenotypic variability and host range for ZYMV, WMV and CMV..

Begomovirusi na krompirju

Virusi iz rodu begomovirusov (družina Geminiviridae) lahko okužijo številne vrste rastlin. Opisanih je več kot 400 različnih begomovirusov, od tega lahko nekateri okužijo tudi krompir

Kompleks virusa prouzrokovača uvijenosti lišća vinove loze

The number of virus species infecting grapevine has increased continuously and more than 60 species are described to infect grapevine. The global expansion of the grape and wine industry has seen a parallel increase in the incidence and economic impact of grapevine leafroll disease (GLD). GLD can cause reduced plant vigor and longevity, and significant losses in both fruit yield and quality. Our knowledge of the nature of the disease is still quite limited due to a variety of challenges related to the complexity of this virus disease, the association of several genetically and serologically distinct GLRaVs, and contrasting symptoms in red- and white-berried cultivars. Within grapevine leafroll complex (Grapevine leafroll-associated viruses, GLRaVs) five different species are classified in the family Closteroviridae: one is classified in the genus Closterovirus (GLRaV-2), three in the genus Ampelovirus (GLRaV-1, GLRaV -3, GLRaV -4, and his strains: GLRaV-4 strain 5, GLRaV-4 strain 6, GLRaV-4 strain 9, GLRaV-4 strain Pr, GLRaV-4 strain De and GLRaV-4 strain Car) and one in the genus Velarivirus (GLRaV-7). GLRaVs are transmitted by grafting and propagation material (rootstock and scion) and some species by mealybugs. Grapevine leafroll-associated viruses usually occur in a mixed infection, and are often present in a mixed infection with other grape viruses. Early research carried out in vineyards in Serbia determined the presence of GLRV-1, -2 and -3, with GLRaV-3 being the most prevalent. If spread of the viruses transsmited by vectors within plantings exists, roguing or removal of infected vines, mealybug control, and sanitation measures can reduce new infections.Brojnost virusa infektivnih za vinovu lozu stalno se povećava, tako da je do sada opisano više od 60 vrsta koji mogu da zaraze vinovu lozu. Paralelno sa globalnom ekspanzijom vinogradarske i vinarske industrije zapaženo je povećanje učestalosti i ekonomskog značaja oboljenja uvijenosti lišća vinove loze (Grapevine Leafroll Disease, GLD). GLD, osim što smanjuje vitalnost i dugovečnost vinove loze, značajno smanjuje prinos i kvalitet grožđa. Epidemiologija oboljenja je nedovoljno istražena zbog kompleksnosti ove bolesti, genetički i serološki različitih vrsta unutar kompleksa i različitosti simptoma kod crvenih i belih sorti vinove loze. Кompleks virusa prouzrokovača uvijenosti lišća vinove loze (Grapevine leafrollassociated viruses, GLRaVs) čini pet različitih virusa koji pripadaju familiji Closteroviridae: jedna vrsta koja pripada rodu Closterovirus (GLRaV-2), tri rodu Ampelovirus (GLRaV-1, GLRaV -3, GLRaV -4, i njegovi sojevi: GLRaV-4 soj 5, GLRaV-4 soj 6, GLRaV-4 soj 9, GLRaV-4 soj Pr, GLRaV-4 soj De i GLRaV-4 soj Car) i jedna vrsta iz roda Velarivirus (GLRaV-7). GLRaVs se prenose kalemljenjem i propagativnim materijalom (podloga i vijoka), a neke vrste i štitastim vašima. Virusi koji izazivaju uvijenost lišća se obično javljaju u mešanoj infekciji, a često su prisutni i u mešanoj infekciji sa drugim virusima vinove loze. Ranija istraživanja sprovedena u vinogorjima u Srbiji utvrdila su prisustvo GLRV-1, -2 i -3, pri čemu je GLRaV-3 bio najzastupljeniji. Ukoliko je širenje virusa unutar zasada putem vektora izraženo, krčenje ili uklanjanje zaraženih čokota, kontrola štitastih vaši i adekvatne sanitarne mere mogu redukovati nove infekcije

First Report of Tomato spotted wilt virus on Gerbera hybrida in Serbia.

In May 2009, approximately 30% of plants within a greenhouse-grown Gerbera hybrida crop in Vranjska Banja (Pčinj District) in Serbia displayed chlorotic oak-leaf patterns followed by necrosis and distortion of leaves. Symptoms on naturally infected gerbera plants and local necrotic spots on Petunia × hybrida mechanically inoculated with infected gerbera sap using chilled 0.05 M phosphate buffer (pH 7) containing 1 mM Na-EDTA, 5 mM Na-DIECA, and 5 mM Na-thioglycolate (4) suggested the presence of a Tospovirus. Symptomatic leaves were tested for the presence of Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV), and Chrysanthemum stem necrosis virus (CSNV) by commercial double-antibody sandwich (DAS)-ELISA diagnostic kits (Loewe Biochemica, Sauerlach, Germany). Commercial positive and negative controls and extract from healthy gerbera tissue were included in each ELISA. All 20 tested plants were negative for INSV and CSNV. TSWV was detected serologically in 18 of 20 gerbera samples. The presence of TSWV in ELISA-positive symptomatic gerbera plants was further confirmed by conventional reverse transcription (RT)-PCR. Total RNAs were extracted with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and RT-PCR was conducted with the OneStep RT-PCR Kit (Qiagen) using Serbian tobacco TSWV isolate (GQ279731) and RNA extract from healthy gerbera as positive and negative controls, respectively. Two different sets of TSWV-specific primers, L1 TSWVR/L2 TSWVF (2) and M962/M66 (3), for a 276-bp fragment of the RNA-dependent RNA polymerase (RdRp) gene and a 897-bp fragment of the NSm gene, respectively, were used for both amplification and sequencing. RT-PCR analyses of each tested plant detected the presence of amplification fragments of expected size. The amplified products corresponding to part of the RdRp and NSm genes derived from the isolate 158-Gerb were purified (QIAquick PCR Purification Kit, Qiagen) and sequenced in both directions (GenBank Accession Nos. HQ246452 and HQ246453, respectively). Sequence analysis of the partial RdRp gene, conducted using MEGA4 software, revealed 91.1 to 98% nt identity (95.1 to 98.8% amino acid [aa] identities) with corresponding sequences of TSWV L RNA deposited in GenBank. The highest identity was found with an isolate from globe artichoke (AM940436) in Greece, and isolates from tomato (GQ279732), impatiens (GQ132190), and tobacco isolates (GQ279731, FJ189392, and FJ189393) found within Serbia. Analysis of the NSm sequence of isolate 158-Gerb demonstrated nucleotide identities varying between 90.6 and 99.6% (80.9 and 99.6% aa identities) with those of previously reported TSWV isolates. The highest identity was with tobacco isolate GQ373174 from Serbia. Therefore, while gerbera is one of the principal ornamental hosts of TSWV in the EPPO region (1), to our knowledge, this is the first report infecting gerbera in Serbia, which may have a devastating influence on its production. References: (1) Anonymous. OEPP/EPPO Bull. 29:465, 1999. (2) R. A. Mumford et al. J. Virol. Methods 46:303, 1994. (3) W. P. Qiu et al. Virology 244:186, 1998. (4) P. Roggero et al. Plant Dis. 86:950, 2002. </jats:p

Status of tobacco viruses in Serbia and molecular characterization of tomato spotted wilt virus isolates

In a four-year survey to determine the presence and distribution of viruses in tobacco crops at 17 localities of the Vojvodina Province and Central Serbia, 380 samples were collected and analyzed by DAS-ELISA. Out of the seven viruses tested, tomato spotted wilt virus (TSWV), potato virus Y (PVY), tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), and alfalfa mosaic virus (AMV) were detected in 37.9, 33.4, 28.7, 23.9, and 15.5% of the total tested samples, respectively. TSWV was the most frequently found virus at the localities of Central Serbia, while PVY and CMV were the most frequent viruses in the Vojvodina Province. Single infections were prevalent in years 2005-2007 and the most frequent were those of PVY A triple combination of those viruses was most frequent mixed infection type in 2008. The presence of all five detected viruses was confirmed in selected ELISA-positive samples by RT-PCR and sequencing. The comparisons of obtained virus isolate sequences with those available in NCBI, confirmed the authenticity of serologically detected viruses. Phylogenetic analysis based on partial nucleocapsid gene sequences revealed a joint clustering of Serbian, Bulgarian and Montenegrin TSWV isolates into one geographic subpopulation, which was distinct from the other subpopulation of TSWV isolates from the rest of the European countries. The high incidence of viruses in Serbian tobacco crops highlights the importance of enhancing farmers knowledge towards better implementation of control strategies for preventing serious losses

First Report of Leek yellow stripe virus in Leek in Serbia

Leek yellow stripe virus (LYSV), one of the most important and widespread viruses of leek and garlic worldwide, is endemic in various countries of the Mediterranean basin (Katis et al. 2012). During an October 2013 survey for the presence of Allium viruses in Serbia, commercially grown leek (Allium porrum) plants with virus-like symptoms were observed in Padinska Skela (City of Belgrade District). Initially, the leaf symptoms included irregular chlorotic to light yellow dashes, particularly on the bases of leaves. The lesions later enlarged and coalesced, resulting in large, yellow stripes and the infected leaves turned yellow and flaccid, followed by die-back. Disease incidence in the leek field was estimated at 20%. A total of 15 symptomatic plants were sampled and tested by double-antibody sandwich (DAS)-ELISA test using commercial polyclonal antisera (Bioreba AG, Reinach, Switzerland) for the most important Allium viruses: LYSV, Garlic common latent virus, Onion yellow dwarf virus, and Iris yellow spot virus (Pappu et al. 2005, Katis et al. 2012). Commercial positive and negative controls and extracts from healthy leek leaves were included in each ELISA. All 15 tested leek samples were positive for LYSV and negative for the rest of tested viruses. Five carborundum-dusted plants of each Chenopodium quinoa and A. porrum ‘Varna’ were mechanically inoculated with sap prepared from ELISA-positive sample (277-13) using 0.01 M phosphate buffer (pH 7). Chlorotic local lesions on C. quinoa and streak mosaic on A. porrum ‘Varna’ were observed 5 and 16 days postinoculation, respectively, on all inoculated plants. Serological results were verified with reverse transcription (RT)-PCR. Total RNAs from all naturally and mechanically infected leek plants were extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RT-PCR was performed using One-Step RT-PCR Kit (Qiagen) and specific primer pair 1LYSV and 2LYSV (Fajardo et al. 2001). An approximately 1000-bp fragment corresponding to the part of nuclear inclusion B (NIb) and coat protein (CP) coding region was obtained from all 20 naturally and mechanically infected leek plants, while no amplicon was recorded in the healthy and water controls. RT-PCR product obtained from one selected isolate (277-13) was purified using QIAquick PCR Purification Kit (Qiagen), sequenced directly in both directions using the same primers as for amplification, and submitted to the GenBank (Accession No. KR075504). Sequence analysis, conducted by MEGA5 software (Tamura et al. 2011), revealed that the leek isolate from Serbia showed the highest nucleotide identity of 94.8% (94.6% amino acid identity) with the sequence of LYSV isolate from leek (X89711). To our knowledge, this is the first report of natural infection of leek with LYSV in Serbia. Leek is an important and traditionally grown vegetable crop in Serbia and the presence of LYSV could cause considerable damage and severe yield losses, resulting in significant economic impact on leek production

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 RTPCR 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 Serbi

First Report of Tomato spotted wilt virus on Chrysanthemum in Serbia

In July 2011, greenhouse-grown chrysanthemum hybrid plants (Chrysanthemum × morifolium) with symptoms resembling those associated with tospoviruses were observed in the Kupusina locality (West Bačka District, Serbia). Disease incidence was estimated at 40%. Symptomatic plants with chlorotic ring spots and line patterns were sampled and tested by double antibody sandwich (DAS)-ELISA using polyclonal antisera (Bioreba AG, Reinach, Switzerland) against the two of the most devastating tospoviruses in the greenhouse floriculture industry: Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV) (2). Commercial positive and negative controls and extracts from healthy chrysanthemum tissue were included in each ELISA. TSWV was detected serologically in 16 of 20 chrysanthemum samples and all tested samples were negative for INSV. The virus was mechanically transmitted from ELISA-positive chrysanthemum samples to five plants each of both Petunia × hybrida and Nicotiana tabacum ‘Samsun’ using chilled 0.01 M phosphate buffer (pH 7) containing 0.1% sodium sulfite. Inoculated plants produced local necrotic spots and systemic chlorotic/necrotic concentric rings, consistent with symptoms caused by TSWV (1). The presence of TSWV in ELISA-positive chrysanthemum plants and N. tabacum‘Samsun’ was further confirmed by conventional reverse transcription (RT)-PCR. Total RNAs were extracted with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) using primers TSWVCP-f/TSWVCP-r specific to the nucleocapsid protein (N) gene (4). A Serbian isolate of TSWV from tobacco (GenBank Accession No. GQ373173) and RNA extracted from a healthy chrysanthemum plant were used as positive and negative controls, respectively. An amplicon of the correct predicted size (738-bp) was obtained from each of the plants assayed, and that derived from chrysanthemum isolate 529-11 was purified (QIAqick PCR Purification Kit, Qiagen) and sequenced (JQ692106). Sequence analysis of the partial N gene, conducted with MEGA5 software, revealed the highest nucleotide identity of 99.6% (99% amino acid identity) with 12 TSWV isolates deposited in GenBank originating from different hosts from Italy (HQ830186-87, DQ431237-38, DQ398945), Montenegro (GU355939-40, GU339506, GU339508), France (FR693055-56), and the Czech Republic (AJ296599). The consensus maximum parsimony tree obtained on a 705-bp partial N gene sequence of TSWV isolates available in GenBank revealed that Serbian TSWV isolate 529-11 from chrysanthemum was clustered in the European subpopulation 2, while the Serbian isolates from tomato (GU369723) and tobacco (GQ373172-73 and GQ355467) were clustered in the European subpopulation 1 denoted previously (3). The distribution of TSWV in commercial chrysanthemum crops is wide (2). To our knowledge, this is the first report of TSWV infecting chrysanthemum in Serbia. Since chrysanthemum popularity and returns have been rising rapidly, the presence of TSWV may significantly reduce quality of crops in Serbia. References: (1) Anonymous. OEPP/EPPO Bull. 34:271, 2004. (2) Daughtrey et al. Plant Dis. 81:1220, 1997. (3) I. Stanković et al. Acta Virol. 55:337, 2011. (4) A. Vučurović et al. Eur. J. Plant Pathol. 133:935, 2012. </jats:p