Biological and molecular characterization of Cucumber mosaic virus in Serbia

Serološkim analizama primenom DAS-ELISA testa, u periodu od 2010. do 2013. godine, prisustvo virusa mozaika krastavca (Cucumber mosaic virus, CMV) utvrđeno je u 29,68% testiranih uzoraka poreklom iz 32 različite vrste, varijeteta ili sorte povrtarskih, industrijskih, ukrasnih i korovskih biljaka sa 74 lokaliteta. U okviru ovih ispitivanja Tulipa sp. i Peperomia tuisana prvi put su otkrivene kao domaćini u našoj zemlji, a Wisteria sinensis i Stenactis annua su otkrivene kao novi domaćini CMV ne samo kod nas, već i u svetu. Analiza genetičke strukture prirodne populacije CMV u Srbiji na osnovu biološke i molekularne karakterizacije obuhvatila je 44 odabrana izolata poreklom sa različitih biljnih vrsta, njihovih varijeteta ili sorti sakupljenih na 30 lokaliteta tokom ovih istraživanja, kao i izolate iz prethodnih istraživanjanja iz perioda 2007-2009. godine. Molekularna karakterizacija na osnovu analize sekvence CP gena obuhvatila je 44 izolata, dok su molekularna proučavanja na osnovu analize sekvenci 2a, 2b i MP gena obuhvatila 19 izolata, a 1a gena 15 izolata CMV. Tokom ovih istraživanja uspostavljeni su ili unapređeni protokoli za amplifikaciju svih pet genskih regiona izolata CMV poreklom iz naše zemlje. Stepen nukleotidne i aminokiselinske sličnosti, kao i filogenetske analize pokazale su varijabilnost prirodne populacije CMV u Srbiji kod četiri od pet genskih regiona. Najveća varijabilnost utvrđena je analizom dela 1a gena (grupisanje u tri podgrupe, IA, IB i II), delova MP i CP gena kao i celog 2b gena (grupisanje u dve podgrupe, IA i II), dok je analiza dela 2a gena pokazala homogenost populacije CMV i grupisanje svih ispitivanih izolata u IA podgrupu. Na osnovu filogenetskih analiza sekvenci pojedinačnih genskih regiona RNK 1, 2 i 3, kao i sekvenci RNK 3 i RNK 2 segmenta utvrđeno je prisustvo rekombinantnih i pseudorekombinantnih izolata u populaciji ovog virusa u Srbiji i identifikovano šest različitih haplotipova. Najveći broj izolata po svim genskim regionima pripada IA podgrupi (haplotip: IA; IA-IA; IA-IA). Po brojnosti sledi haplotip: II; IA-II; II-II zastupljen sa tri izolata, dok su preostala četiri haplotipa utvrđeni kod pojedinačnih izolata (haplotip: IA; IA-IA; IA-II, haplotip: IB; IA-IA; IA-IA, haplotip: II; IA-IA; II-II i haplotip: IB; IA-II; IA-IA)...During the period from 2010 to 2013, the serological analysis, using DAS-ELISA, detected the presence of Cucumber mosaic virus (CMV) in 29,68% of tested samples originating from 32 different species, varieties or cultivars of vegetables, industrial crops, ornamentals and weeds collected at 74 localities. Tulipa sp. and Peperomia tuisana are discovered as new host plants of CMV in Serbia, while Wisteria sinensis and Stenactis annua are detected as two new natural host plants worldwide. Analysis of genetic structure of CMV population in Serbia is based on biological and molecular characterization of 44 selected isolates from different host plants collected during this research at 30 localities, or collected in previous investigation during from 2007 to 2009. The molecular characterization based on the sequence analysis of CP gene included 44 isolates, while analysis of 2a, 2b and MP genes included 19 isolates and the analysis of 1a gene included 15 isolates of CMV. During this studies, the protocols for molecular amplification of all five genomic regions of CMV isolates from Serbia were either established or improved. Nucleotide and amino acid sequence identity, as well as phylogenetic analyses showed variability in four out of five genomic regions of CMV population in Serbia. The highest variability was determined within the partial 1a gene (grouping into three subgroups, IA, IB and II), partial MP and CP genes and the complete 2b gene (grouping into two subgroups IA and II), while sequence analysis of the partial gene 2a showed that CMV population is homogeneous, and all isolates grouped within IA subgroup. Phylogenetic analysis of particular genetic regions of RNA 1, 2 and 3, as well as combined genetic regions of RNA 3 and RNA 2, revealed the presence of recombinats and reassortments within the population of CMV in Serbia and identified the presence of six different haplotypes. Based on the analysis of all genes, the majority of the isolates from Serbia belong to subgroup IA (haplotype: IA; IA-IA; IA-IA), followed by haplotype: II; IA-II; II-II represented by three isolates, while other haplotypes were represented by single isolates (haplotype: IA; IA-IA; IA-II, haplotype: IB; IA-IA; IA-IA, haplotype: II; IA-IA; II-II, and haplotype: IB; IA-II; IA-IA). Based on the analysis of all five genes, neither of the investigated isolates belongs to IB or II subgroup..

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 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

Pegavost i sušenje lista - značajna bolest peršuna u Srbiji

In the period 2005-2014, total of 19 isolates of Alternaria petroselini originating from parsley leaves, taproot and seed, as well as infested soil were collected and analyzed on the bases of morphological, pathogenic and molecular features. All isolates formed dark gray colonies on PDA and subsurfacely produced microsclerotia, as well as pigmented, broadly ellipsoidal, singly feodyctiosporic conidia. Pathogenicity and host range studies using spore suspension spray inoculation, revealed that isolates were pathogenic for parsley, parsnip, celery and coriander, weakly pathogenic for carrot, fennel and anise, and not pathogenic for onion, cabbage, pepper, tomato, and cucumber. Molecular detection utilizing A. radicina specific primers failed to demonstrate difference between two closely related but diffe­rent species, A. radicina and A. petroselini. For further confirmation of idendty of Serbian A. petroselini isolates, sequencing of selected genomic fragments including ITS rDNA, Alt a1 and EF1a protein coding genes was performed. BLAST search revealed that the most reliable molecular delineation between A. petroselini and closely related species could be achieved on the bases of sequence analysis of Alt a1 and EF1a genes (25-27, and 10-12 nucleotide differences from A. radicina isolates, respectively), while sequence of ITS rDNA were helpful, but not completely informative for identification of A. petroselini isolates.U periodu od 2005. do 2014. godine, sakupljeno je 19 izolata Alternaria petroselini poreklom iz lista, korena i semena peršuna, kao i infestiranog zemljišta i analizirano na osnovu morfoloških, patogenih i molekularnih osobina. Svi izolati formirali su tamnosive kolonije na PDA i supstratno obrazovali mikrosklerocije, kao i obojene, elipsoidno okruglaste, feodiktiosporne konidije pojedinačno na kratkim konidioforama. Ispitujući patogenost i krug domaćina inokulacijom suspenzijom spora, svi ispitivani izolati ispoljili su patogenost za peršun, paštrnak, celer i korijander, slabu patogenost za mrkvu, morač i anis, a nisu bili infektivni za crni luk, kupus, papriku, paradajz i krastavac. Molekularna detekcija korišćenjem prajmera specifičnih za detekciju A. radicina nije uspela da napravi razliku između izolata dve srodne, ali različite vrste, A. radicina i A. petroselini. Dalja karakterizacija izolata A. petroselini poreklom iz Srbije, obavljena je sekvenciranjem različitih genskih segmenata uključujući ITS rDNK, kao i Alt a1 i EF1a gene. BLAST analiza pokazala je da su sekvence Alt a1 i EF1a gena najpouzdanije za razlikovanje A. petroselini od drugih blisko srodnih vrsta (25-27 odnosno 10-12 nukleotida razlike od izolata A. radicina). Sekvence ITS rDNK regiona bile su korisne, ali ne i potpuno informativne za identifikaciju izolata A. petroselini

First Report of Cucumber mosaic virus Infecting Watermelon in Serbia.

In June 2012, field-grown watermelon plants (Citrullus lanatus L.) with virus-like symptoms were observed in Silbaš locality, South Backa District of Serbia. Plants infected early in the growing season showed severe symptoms including stunting, mosaic, mottling, blistering, and leaf curling with reduced leaf size, while those infected at later stages exhibited only a mild mosaic. Affected plants were spread across the field and disease incidence was estimated at 40%. Thirteen symptomatic watermelon plants were sampled and analyzed by double-antibody sandwich (DAS)-ELISA using a commercial diagnostic kit (Bioreba AG, Reinach, Switzerland) against the most important watermelon viruses: Cucumber mosaic virus (CMV), Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV), Papaya ringspot virus (PRSV), and Squash mosaic virus (SqMV) (1). Commercial positive and negative controls and an extract from healthy watermelon tissue were included in each ELISA. Serological analyses showed that all plants were positive for CMV and negative for ZYMV, WMV, PRSV, and SqMV. The virus was mechanically transmitted from an ELISA-positive sample (449-12) to five plants of each Citrullus lanatus ‘Creamson sweet’ and Chenopodium amaranticolor using 0.01 M phosphate buffer (pH 7) with Serbian CMV isolate from Cucurbita pepo ‘Olinka’ (GenBank Accession No. HM065510) and healthy watermelon plants as positive and negative controls, respectively. Small necrotic lesions on C. amaranticolor and mild mosaic with dark green vein banding on watermelon leaves were observed on all inoculated plants 5 and 14 days post-inoculation, respectively. For further confirmation of CMV infection, reverse transcription (RT)-PCR was performed with the One-Step RT-PCR Kit (Qiagen, Hilden, Germany) using specific primers CMVCPfwd (5′-TGCTTCTCCRCGARWTTGCGT-3′) and CMVCPrev (5′-CGTAGCTGGATGGACAACCCG-3′), designed to amplify an 871-bp fragment of the RNA3 including the whole CP gene. Total RNA from 12 naturally infected and five mechanically infected watermelon plants was extracted with the RNease Plant Mini Kit (Qiagen). Total RNA obtained from the Serbian CMV isolate (HM065510) and healthy watermelon plants were used as positive and negative controls, respectively. The expected size of RT-PCR products were amplified from all naturally and mechanically infected watermelon plants but not from healthy tissues. The PCR product derived from isolate 449-12 was purified and directly sequenced using the same primer pair as in RT-PCR (JX280942) and analyzed by MEGA5 software (3). Sequence comparison of the complete CP gene (657 nt) revealed that the Serbian isolate 449-12 shared the highest nucleotide identity of 98.9% (99.1% amino acid identity) with the Spanish melon isolate (AJ829777) and Syrian tomato isolate (AB448696). To our knowledge, this is the first report of CMV on watermelon in Serbia. CMV is widely distributed within the Mediterranean basin where it has a substantial impact on many agricultural crops (2) and is often found to be prevalent during pumpkin and squash surveys in Serbia (4). The presence of CMV on watermelon could therefore represent a serious threat to this valuable crop in Serbia. References: (1) L. M. da Silveira et al. Trop. Plant Pathol. 34:123, 2009. (2) M. Jacquemond. Adv. Virus Res. 84:439, 2012. (3) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011. (4) A. Vucurovic et al. Eur. J. Plant Pathol. 133:935, 2012. </jats:p

Plasmopara obducens - nova pretnja proizvodnji Impatiens Walleriana u Srbiji

During 2010, Impatiens walleriana plants with symptoms of downy mildew were collected in a greenhouse in the vicinity of Mionica, Kolubara District. Disease incidence was extremely high, approaching 100%, and wilting and collapse of affected plants was very rapid, resulting in losses of more than 90%. White downy growth produced on the lower leaf surface consisted of hyaline, thin-walled sporangiophores with monopodial branching and numerous, ovoid and hyaline sporangia. Apical branchlets of sporangiophores were at right angles to the main axis, with no apical thickening. Pathogenicity tests included inoculation of young I. walleriana plants by spraying with a sporangial suspension, and downy mildew symptoms were observed after 13 to 15 days. The absence of well-defined spots on the infected impatiens leaves and straight sporangiophores indicated that the pathogen was P. obducens, which was further supported by molecular identification, the 5'-end of the nuclear DNA coding for the large ribosomal subunit (LSU rDNA) was amplified by PCR, using primers NL1 and NL4. A representative isolate, 28-10, was sequenced and phylogenetic analysis showed its grouping with other P. obducens isolates of different origin. Considering that impatiens downy mildew in Serbia is proved to be caused by P. obducens it is necessary to employ adequate phytosanitary measures to prevent further spread of the pathogen.Tokom 2010. godine biljke Impatiens walleriana sa simptomima plamenjače sakupljene su u stakleniku u okolini Mionice, Kolubarski okrug. Učestalost oboljenja bila je izuzetno visoka, blizu 100%, a sušenje i propadanje zaraženih biljaka veoma brzo, što je dovelo do šteta većih od 90%. Bela prevlaka patogena koja se razvijala na naličju listova sastojala se od hijalinskih monopodijalno razgranatih sporangiofora sa tankim zidovima i brojnih, ovoidnih i hijalinskih sporangija. Vrhovi grana sporangiofora granali su se pod pravim uglom u odnosu na glavnu osu, bez vršnog zadebljanja. Testovi patogenosti uključili su inokulacije mladih biljaka I. walleriana prskanjem suspenzijom sporangija, a simptomi plamenjače razvili su se nakon 13-15 dana. Odsustvo definisanih pega na zaraženim listovima impatiensa i prisustvo pravih sporangiofora ukazalo je da je prouzrokovač P. obducens, što je dalje potvrđeno molekularnom identifikacijom. Primenom lančane reakcije polimeraze (polymerase chain reaction, PCR), pomoću prajmera NL1 i NL4, umnožen je kodirajući 5'-kraj DNA velike ribozomalne podjedinice (LSU rDNA). Amplifikovani produkt odabranog izolata 28-10 je sekvencioniran, a filogenetske analize pokazale su njegovo grupisanje sa ostalim izolatima P. obducens različitog porekla. Uzimajući u obzir da je ovim istraživanjima dokazano da je prouzrokovač plamenjače na Impatiens walleriana u Srbiji P. obducens, neophodno je preduzimanje odgovarajućih fitosanitarnih mera u cilju sprečavanja daljeg širenja patogena u našoj zemlji

Biological and molecular characterization of Cucumber mosaic virus in Serbia

Serološkim analizama primenom DAS-ELISA testa, u periodu od 2010. do 2013. godine, prisustvo virusa mozaika krastavca (Cucumber mosaic virus, CMV) utvrđeno je u 29,68% testiranih uzoraka poreklom iz 32 različite vrste, varijeteta ili sorte povrtarskih, industrijskih, ukrasnih i korovskih biljaka sa 74 lokaliteta. U okviru ovih ispitivanja Tulipa sp. i Peperomia tuisana prvi put su otkrivene kao domaćini u našoj zemlji, a Wisteria sinensis i Stenactis annua su otkrivene kao novi domaćini CMV ne samo kod nas, već i u svetu. Analiza genetičke strukture prirodne populacije CMV u Srbiji na osnovu biološke i molekularne karakterizacije obuhvatila je 44 odabrana izolata poreklom sa različitih biljnih vrsta, njihovih varijeteta ili sorti sakupljenih na 30 lokaliteta tokom ovih istraživanja, kao i izolate iz prethodnih istraživanjanja iz perioda 2007-2009. godine. Molekularna karakterizacija na osnovu analize sekvence CP gena obuhvatila je 44 izolata, dok su molekularna proučavanja na osnovu analize sekvenci 2a, 2b i MP gena obuhvatila 19 izolata, a 1a gena 15 izolata CMV. Tokom ovih istraživanja uspostavljeni su ili unapređeni protokoli za amplifikaciju svih pet genskih regiona izolata CMV poreklom iz naše zemlje. Stepen nukleotidne i aminokiselinske sličnosti, kao i filogenetske analize pokazale su varijabilnost prirodne populacije CMV u Srbiji kod četiri od pet genskih regiona. Najveća varijabilnost utvrđena je analizom dela 1a gena (grupisanje u tri podgrupe, IA, IB i II), delova MP i CP gena kao i celog 2b gena (grupisanje u dve podgrupe, IA i II), dok je analiza dela 2a gena pokazala homogenost populacije CMV i grupisanje svih ispitivanih izolata u IA podgrupu. Na osnovu filogenetskih analiza sekvenci pojedinačnih genskih regiona RNK 1, 2 i 3, kao i sekvenci RNK 3 i RNK 2 segmenta utvrđeno je prisustvo rekombinantnih i pseudorekombinantnih izolata u populaciji ovog virusa u Srbiji i identifikovano šest različitih haplotipova. Najveći broj izolata po svim genskim regionima pripada IA podgrupi (haplotip: IA; IA-IA; IA-IA). Po brojnosti sledi haplotip: II; IA-II; II-II zastupljen sa tri izolata, dok su preostala četiri haplotipa utvrđeni kod pojedinačnih izolata (haplotip: IA; IA-IA; IA-II, haplotip: IB; IA-IA; IA-IA, haplotip: II; IA-IA; II-II i haplotip: IB; IA-II; IA-IA)...During the period from 2010 to 2013, the serological analysis, using DAS-ELISA, detected the presence of Cucumber mosaic virus (CMV) in 29,68% of tested samples originating from 32 different species, varieties or cultivars of vegetables, industrial crops, ornamentals and weeds collected at 74 localities. Tulipa sp. and Peperomia tuisana are discovered as new host plants of CMV in Serbia, while Wisteria sinensis and Stenactis annua are detected as two new natural host plants worldwide. Analysis of genetic structure of CMV population in Serbia is based on biological and molecular characterization of 44 selected isolates from different host plants collected during this research at 30 localities, or collected in previous investigation during from 2007 to 2009. The molecular characterization based on the sequence analysis of CP gene included 44 isolates, while analysis of 2a, 2b and MP genes included 19 isolates and the analysis of 1a gene included 15 isolates of CMV. During this studies, the protocols for molecular amplification of all five genomic regions of CMV isolates from Serbia were either established or improved. Nucleotide and amino acid sequence identity, as well as phylogenetic analyses showed variability in four out of five genomic regions of CMV population in Serbia. The highest variability was determined within the partial 1a gene (grouping into three subgroups, IA, IB and II), partial MP and CP genes and the complete 2b gene (grouping into two subgroups IA and II), while sequence analysis of the partial gene 2a showed that CMV population is homogeneous, and all isolates grouped within IA subgroup. Phylogenetic analysis of particular genetic regions of RNA 1, 2 and 3, as well as combined genetic regions of RNA 3 and RNA 2, revealed the presence of recombinats and reassortments within the population of CMV in Serbia and identified the presence of six different haplotypes. Based on the analysis of all genes, the majority of the isolates from Serbia belong to subgroup IA (haplotype: IA; IA-IA; IA-IA), followed by haplotype: II; IA-II; II-II represented by three isolates, while other haplotypes were represented by single isolates (haplotype: IA; IA-IA; IA-II, haplotype: IB; IA-IA; IA-IA, haplotype: II; IA-IA; II-II, and haplotype: IB; IA-II; IA-IA). Based on the analysis of all five genes, neither of the investigated isolates belongs to IB or II subgroup..

Biological and molecular characterization of Cucumber mosaic virus in Serbia

Serološkim analizama primenom DAS-ELISA testa, u periodu od 2010. do 2013. godine, prisustvo virusa mozaika krastavca (Cucumber mosaic virus, CMV) utvrđeno je u 29,68% testiranih uzoraka poreklom iz 32 različite vrste, varijeteta ili sorte povrtarskih, industrijskih, ukrasnih i korovskih biljaka sa 74 lokaliteta. U okviru ovih ispitivanja Tulipa sp. i Peperomia tuisana prvi put su otkrivene kao domaćini u našoj zemlji, a Wisteria sinensis i Stenactis annua su otkrivene kao novi domaćini CMV ne samo kod nas, već i u svetu. Analiza genetičke strukture prirodne populacije CMV u Srbiji na osnovu biološke i molekularne karakterizacije obuhvatila je 44 odabrana izolata poreklom sa različitih biljnih vrsta, njihovih varijeteta ili sorti sakupljenih na 30 lokaliteta tokom ovih istraživanja, kao i izolate iz prethodnih istraživanjanja iz perioda 2007-2009. godine. Molekularna karakterizacija na osnovu analize sekvence CP gena obuhvatila je 44 izolata, dok su molekularna proučavanja na osnovu analize sekvenci 2a, 2b i MP gena obuhvatila 19 izolata, a 1a gena 15 izolata CMV. Tokom ovih istraživanja uspostavljeni su ili unapređeni protokoli za amplifikaciju svih pet genskih regiona izolata CMV poreklom iz naše zemlje. Stepen nukleotidne i aminokiselinske sličnosti, kao i filogenetske analize pokazale su varijabilnost prirodne populacije CMV u Srbiji kod četiri od pet genskih regiona. Najveća varijabilnost utvrđena je analizom dela 1a gena (grupisanje u tri podgrupe, IA, IB i II), delova MP i CP gena kao i celog 2b gena (grupisanje u dve podgrupe, IA i II), dok je analiza dela 2a gena pokazala homogenost populacije CMV i grupisanje svih ispitivanih izolata u IA podgrupu. Na osnovu filogenetskih analiza sekvenci pojedinačnih genskih regiona RNK 1, 2 i 3, kao i sekvenci RNK 3 i RNK 2 segmenta utvrđeno je prisustvo rekombinantnih i pseudorekombinantnih izolata u populaciji ovog virusa u Srbiji i identifikovano šest različitih haplotipova. Najveći broj izolata po svim genskim regionima pripada IA podgrupi (haplotip: IA; IA-IA; IA-IA). Po brojnosti sledi haplotip: II; IA-II; II-II zastupljen sa tri izolata, dok su preostala četiri haplotipa utvrđeni kod pojedinačnih izolata (haplotip: IA; IA-IA; IA-II, haplotip: IB; IA-IA; IA-IA, haplotip: II; IA-IA; II-II i haplotip: IB; IA-II; IA-IA)...During the period from 2010 to 2013, the serological analysis, using DAS-ELISA, detected the presence of Cucumber mosaic virus (CMV) in 29,68% of tested samples originating from 32 different species, varieties or cultivars of vegetables, industrial crops, ornamentals and weeds collected at 74 localities. Tulipa sp. and Peperomia tuisana are discovered as new host plants of CMV in Serbia, while Wisteria sinensis and Stenactis annua are detected as two new natural host plants worldwide. Analysis of genetic structure of CMV population in Serbia is based on biological and molecular characterization of 44 selected isolates from different host plants collected during this research at 30 localities, or collected in previous investigation during from 2007 to 2009. The molecular characterization based on the sequence analysis of CP gene included 44 isolates, while analysis of 2a, 2b and MP genes included 19 isolates and the analysis of 1a gene included 15 isolates of CMV. During this studies, the protocols for molecular amplification of all five genomic regions of CMV isolates from Serbia were either established or improved. Nucleotide and amino acid sequence identity, as well as phylogenetic analyses showed variability in four out of five genomic regions of CMV population in Serbia. The highest variability was determined within the partial 1a gene (grouping into three subgroups, IA, IB and II), partial MP and CP genes and the complete 2b gene (grouping into two subgroups IA and II), while sequence analysis of the partial gene 2a showed that CMV population is homogeneous, and all isolates grouped within IA subgroup. Phylogenetic analysis of particular genetic regions of RNA 1, 2 and 3, as well as combined genetic regions of RNA 3 and RNA 2, revealed the presence of recombinats and reassortments within the population of CMV in Serbia and identified the presence of six different haplotypes. Based on the analysis of all genes, the majority of the isolates from Serbia belong to subgroup IA (haplotype: IA; IA-IA; IA-IA), followed by haplotype: II; IA-II; II-II represented by three isolates, while other haplotypes were represented by single isolates (haplotype: IA; IA-IA; IA-II, haplotype: IB; IA-IA; IA-IA, haplotype: II; IA-IA; II-II, and haplotype: IB; IA-II; IA-IA). Based on the analysis of all five genes, neither of the investigated isolates belongs to IB or II subgroup..

Alternaria petroselini pathogen of parsley in Serbia

After its first detection in 2005, Alternaria petroselini, the causal agent of black leaf spot, has become one of the most important pathogens of parsley (Petroselinum crispum (Mill.) Nym.) in Serbia. During almost a decade, isolates of A. petroselini originating from parsley leaves, seed and infested soil were collected and identified on the bases of morphological, pathogenic and molecular features. All morphological features were consistent with those described previously, including dark brown colonies on potato dextrose agar (PDA) and subsurface microsclerotia production, and the presence of pigmented, broadly ellipsoidal, singly produced feodyctiosporic conidia. Pathogenicity and host range studies using spray inoculation of spore suspensions, revealed that isolates were pathogenic for parsley, parsnip, celery and coriander, weakly pathogenic for carrot, fennel and anise, and not pathogenic for onion, cabbage, pepper, tomato, and cucumber. Molecular detection utilizing polymerase chain reaction (PCR) and A. radicina specific primers failed to demonstrate a difference between A. petroselini and A. radicina, amplifying isolates belonging to both species. For further confirmation of Serbian A. petroselini isolates, sequencing of selected genomic fragments including ITS and mitochondrial small subunit (mtSSU) rDNA and major allergen Alt a1, b-tubulin, and translation elongation factor-1 alpha (EF1a) protein coding genes was performed. All obtained sequences were deposited in GenBank and BLAST search for the closest matching revealed several A. petroselini isolates from GenBank to share 99-100% nucleotide identity. Parsley is traditionally grown herb and vegetable plant in Serbia and A. petroselini causes considerable damage every year and occasionally very severe yield losses, resulting in significant economic impact, especially in parsley leaf production. Further investigation of the epidemiology and population structure of A. petroselini is needed in order to establish efficient control measures

Galectin-3 Involvement in Cognitive Processes for New Therapeutic Considerations

Cognitive impairment may be a consequence of the normal aging process, but it may also be the hallmark of various neurodegenerative and psychiatric diseases. Early identification of individuals at particular risk for cognitive decline is critical, as it is imperative to maintain a cognitive reserve in these neuropsychiatric entities. In recent years, galectin-3 (Gal-3), a member of the galectin family, has received considerable attention with respect to aspects of neuroinflammation and neurodegeneration. The mechanisms behind the putative relationship between Gal-3 and cognitive impairment are not yet clear. Intrigued by this versatile molecule and its unique modular architecture, the latest data on this relationship are presented here. This mini-review summarizes recent findings on the mechanisms by which Gal-3 affects cognitive functioning in both animal and human models. Particular emphasis is placed on the role of Gal-3 in modulating the inflammatory response as a fine-tuner of microglia morphology and phenotype. A review of recent literature on the utility of Gal-3 as a biomarker is provided, and approaches to strategically exploit Gal-3 activities with therapeutic intentions in neuropsychiatric diseases are outlined