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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 esta

Propolis, the mixture of the nectar of flowers collected by honeybees, showed antiviral activity against human and plant viruses. Since Zucchini yellow mosaic virus (ZYMV, Potyvirus, Potyviridae) is the most important pathogen of oilseed pumpkin (Cucurbita pepo var. styriaca) production worldwide, the aim of this study was to evaluate the effect of propolis extract on ZYMV infection in oilseed pumpkin. Propolis extracted by 95% ethanol was diluted in distilled water to concentration of 1, 2.5, 5 and 10%. Effects of different propolis concentration were evaluated in in vitro and in vivo experiments. In vitro, ZYMV inoculum was mixed with different propolis concentrations and oilseed pumpkin plants were inoculated immediately, 1 and 2 hours after mixing. In vivo, oilseed pumpkin plants were sprayed with different propolis concentrations before and after ZYMV inoculation immediately, 1 and 2 h later. In each experiment 10 oilseed pumpkin plants in one true leaf stage were inoculated. Plants inoculated with ZYMV and sprayed with propolis were used as a positive and negative control, respectively. Symptom appearance was checked 10 days post-inoculation. Only 5 and 10% propolis concentration showed effect in reducing symptoms of ZYMV infection, in all experiments. In vitro, number of symptomatic plants was reduced to 80 and 70% after one-hour period, and to 70 and 60% after two-hour period. In vivo, propolis concentrations of 5 and 10% sprayed before inoculation reduced number of symptomatic plants to 70 and 60% after one-hour period, and to 60 and 50% after two-hour period, while concentrations of 5 and 10% sprayed after inoculation, reduced number of symptomatic plants to 80% each after one-hour period, and to 70 and 80% after two-hour period. In this study propolis exhibited a positive effect on reduction of ZYMV infection despite the fact that plants were challenged with higher levels of the virus than it occurs naturally. Encouraging results of propolis capacity for

Investigation of the presence and distribution of Cucumber mosaic virus in watermelon crops in Serbia during 2012 revealed that the virus was present in five out of six surveyed localities. CMV was detected in 24.05% of total serologically tested samples using DAS-ELI­SA method and the virus was the most prevalent in the locality of Silbaš. During this investigation, Cucumber mosaic virus was present more often in mixed infections with ZYMV and/or WMV (12.66%), while the single infection was detected in lower percentage, 11.39%. Five CMV isolates were obtained by mechanical inoculations of N. glutinosa and two of them were selected for further biological characterization. Concerning type and severity of symptoms on test plants, the investigated isolates exhibited different phenotypic features. Molecular detection was performed by amplification of a fragment of 871 bp in all tested isolates, using the primer pair CMVCPfwd/CPrev that amplify the entire coat protein gene and part of 5' and 3' UTR regions. The affiliation of Serbian CMV isolates from watermelon to subgroup IA, the group that includes most of the isolates selected for phylogenetic analysis, was determined by sequence analysis of the complete CP gene and reconstruction of phylogenetic tree., Proučavanjem pojave i rasprostranjenosti virusa mozaika krastavca u usevu lubenice u Srbiji tokom 2012. godine, prisustvo virusa utvrđeno je u pet od šest pregledanih lokaliteta. Prisustvo virusa ustanovljeno je u 24,05% serološki testiranih uzoraka (DAS-ELISA), a najzastupljeniji je bio na lokalitetu Silbaš. Tokom ovih istraživanja virus mozaika krastavca češće je bio prisutan u mešanim infekcijama sa ZYMV i/ili WMV (12,66%), dok je prisustvo pojedinačne infekcije utvrđeno u nešto nižem procentu, 11,39%. Mehaničkim inokulacijama N. glutinosa, dobijeno je pet izolata, od kojih su dva odabrana za dalju biološku karakterizaciju. Na osnovu tipa i jačine simptoma na test-biljkama, utvrđena je fenotipska varijabilnost ispitivanih izolata. Molekularna detekcija obavljena je amplifikacijom fragmenta dužine 871 bp kod svih ispitivanih izolata korišćenjem para prajmera CMVCPfwd/CPrev, koji omogućavaju umnožavanje gena za protein omotača i deo 5' i 3' UTR regiona. Analizom sekvenci kompletnog gena za protein omotača i rekonstrukcijom filogenetskog stabla, utvrđena je pripadnost izolata CMV iz lubenice iz Srbije podgrupi IA, koja obuhvata većinu izolata odabranih za filogenetsku analizu.

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 region, 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

Tomato spotted wilt virus is an economically important pathogen in several potato producing areas in the world. Although natural infections of potato in Serbia have not been detected, two TSWV isolates from tobacco, with different biological features were compared based on NSm gene sequence analysis and they were used for mechanical and graft inoculations to evaluate the susceptibility of five popular potato cultivars: 'Riviera', 'Arnova', 'Curoda', 'Kondor' and 'Aladin'. Both TSWV isolates infected all tested potato cultivars. Statistical analyses showed that the rate of TSWV transmission from infected foliage to tubers and from infected tubers to progeny plants was affected by cultivars, while virus transmission efficiency was independent of investigated isolates. Different potato tissues used for sample preparation did not influence virus detection by ELISA, but composite sampling from six different tuber parts is recommended since it was the most reliable.

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.

A survey was conducted in 2012 and 2013 to detect the presence and distribution of Alfalfa mosaic virus (AMV) in lavandin crops growing in continental parts of Croatia. A total of 73 lavandin samples from six crops in different localities were collected and analyzed for the presence of AMV and Cucumber mosaic virus (CMV) using commercial double-antibody sandwich (DAS)-ELISA kits. AMV was detected serologically in 62 samples collected at three different localities, and none of the samples tested positive for CMV. For further analyses, six selected samples of naturally infected lavandin plants originating from different localities were mechanically transmitted to test plants: Chenopodium quinoa, C. amaranticolor, Nicotiana benthamiana and Ocimum basilicum, confirming the infectious nature of the disease. Molecular detection was performed by amplification of a 751 bp fragment in all tested samples, using the specific primers CP AMV1/CP AMV2 that amplify the part of the coat protein (CP) gene and 3'-UTR. The RT-PCR products derived from the isolates 371-13 and 373-13 were sequenced (KJ504107 and KJ504108, respectively) and compared with the AMV sequences available in GenBank. CP sequence analysis, conducted using the MEGA5 software, revealed that the isolate 371-13 had the highest nucleotide identity of 99.5% (100% amino acid identity) with an isolate from Argentina originating from Medicago sativa (KC881010), while the sequence of isolate 373-13 had the highest identity with an Italian AMV isolate from Lavandula stoechas (FN667967) of 98.6% (99% amino acid identity). Phylogenetic analysis revealed the clustering of selected isolates into four molecular groups and the lavandin AMV isolates from Croatia grouped into two distinct groups, implying a significant variability within the AMV lavandin population., Tokom 2012. i 2013. godine sprovedena su istraživanja u cilju utvrđivanja prisustva i rasprostranjenosti virusa mozaika lucerke (Alfalfa mosaic virus, AMV) u usevu lavande u kontinentalnom delu Hrvatske. Sa šest lokaliteta gajenja ukupno je sakupljeno 73 uzorka lavande koji su serološki testirani na prisustvo AMV i virusa mozaika krastavca (Cucumber mosaic virus, CMV) korišćenjem komercijalno dostupnih kitova za DAS-ELISA test. Prisustvo AMV dokazano je u 62 uzorka lavande sakupljena sa tri različita lokaliteta, dok prisustvo CMV nije dokazano ni u jednom od testiranih uzoraka. Za dalja istraživanja odabrano je šest uzoraka prirodno zaraženih biljaka lavande poreklom iz različitih lokaliteta, koji su uspešno preneti mehaničkim inokulacijama na test biljke Chenopodium quinoa, C. amaranticolor, Nicotiana benthamiana, i Ocimum basilicum, čime je potvrđena infektivna priroda oboljenja. Molekularna detekcija obavljena je amplifikacijom fragmenta dužine 751 bp kod svih ispitivanih izolata korišćenjem specifičnih prajmera CP AMV1/CP AMV2 koji omogućavaju umnožavanje dela gena za proteinski omotač i 3' neprepisujućeg regiona. U cilju dalje identifikacije, RT-PCR produkti izolata 371-13 i 373-13 su sekvencionisani (KJ504107 i KJ504108) i upoređeni sa AMV sekvencama dostupnim u GenBank bazi podataka. Analizom sekvenci gena za protein omotača, korišćenjem MEGA5 softverskog paketa, najviši stepen nukleotidne sličnosti od 99.7% (100% aminokiselinska sličnost) izolat 371-13 pokazao je sa izolatom AMV iz Argentine poreklom iz Medicago sativa (KC881010), dok sekvenca izolata 373-13 najveću sličnost od 98.6% (99% aminokiselinska sličnost) deli sa italijanskim izolatom AMV iz Lavandula stoechas (FN667967). Filogenetska analiza pokazala je grupisanje izolata u četiri molekularne grupe, a izolati AMV iz lavande iz Hrvatske grupišu se u dve odvojene grupe, ukazujući na postojanje značajne varijabilnosti u populaciji AMV poreklom iz lavande.

The presence of Zucchini yellow mosaic virus (ZYMV) in two out of seven watermelon production localities in Serbia during 2011 was investigated by analyzing leaves sampled from symptomatic and asymptomatic watermelon plants and utilizing DAS-ELISA test. In the locality of Gornji Tavankut, ZYMV was detected in 23.08% of tested plants in single infections, and in the locality of Silbas it was detected in 35.29% of tested plants in mixed infections with Cucumber mosaic virus and Alfalfa mosaic virus. ZYMV was successfully mechanically transmitted from naturally infected watermelon plants to Cucurbita pepo 'Ezra F1'. Molecular detection was performed by RT-PCR and amplification of part of the gene for nuclear inclusions, gene of coat protein and part of 3' non-coding region, which confirmed the identification of the ZYMV isolates. Phylogenetic analysis revealed grouping of the isolate originating from watermelon with other isolates from Serbia and Central Europe within A-I subgroup. Analysis of amino acid sequences of the N terminal end of the CP gene revealed that isolate 550-11 belongs to the Central European branch., Prisustvo virusa žutog mozaika cukinija (Zucchini yellow mosaic virus, ZYMV) na dva od sedam lokaliteta gajenja lubenice u Srbiji tokom 2011. godine utvrđeno je analizom uzoraka lišća simptomatičnih i asimptomatičnih biljaka lubenice primenom DAS-ELISA metode. Na lokalitetu Gornji Tavankut, ZYMV je dokazan u 23,08% testiranih biljaka u pojedinačnim infekcijama, a na lokalitetu Silbaš u 35,29% testiranih biljaka u mešanim infekcijama sa virusom mozaika krastavca i virusom mozaika lubenice. ZYMV je uspešno mehanički prenesen sa prirodno zaraženih biljaka lubenice na Cucurbita pepo 'Ezra F1'. Molekularna detekcija obavljena je RT-PCR metodom umnožavanjem dela gena za nuklearne inkluzije, gena za protein omotača i dela 3' neprepisujućeg regiona, čime je potvrđena identifikacija izolata. Filogenetske analize pokazale su grupisanje izolata poreklom iz lubenice sa drugim izolatima iz Srbije i Centralne Evrope u okviru A-I podgrupe. Analize aminokiselinske sekvence N-terminalnog kraja CP gena, takođe su pokazale pripadnost izolata 550-11 centralnoevropskoj grani.

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.

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.