A. Vučurović

Incidence and Distribution of Iris yellow spot virus on Onion in Serbia

In a survey to determine the presence and distribution of Iris yellow spot virus (IYSV) in greenhouse ornamentals and onion field crops in 14 districts of Serbia as well as on imported ornamental plants, 1,574 samples were collected and analyzed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). IYSV was not detected in nearly 1,200 plant samples collected from 39 genera of ornamentals grown in greenhouses in Serbia or imported from other countries during 2005 to 2007. The virus was detected in samples from an onion seed crop in the Sirig locality (South Bačka District) that showed symptoms resembling those caused by IYSV and in samples without IYSV-like symptoms from an onion bulb crop in the Obrenovac locality (City of Belgrade District). Mechanical transmission of IYSV isolates was difficult, and only the isolate 605-SRB could infect four plant species, but not in all replications. No virus transmission could be demonstrated in 5,000 tested seeds originating from IYSV-infected onion crops. For further confirmation of IYSV, the nucleotide sequence of its nucleocapsid (NC) gene was obtained by reverse transcription-polymerase chain reaction (RT-PCR) in symptomatic onion samples as well as in symptomless leaves of Nicotiana benthamiana. Four previously developed primers were tested to determine their suitability for routine detection of Serbian IYSV isolates. Phylogenetic analysis showed clustering of isolates 605-SRB and 622-SRB from the onion seed crop and isolate 283-SRB from the onion bulb crop into two distant clades. The analysis indicated that Serbian isolates of IYSV do not share a recent common ancestor and that they represent two distinct lineages of IYSV in Serbia. Considering that onion is one of the most important and traditionally grown vegetable crops in Serbia, IYSV represents a potentially devastating pathogen in this country.

Non-persistently aphid-borne viruses infecting pumpkin and squash in Serbia and partial characterization of Zucchini yellow mosaic virus isolates

Cucurbit species grown in the Vojvodina Province, Serbia, were surveyed for the incidence of Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV), Cucumber mosaic virus (CMV), Squash mosaic virus (SqMV), Papaya ringspot virus (PRSV) and Tobacco ringspot virus (TRSV) from 2007 to 2009. Samples from more than 700 pumpkin, squash and bottle gourd plants with virus-like symptoms were analyzed by double-antibody sandwich (DAS)-ELISA. ZYMV, WMV and CMV were detected in 79.2, 32.2, and 12.8% of tested samples, respectively. WMV was prevalent in 2007 and ZYMV in 2008–09. Mixed infections were the most frequent type in 2007–08 in contrast to 2009 when single infection of ZYMV prevailed. ZYMV was the most widespread being found in 33 out of 39 inspected fields. Virus species identification was confirmed in selected samples by conventional reverse transcription-polymerase chain reaction (RT-PCR) and sequencing of their coat protein genes. By comparing the obtained virus isolate sequences with those available in GenBank, the identification of serologically detected viruses was confirmed. Phylogenetic analysis based on complete coat protein (CP) sequences highlighted that Serbian ZYMV isolates were closely related to other Central European ZYMV isolates. Finally, additional testing of ELISA-negative samples by RT-PCR using primers specific to six other mosaic viruses revealed the presence of Tomato spotted wilt virus (TSWV) in winter (Cucurbita maxima) and summer (C. pepo ‘Beogradska’) squash. This is the first report of TSWV natural occurrence on cucurbits in Serbia and on winter squash worldwide.

Phytophthora ramorum occurrence in ornamentals in Serbia.

In a survey to determine the presence of Phytophthora ramorum in Serbia, ornamentals from garden centers, nurseries, and private and public gardens, as well as imported plant material, were inspected. In total, 577 plant, soil, and potting media samples were tested using various detection methods: lateral flow diagnostic test, enzyme-linked immunosorbent assay, conventional polymerase chain reaction, and isolation, followed by identification based on growth characteristics in culture and morphological features. P. ramorum was not detected in any of the 162 soil or potting media tested by the baiting method. P. ramorum was detected in 12 Rhododendron samples from one private garden in Zemun (City of Belgrade District) exhibiting symptoms of leaf necrosis and blight and petiole necrosis, and in three samples of Pieris spp. from one garden center exhibiting symptoms of leaf necrosis. Eight Phytophthora isolates were obtained from the positive Rhododendron plants and three isolates from Pieris plants, and all were identified as P. ramorum on the basis of their uniform morphological and growth characteristics. P. ramorum conformation was also made by sequencing of the internal transcribed spacer regions for a single isolate taken from one infected rhododendron and one pieris plant. Serbian isolates were determined as A1 mating type, due to formation of a few typical sexual structures when crossed with the A2 mating type of P. cinnamomi and P. cryptogea. Pathogenicity test on nonwounded detached leaves of 19 popular ornamentals, as well as the most frequently imported ones, revealed that 10 host species were susceptible, including Robinia pseudoacacia, which is widely distributed in Serbia. During this study, Cotoneaster horizontalis and C. dammeri were determined to be new experimental hosts of P. ramorum. This article provides evidence of P. ramorum introduction into Serbia. Although P. ramorum has not been detected in Serbian production nurseries, its presence outdoors might cause severe damages on susceptible common urban plants in public green and natural ecosystems.

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.

First report of foliar and stem blight on sunflower caused by Alternaria helianthiinficiens in Croatia.

Sunflower (Helianthus annus L.) is the most important oilseed crop in Croatia. In August 2009, in six localities of eastern Croatia, severe foliar and stem blight symptoms were observed on several genotypes with disease incidence ranging from 10 to 50%. At the initial stage of the infection, irregular to oval, brown spots different in size, surrounded by a chlorotic halo, appeared on the leaves that gradually became enlarged and coalesced, and whole leaves turned yellow and necrotic, followed by defoliation. Lesions on the stems were light to dark brown, randomly distributed, rounded and tapered on the ends; later becoming large and elongated causing stem breakage. Tissue within the lesion was reddish on the cross section. To determine the causal agent, small pieces of symptomatic leaves and stem tissue of sunflower were surface disinfested and placed on potato dextrose agar. A total of 17 isolates from leaves as well as six from stems were obtained and all formed cottony, dark olivaceous to black colonies under 12 h of fluorescent light per day. All isolates formed uniform solitary, pale brown to brown, long ovoid conidia with five to eight transverse and one to two longitudinal septa. The conidia of all isolates were slightly constricted at the transverse septa, measuring 55 to 90 × 14 to 20 μm. Based on the morphological characteristics, the pathogen was identified as Alternaria helianthiinficiens E.G. Simmons, Walcz & R.G. Roberts (4). The pathogenicity was tested with one representative isolate (Alt5) by injection of a conidial suspension (106 conidia/ml) into stems of 20 healthy sunflower seedlings and by spraying 20 non-wounded detached leaves with a suspension of spores. Small necrotic spots on all inoculated seedlings and leaves formed 5 and 9 days after inoculation, respectively. The control sunflower seedlings and detached leaves, inoculated with sterile water, showed no reactions. The identity of isolate Alt5 was futher confirmed by amplification and sequencing of the internal transcribed spacer (ITS) region of rDNA. Because there are no available corresponding ITS sequences of A. helianthiinficiens in the GenBank, reference type strain CBS 208.86 (publicly purchased, CBS, Utrecht, Netherlands) was also sequenced in this study. Total DNA was extracted directly from fungal mycelium and PCR amplification and sequencing were performed with primers ITS1F/ITS4. Sequence analysis of ITS region revealed 100% nucleotide identity between isolate Alt5 (GenBank Accession No. JX101648) and isolate CBS 208.86 (GenBank Accession No. JX101649). The nucleotide identity of both isolates compared with A. helianthi (HM449991), another sunflower pathogenic fungus, was only 80%. A. helianthiinficiens has previously been reported on sunflower in Hungary and the USA (3), Serbia (1), and Korea (2). However, to our knowledge, this is the first report of A. helianthiinficiens occurrence in Croatia as a new and harmful parasite of sunflower, illustrating an expansion of its geographical range and underscoring the need for phytosanitary control because it is a seedborne fungus. References: (3) M. Aćimović and N. Lačok. Helia 14:129, 1991. (4) H. S. Cho and S. H. Yu. Plant Pathol. J. 16:331, 2000. (2) E. G. Simmons. Mycotaxon 25:203, 1986. (1) E. G. Simmons. Alternaria: An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, the Netherlands, 2007.

First report of Tomato spotted wilt virus on Brugmansia sp. in Serbia.

Brugmansia (Brugmansia spp.), also known as Angels trumpet, is a perennial shrub in the Solanaceae that is a popular landscape plant in the tropics and subtropics, and potted plant in temperate regions. In April 2012, virus-like symptoms including chlorotic leaf patterns and curling followed by necrosis and distortion of leaves were observed on five outdoor-grown brugmansia plants in a private garden in Mackovac, Rasina District, Serbia. Symptomatic leaves were tested for the presence of several common ornamental viruses including Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV), Cucumber mosaic virus (CMV), and Tobacco mosaic virus (TMV) by commercial double-antibody sandwich (DAS)-ELISA diagnostic kits (Bioreba AG, Reinach, Switzerland). Commercial positive and negative controls and extract from healthy brugmansia leaves were included in each ELISA. TSWV was detected serologically in all five brugmansia samples and all tested samples were negative for INSV, CMV, and TMV. The virus was mechanically transmitted from an ELISA-positive sample (41-12) to five plants of each Petuina × hybrida and Nicotiana glutinosa. Inoculated P. × hybrida plants showed local necrotic lesions and N. glutinosa showed mosaic and systemic necrosis 4 and 12 days post-inoculation, respectively, which were consistent with symptoms caused by TSWV (1). For further confirmation of TSWV infection, reverse transcription (RT)-PCR was performed with the OneStep RT-PCR (Qiagen, Hilden, Germany) using a set of TSWV-specific primers, TSWV CP-f and TSWV CP-r (4), designed to amplify a 738-bp fragment of the nucleocapsid protein (N) gene. Total RNAs from naturally infected brugmansia and symptomatic N. glutinosa plants were extracted using the RNeasy Plant Mini Kit (Qiagen). Total RNAs obtained from the Serbian tobacco isolate of TSWV (GenBank Accession No. GQ373173) and healthy brugmansia plants were used as positive and negative controls, respectively. The expected size of the RT-PCR product was amplified from symptomatic brugmansia and N. glutinosa but not from healthy tissues. The amplified product derived from the isolate 41-12 was sequenced directly after purification with the QIAquick PCR Purification kit (Qiagen), deposited in GenBank (JX468080), and subjected to sequence analysis by MEGA5 software (3). Sequence comparisons revealed that the Serbian isolate 41-12 shared the highest nucleotide identity of 99.9% (99.5% amino acid identity) with an Italian TSWV isolate P105/2006RB (DQ915946) originating from pepper. To our knowledge, this is the first report of TSWV on brugmansia in Serbia. Due to the increasing popularity and economic importance of brugmansia as an ornamental crop, thorough inspections and subsequent testing for TSWV and other viruses are needed. This high-value ornamental plant may act also as reservoir for the virus that can infect other ornamentals and cultivated crops, considering that TSWV has a very broad host range (2). References: (1) Anonymous. OEPP/EPPO Bull. 34:271, 2004. (2) G. Parrella et al. J. Plant Pathol. 85:227, 2003. (3) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011. (4) A. Vučurović et al. Eur. J. Plant Pathol. 133:935, 2012.

Plasmopara obducens: A new threat to the production of Impatiens Walleriana in Serbia

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.

Viruses affecting tomato crops in Serbia

In a two-year survey (2011–2012), 3220 samples were collected and analyzed in order to determine the presence and distribution of viruses in tomato crops at 56 localities of 18 districts in Serbia. Out of 12 viruses tested, Cucumber mosaic virus (CMV), Potato virus Y (PVY), Alfalfa mosaic virus (AMV), Tomato spotted wilt virus (TSWV), Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV) were detected in 42.1, 40, 11, 8.6, 2.3 and 1.3% of the total tested samples, respectively. The results revealed that CMV was prevalent in 2011 and PVY in 2012. CMV and PVY, apart from being predominant, were also the most widespread viruses. In general, single infections were the most frequent type of infection. Additionally, the most common mixed infections were double infections and the most prevalent combination was CMV and PVY. In 2011, the incidence of diseases and the percentage of all infection types were significantly higher than in 2012. Furthermore, in 2011, regardless of total single infections being prevalent compared to mixed infections, two prevailing viruses were commonly detected in mixed infections. The additional molecular testing of ELISA-negative samples using virus specific primers did not reveal the presence of Pepino mosaic virus (PepMV), Tomato yellow leaf curl virus (TYLC), Tomato infections chlorosis virus (TICV) and Tomato chlorosis virus (ToCV).

Incidence and distribution of leek yellow stripe virus in allium crops in Serbia

Ivan Vučurović1*, Dušan Nikolić2, Nikola Radović2, Ana Vučurović2, Danijela Ristić1, Branka Krstić2 and Ivana Stanković2 1Institute for Plant Protection and Environment, Department of Plant Pathology, Teodora Drajzera 9, 11040 Belgrade, Serbia 2University of Belgrade, Faculty of Agriculture, Institute of Phytomedicine, Department of Phytopathology, Nemanjina 6, 11080 Belgrade, Serbia *Corresponding author: vucurovic.ivan@gmail.com Received: 24 October 2017 Accepted: 14 November 2017

First Report of Septoria Leaf Spot of Lavandin Caused by Septoria lavandulae in Croatia

Lavandula × intermedia Emeric ex Loiseleur, commonly known as lavandin, is an aromatic and medicinal perennial shrub widely and traditionally grown in Croatia. The lavandin essential oil is primarily used in perfumery and cosmetic industries, but also possesses anti-inflammatory, sedative, and antibacterial properties. In June 2012, severe foliar and stem symptoms were observed on approximately 40% of plants growing in a commercial lavandin crop in the locality of Banovo Brdo, Republic of Croatia. Initial symptoms on lower leaves included numerous, small, oval to irregular, grayish brown lesions with a slightly darker brown margin of necrotic tissue. Further development of the disease resulted in yellowing and necrosis of the infected leaves followed by premature defoliation. Similar necrotic oval-shaped lesions were observed on stems as well. The lesions contained numerous, dark, sub-globose pycnidia that were immersed in the necrotic tissue or partly erumpent. Small pieces of infected internal tissues were superficially disinfected with 50% commercial bleach (4% NaOCl) and placed on potato dextrose agar (PDA). A total of 10 isolates from leaves and five from stems of lavandin formed a slow-growing, dark, circular colonies with raised center that produced pycnidia at 23°C, under 12 h of fluorescent light per day. All 15 recovered isolates formed uniform hyaline, elongate, straight or slightly curved conidia with 3 to 4 septa, with average dimensions of 17.5 to 35 × 1.5 to 2.5 μm. Based on the morphological characteristics, the pathogen was identified as Septoria lavandulae Desm., the causal agent of lavender leaf spot (1,2). Pathogenicity of one selected isolate (428-12) was tested by spraying 10 lavandin seedlings (8 weeks old) with a conidial suspension (106 conidia/ml) harvested from a 4-week-old monoconidial culture on PDA. Five lavandin seedlings, sprayed with sterile distilled water, were used as negative control. After 5 to 7 days, leaf spot symptoms identical to those observed on the source plants developed on all inoculated seedlings and the pathogen was successfully re-isolated. No symptoms were observed on any of the control plants. Morphological identification was confirmed by amplification and sequencing of the internal transcribed spacer (ITS) region of rDNA (3). Total DNA was extracted directly from fungal mycelium with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and PCR amplification performed with primers ITS1F/ITS4. Sequence analysis of ITS region revealed at least 99% identity between the isolate 428-12 (GenBank Accession No. KF373078) and isolates of many Septoria species; however, no information was available for S. lavandulae. To our knowledge, this is the first report of Septoria leaf spot of lavandin caused by S. lavandulae in Croatia. Since the cultivation area of lavandin plants has been increasing in many continental parts of Croatia, especially in Slavonia and Baranja counties, the presence of a new and potentially harmful disease may represent a serious constraint for lavandin production and further monitoring is needed. References: (1) T. V. Andrianova and D. W. Minter. IMI Descriptions of Fungi and Bacteria, 142, Sheet 1416, 1999. (2) R. Bounaurio et al. Petria 6:183, 1996. (3) G. J. M. Verkley et al. Mycologia 96:558, 2004.