First Report of Fusarium Root Rot of Stored Carrot Caused by Fusarium avenaceum in Serbia.

Carrot (Daucus carota L. subsp. sativus (Hoffm.) Thell., Apiaceae), a widely consumed antioxidant-rich plant, is among the major vegetable crops grown in Serbia, with average annual production of 65,400 tons on approximately 7,000 ha (4). In May 2013, a severe root rot was observed on approximately 20% of cold-stored carrot roots originating from Gospodinci, South Backa District, Serbia. Symptoms included dry rot of the collar and crown as well as large, brown to dark brown, circular, sunken lesions on the stored roots. Frequently, abundant whitish mycelium was observed covering the surface of the colonized roots. To determine the causal agent, small pieces of infected tissue were surface-disinfested with 2% NaOCl without rinsing, air-dried, and placed on potato dextrose agar. Five single-spore isolates obtained from collar and crown tissue sections, as well as nine isolates from root sections, all formed abundant, cottony white to pale salmon fungal colonies with reddish orange pigment on the reverse surface of the agar medium when grown at 25°C under 12 h of fluorescent light per day. All recovered isolates formed numerous, three- to six-septate, hyaline, needle-like, straight to slightly curved, fusoid macroconidia (30 to 80 × 4 to 5.5 µm, average 58.3 × 4.9 µm, n = 100 spores) each with a tapering apical cell. Microconidia of all isolates were generally scarce, two- to four-septate, spindle-shaped, and 15 to 35 × 3 to 5 µm (average 21.3 × 4.2 µm). Chlamydospores were not observed. Based on these morphological characteristics, the pathogen was identified as Fusarium avenaceum (Fries) Saccardo (1). The pathogenicity on carrot was tested for isolate 19-14 by inoculating each of five carrot roots surface-disinfected with 2% NaOCl, by placing a mycelial plug into the surface of a wound created with a cork borer. Carrot roots inoculated with sterilized PDA plugs served as a negative control treatment. After 5 days of incubating the roots at 25°C, root rot symptoms identical to those observed on the source carrot plants developed on all inoculated roots, and the pathogen was re-isolated from each of these roots using the same procedure descibed above. There were no symptoms on the control roots. Morphological species identification was confirmed by sequencing the translation elongation factor (EF-1α) gene (2). Total DNA was extracted directly from fungal mycelium of isolate 19-14 with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany), and PCR amplification was performed with primer pair EF-1/EF-2 (2). Sequence analysis of the EF-1α gene revealed 100% nucleotide identity of isolate 19-14 (GenBank Accession No. KM102536) with the EF-1α sequences of two F. avenaceum isolates from Canada (KC999504 from rye and JX397864 from Triticum durum). To our knowledge, this is the first report of F. avenaceum causing collar, crown, and root rots of stored carrot in Serbia. Since F. avenaceum can produce several mycotoxins, including moniliformin, acuminatopyrone, and chrysogine (3), the presence of this pathogen on stored carrots could represent a significant constraint for carrot production in Serbia, for both direct yield losses and potential mycotoxin contamination. References: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual, Blackwell Publishing, London, UK, 2006. (2) K. O'Donnell et al. Proc. Natl. Acad. Sci. U.S.A. 95:2044, 1998. (3) J. L. Sorenson. J. Agric. Food Chem. 57:1632, 2009. (4) Statistical Office, Republic of Serbia. Retrieved from http://webrzs.stat.gov.rs in May 2014.

First Report of Watermelon mosaic virus Infecting Melon and Watermelon in Bosnia and Herzegovina.

Hereby the expansion of host range of Watermelon mosaic virus (WMV, Potyvirus, Potyviridae), found previously on zucchini in Bosnia and Herzegovina (3), to two new hosts is reported. Also, this is the first finding of WMV "emerging" (EM) isolate causing more severe symptoms in some cucurbits than "classic" (CL) isolates (1). During a July 2013 survey to determine the presence of WMV on cucurbits in Bosnia and Herzegovina, in the Kosijerovo locality (Laktasi Municipality, Bosnia and Herzegovina), virus-like symptoms were observed on 10% of plants. Severe mosaic, puckering, and leaf deformation as well as necrosis and leaf distortion were observed in a melon (Cucumis melo L.) crop, while mosaic, green vein banding, and leaf curling with reduced leaf size were observed in watermelon (Citrullus lanatus [Thunb.] Matsum and Nakai). Sampled melon and watermelon plants were tested for the presence of WMV with commercial double-antibody sandwich (DAS)-ELISA kit (Bioreba, AG, Reinach, Switzerland). Commercial positive and negative controls were included in each assay. Out of the 30 melon and 25 watermelon plants tested, 24 and 23 samples were positive for WMV, respectively, while no other cucurbit viruses were detected. The virus was mechanically transmitted from one of each of ELISA-positive melon (309-13) and watermelon (314-13) samples to five plants of each Cucurbita pepo 'Ezra F1', C. melo 'Ananas,' and C. lanatus 'Creamson sweet' using 0.01 M phosphate buffer (pH 7). Mild to severe mosaic and bubbling followed by leaf deformation were observed in all inoculated plants 10 to 14 days post-inoculation, regardless the isolate. Serological detection was verified with reverse transcription (RT)-PCR using the One-Step RT-PCR Kit (Qiagen, Hilden, Germany) with primers WMV 5' and WMV 3' (1), designed to amplify a 402- to 408-bp fragment overlapping the N-terminal part of the coat protein (CP) gene. Total RNAs were extracted with the RNeasy Plant Mini Kit (Qiagen). Total RNAs from the Serbian WMV oil pumpkin isolate (GenBank Accession No. JF325890) and RNA from healthy melon and watermelon plants were used as positive and negative controls, respectively. An amplicon of the expected size was produced from all serologically positive melon and watermelon plants, but not from healthy tissues. The RT-PCR products derived from isolates 309-13 and 314-13 were sequenced directly (KJ603311 and KM212956, respectively) and compared with WMV sequences available in GenBank. Sequence analysis revealed 91.5% nucleotide (nt) identity (94.6% amino acid [aa] identity) between the two WMV isolates. The melon WMV isolate shared the highest nt identity of 100% with four WMV isolates from Slovakia (GQ241712 to 13), Serbia (FJ325890), and Bosnia and Herzegovina (KF517099), while the sequence of isolate 314-13 had the highest nt identity with three Serbian isolates (JX262104 to 05 and JX262114) of 99.7% (99.2% aa identity). Phylogenetic analyses placed isolate 309-13 with CL isolates, while isolate 314-13 clustered with EM isolates (1,2). To our knowledge, this is the first report of WMV on melon and watermelon and the first report on EM isolates in Bosnia and Herzegovina. This could cause significant economic losses and become a limiting factor for cucurbit production with the potential of EM isolates to rapidly replace CL (2). References: (1) C. Desbiez et al. Arch. Virol. 152:775, 2007. (2) C. Desbiez et al. Virus Res. 152:775, 2009. (3) V. Trkulja et al. Plant Dis. 98:573, 2014.

First Report of Cucumber mosaic virus in Tulipa sp. in Serbia.

Tulips (Tulipa sp. L.), popular spring-blooming perennials in the Liliaceae family, are one of the most important ornamental bulbous plants, which have been cultivated for cut flower, potted plant, garden plant, and for landscaping. In May 2013, during a survey to determine the presence of Cucumber mosaic virus (CMV, Cucumovirus, Bromoviridae) on ornamentals in Serbia, virus-like symptoms, including the presence of bright streaks, stripe and distortion of leaves, and reduced growth and flower size, were observed in an open field tulip production in the Krnjaca locality (a district of Belgrade, Serbia). Disease incidence was estimated at 20%. Symptomatic tulip plants were collected and tested for the presence of CMV by double-antibody sandwich (DAS)-ELISA using commercial diagnostic kit (Bioreba, AG, Reinach, Switzerland). Commercial positive and negative controls were included in each ELISA. Of the six tulip plants tested, all were positive for CMV. In bioassay, five plants of each Chenopodium quinoa, Nicotiana tabacum 'Samsun,' and N. glutinosa were mechanically inoculated with sap from selected ELISA-positive sample (79-13) using 0.01 M phosphate buffer (pH 7). Chlorotic local lesions on C. quinoa, and severe mosaic and leaf malformations on N. tabacum 'Samsun' and N. glutinosa, were observed 5 and 14 days post-inoculation, respectively. All mechanically inoculated plants were positive for CMV in DAS-ELISA testing. For further confirmation of CMV presence in tulip, total RNAs from all ELISA-positive symptomatic tulip plants were extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Reverse transcription (RT)-PCR was performed with the One-Step RT-PCR Kit (Qiagen) using specific primer pair CMVCPfwd and CMVCPrev (1), which flank conserved fragment of the RNA3 including the entire coat protein (CP) gene and part of 3'- and 5'-UTRs. Total RNAs obtained from the Serbian watermelon CMV isolate (GenBank Accession No. JX280942) and healthy tulip leaves served as the positive and negative controls, respectively. The RT-PCR products of 871 bp were obtained from all six samples that were serologically positive to CMV, as well as from the positive control. No amplicon was recorded in the healthy control. The amplified product which derived from isolate 79-13 was purified (QIAquick PCR Purification Kit, Qiagen), directly sequenced in both directions using the same primer pair as in RT-PCR, deposited in GenBank (KJ854451), and analyzed by MEGA5 software (4). Sequence comparison of the complete CP gene (657 nt) revealed that the Serbian isolate 79-13 shared the highest nucleotide identity of 99.2% (99% amino acid identity) with CMV isolates from Japan (AB006813) and the United States (S70105). To our knowledge, this is the first report on the occurrence of CMV causing mosaic on Tulipa sp. in Serbia. Taking into account vegetative reproduction of tulips and the large scale of international trade with tulip seeding material, as well as wide host range of CMV including a variety of ornamentals (2,3), this is a very important discovery representing a serious threat for the floriculture industry in Serbia. References: (1) K. Milojevic et al. Plant Dis. 96:1706, 2012. (2) M. Samuitiene and M. Navalinskiene. Zemdirbyste-Agriculture 95:135, 2008. (3) D. Sochacki. J. Hortic. Res. 21:5, 2013. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

First Report of Fusarium Wilt of Strawberry Caused by Fusarium oxysporum in Serbia.

Strawberry (Fragaria × ananassa Duch.) is the third most important berry crop in Serbia with average production ranging from 30,000 to 35,000 t on approximately 5,000 ha (2). In June 2013, symptoms of wilt and whole plant collapse were observed on approximately 25% plants growing in commercial strawberry crop of cv. Alba in the locality of Zablace (Moravica district). Initial symptoms included leaf chlorosis and wilt, followed by withering and necrosis of older leaves and reduced fruit production, eventually leading to plant collapse and desiccations. Internal vascular tissues of the crown showed distinct brown reddish discoloration. Three small pieces of infected roots, petioles, or crown vascular tissues were surface disinfested with 2% NaOCl and placed on five potato dextrose agars (PDA) per sample. After 7 days incubation at 23°C under 12 h of fluorescent light, nine monoconidial isolates were obtained (1) forming colonies with light purple mycelia. Colonies produced numerous hyaline, oval to ellipsoid microconidia (5 to 15 × 2.5 to 4.5 µm, average 8.45 × 2.25 µm), 3 to 5 septate fusoid macroconidia with pedicellate bases (20 to 50 × 2.70 to 6 µm, average 32.35 × 3.25 µm from 100 measured) and chlamydospores. Morphological and growth features were similar to the descriptions of Fusarium oxysporum Schlechtend emend. Snyder & Hansen (1). Pathogenicity of one selected isolate (97-13) was tested by dipping for 15 min the roots of five plants of each cultivar: Alba, Arosa, Clery, and Roxana into a conidial suspension (1 × 106 conidia/ml) harvested from a 7-day-old culture on PDA. Control plants were dipped in sterile distilled water. The inoculated plants were transplanted into pots containing sterilized peat and maintained in the greenhouse at 25°C. Thirty to thirty-five days post-inoculation, all plants developed wilt symptoms and vascular discoloration of crown tissues from which F. oxysporum was successfully re-isolated using the same method as for isolation. No symptoms were observed on any of the control plants. Morphological identification was confirmed by amplification and sequencing of a portion of the translation elongation factor-1 alpha (EF-1α) gene. Total DNA was extracted directly from fungal mycelium with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and PCR amplification performed with primers EF-1/EF-2 (4). Sequence analysis of EF-1α region revealed that Serbian isolate 97-13 (GenBank Accession No. KJ647280) shared 99 to 100% identity with the F. oxysporum sequences in GenBank. To our knowledge, this is the first report of Fusarium wilt on strawberry in Serbia. The presence of a new and potentially harmful disease may represent a serious constraint for strawberry production in Serbia. References: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual, Blackwell Publishing, London, UK, 2006. (2) M. Nikolic et al. Acta Hort. 842:615, 2009. (3) K. O'Donnell et al. Proc. Natl. Acad. Sci. USA 95:2044, 1998.

First Report of Zucchini yellow mosaic virus in Watermelon in Bosnia and Herzegovina.

Several potyvirus species cause severe economic losses in cucurbit crops in the Mediterranean region, but Zucchini yellow mosaic virus (ZYMV) is regarded as one of the most destructive (2,3). In June 2012, field-grown watermelon plants (Citrullus lanatus [Thunb.] Matsum and Nakai) showing mild to severe mosaic, mottling, and bubbling followed by leaf deformation with blistering were observed in the Kukulje locality (Region of Banja Luka) in Bosnia and Herzegovina. Incidence of virus infection in the field was visually estimated at 15%. Symptomatic watermelon plants were collected and tested for the presence of the most prevalent watermelon viruses including ZYMV, Cucumber mosaic virus (CMV), Watermelon mosaic virus (WMV), Papaya ringspot virus (PRSV), and Squash mosaic virus (SqMV) (1) using commercial double-antibody sandwich (DAS)-ELISA diagnostic kits (Bioreba AG, Reinach, Switzerland). Commercial positive and negative controls were included in each assay. Of the 14 watermelon plants tested, all were positive for ZYMV and negative for WMV, CMV, PRSV, and SqMV. Sap prepared from an ELISA-positive sample (isolate 314-12) and healthy watermelon plants, using 0.01 M phosphate buffer (pH 7) was mechanically inoculated onto five carborundum-dusted plants of each Chenopodium quinoa and Citrullus lanatus 'Creamson sweet'. Mechanically inoculated C. quinoa plants exhibited chlorotic spots 5 days post-inoculation, while severe mosaic accompanied by crinkling and leaf deformation were observed on all inoculated watermelon plants 12 days post-inoculation. For further confirmation of the virus identity, total RNAs from all 14 naturally and 5 mechanically infected watermelon plants were extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and subjected by reverse transcription (RT)-PCR. RT-PCR was carried out with One-Step RT-PCR Kit (Qiagen) using ZYMV-specific primer pair, CPfwd and CPrev (4), designed to amplify an 1,100-bp fragment covering the entire coat protein (CP) gene and part of the nuclear inclusion (NIb) and 3'-UTR. Total RNAs obtained from the Serbian ZYMV isolate from winter squash (GenBank Accession No. JN315861) and tissue sample from healthy watermelon leaves were used as positive and negative controls, respectively. The expected size of the RT-PCR product was amplified from each of the watermelon plants assayed confirming serological virus identification. One amplicon derived from isolate 314-12 was purified (QIAquick PCR Purification Kit, Qiagen) and sequenced directly (KF836440). Sequence analysis of the complete CP gene, conducted by MEGA5 software, revealed that watermelon isolate from Bosnia and Herzegovina showed the highest nucleotide identity of 99.8% (99.6% amino acid identity) with 14 ZYMV isolates originating from different hosts from Serbia (HM072431, JF308189 to 90, JN315856 to 57, JN315859 to 61) and Austria (AJ420012 to 17). To our knowledge, this is the first report of ZYMV in Bosnia and Herzegovina, which is an important discovery. It represents expansion of this virus to new geographical area. Considering that the ZYMV is among the most devastating pathogens of cucurbits (3), further survey is needed to determine its distribution in Bosnia and Herzegovina. References: (1) L. M. da Silveira et al. Trop. Plant Pathol. 34:123, 2009. (2) H. Lecoq et al. Virus Res. 141:190, 2009. (3) H. Lecoq and C. Desbiez. Adv. Virus Res. 84:67, 2012. (4) M. F. Pfosser and H. Baumann. Arch. Virol. 147:1599, 2002.

First Report of Watermelon mosaic virus in Zucchini Squash in Bosnia and Herzegovina.

Aphid-borne Watermelon mosaic virus (WMV; genus Potyvirus, family Potyviridae) is widely distributed in the Mediterranean area and is one of the most prevalent cucurbit viruses in the region (4). In July 2012, approximately 20% of zucchini squash (Cucurbita pepo L.) plants showing virus-like symptoms were observed in one field in Kukulje locality (region of Banja Luka), Bosnia and Herzegovina. Infected plants exhibited mild to severe mosaic, chlorotic mottling, and dark green vein banding, as well as puckering and leaf deformation. Symptoms mostly developed on leaves, while fruits usually only failed to develop a normal coloration. Leaves from 15 symptomatic zucchini squash plants were sampled and analyzed utilizing double-antibody sandwich (DAS)-ELISA kits (Bioreba, AG, Reinach, Switzerland) with commercial antisera specific for five commonly occurring cucurbit-infecting viruses: WMV, Zucchini yellow mosaic virus (ZYMV), Papaya ringspot virus (PRSV), Cucumber mosaic virus (CMV), and Squash mosaic virus (SqMV) (1,3,4). Commercial positive and negative controls were included in each test. WMV was detected serologically in all tested zucchini squash samples, while no presence of other tested viruses were found. Crude sap extracted from leaves of a serologically positive sample (307-12) using 0.01 M phosphate buffer (pH 7) was mechanically inoculated onto five plants of C. pepo 'Ezra F1' and severe mosaic accompanied by bubbling and leaf malformation was observed 14 days post-inoculation. Viral identification in all naturally and mechanically infected plants was further confirmed by conventional reverse transcription (RT)-PCR. Total RNAs were extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and RT-PCR was performed using the One-Step RT-PCR Kit (Qiagen) with specific primers WMV 5' and WMV 3' (4), yielding a 402- to 408-bp fragment corresponding to the N-terminal part of the coat protein (CP) gene (2). Total RNAs obtained from the Serbian WMV isolate from oil pumpkin (GenBank Accession No. JF325890) and healthy zucchini squash leaves were used as positive and negative controls, respectively. A product of the correct predicted size was obtained in all naturally and mechanically infected plants as well as positive control. No amplicon was recorded in healthy control. After purification (QIAquick PCR Purification Kit, Qiagen) the amplicon obtained from one selected isolate 307-12 was sequenced directly in both direction, aligned and compared by MEGA5 software with WMV sequences available in GenBank. Sequence comparisons revealed that the zucchini squash isolate from Bosnia and Herzegovina (KF517099) showed the highest nucleotide identity of 100% with one isolate from Serbia (FJ325891) and two Slovakian WMV isolates (GQ241712 to 13), all belonging to the classical group of WMV isolates (4). To our knowledge, this is the first report of WMV infecting zucchini squash in Bosnia and Herzegovina. Since squash and other cucurbit species represent valuable crops in Bosnia and Herzegovina, with annual production close to US$8.5 million ( http://faostat.fao.org ) and rising rapidly, the presence of a devastating pathogen like as WMV could be a serious constraint for their production. References: (1) A. Ali et al. Plant Dis. 96:243, 2012. (2) C. Desbiez et al. Arch. Virol. 152:775, 2007. (3) S. Jossey and M. Babadoost. Plant Dis. 92:61, 2008. (4) H. Lecoq and C. Desbiez. Adv. Virus Res. 84:67, 2012.

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.

First Report of Iris yellow spot virus Infecting Onion in Bosnia and Herzegovina.

In July 2012, a survey was conducted to determine the presence of tospoviruses in Bosnia and Herzegovina, symptoms resembling those caused by Iris yellow spot virus (IYSV; genus Tospovirus, family Bunyaviridae) were observed in an onion (Allium cepa) seed crop in the Gornji Karajzovci locality (Region of Banja Luka). Symptoms included chlorotic to necrotic, straw-colored, spindle- and diamond-shaped lesions, variable in size and randomly distributed on the leaves and particularly on the scapes. Later the lesions enlarged and coalesced, causing scape breakage. Affected plants occurred throughout the field and disease incidence was estimated at 20%. Symptomatic plants were sampled and assayed by double-antibody sandwich (DAS)-ELISA test using commercial polyclonal antisera (Bioreba AG, Reinach, Switzerland) against IYSV and two other tospoviruses, Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV). Commercial positive and negative controls were included in each test. IYSV was detected serologically in 19 of 20 screened samples and none of the samples tested positive for TSWV or INSV. The virus was mechanically transmitted from an ELISA-positive sample (302-12) to five of each Petunia × hybrida and Nicotiana benthamiana using chilled 0.01 M phosphate buffer (pH 7) containing 0.1% sodium sulfite (1). All inoculated P. × hybrida showed local necrotic spots, while N. benthamiana developed mild mosaic 4 and 10 days post-inoculation, respectively. However, difficulties were encountered in reproducing the disease symptoms on mechanically inoculated onion plants corroborating a previous study (2). Serological findings were verified with reverse transcription (RT)-PCR. Total RNAs from all naturally infected onion plants as well as mechanically infected N. benthamiana plants were extracted with the RNease Plant Mini Kit (Qiagen, Hilden, Germany). RT-PCR was performed with One-Step RT-PCR Kit (Qiagen) using IYSV-specific primers IYSV56U/IYSV917L (3), designed to amplify an 896-bp fragment of the S RNA which includes whole nucleocapsid (N) gene. Total RNAs from Serbian IYSV isolate from onion (GenBank Accession No. EU586203) and from healthy onion plants were used as positive and negative controls, respectively. An amplicon of the expected size was obtained from each of the plants assayed as well as from positive control, but not from the negative control. The amplified products derived from onion isolate 302-12 was purified (QIAquick PCR Purification Kit, Qiagen), sequenced directly (JX861126), and compared with known IYSV isolates. Sequence analysis of the complete N gene, conducted with MEGA5 software (4), revealed the highest nucleotide identity of 99.5% (100% amino acid identity) with IYSV onion isolate (DQ658242) from Texas. To our knowledge, this is the first report of IYSV in Bosnia and Herzegovina. Onion is an important and traditionally grown vegetable crop in Bosnia and Herzegovina and the presence of IYSV could represent an important constraint to onion and other susceptible host production. The discovery of IYSV on onion should prompt more detailed surveys, thorough inspections and subsequent testing to establish the distribution and incidence of IYSV in Bosnia and Herzegovina. References: (1) A. Kritzman et al. Plant Dis. 85:838, 2001. (2) L. Pozzer et al. Plant Dis. 83:345, 1999. (3) I. Robène-Soustrade et al. Plant Pathol. 55:288, 2006. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

First Report of Tomato spotted wilt virus on Gloxinia in Bosnia and Herzegovina.

In June and July 2012, symptoms resembling those caused by a tospovirus infection were observed on the greenhouse-grown gloxinia (Sinningia speciosa Benth. and Hook.) in the Lijevce polje, in the vicinity of Banja Luka (Bosnia and Herzegovina). Infected plants exhibited chlorotic ring spots and chlorotic and necrotic patterns followed by necrosis and distortion of leaves. Disease symptom incidence was estimated at 30% out of 400 inspected plants. Symptomatic leaves were collected and tested by double-antibody sandwich (DAS)-ELISA test using commercial polyclonal antisera (Bioreba AG, Reinach, Switzerland) for two of the most important tospoviruses in the greenhouse production of ornamentals: Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV) (2). TSWV was detected serologically in 27 out of 30 tested gloxinia samples, and all were negative for INSV. Symptomatic leaves of five selected ELISA-positive gloxinia plants were separately ground in chilled 0.01 M phosphate buffer (pH 7) containing 0.1% w/v sodium sulphite and were mechanically inoculated on five plants of Petunia × hybrida. All inoculated plants produced typical symptoms of TSWV (1), necrotic spots on inoculated leaves in 2 to 5 days post-inoculation. For further confirmation of TSWV infection, total RNAs were extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) from all 27 infected gloxinia plants and tested by reverse transcription (RT)-PCR assay. A 738-bp fragment of TSWV nucleocapsid (N) gene was amplified with One-Step RT-PCR Kit (Qiagen) using primer pairs TSWV CP-f and TSWV CP-r (4). Total RNAs from Serbian tobacco TSWV isolate (GenBank Accession No. GQ373173) and RNA extract from healthy gloxinia plants were used as positive and negative controls, respectively. Amplicons of the expected size were obtained from all 27 naturally infected gloxinia plants, while no amplification products were obtained from the healthy control. After the purification with QIAquick PCR Purification Kit (Qiagen), the RT-PCR product obtained from one selected isolate 160-12 was sequenced directly in both directions and submitted to GenBank (JX468079). Sequence analysis of the partial N gene, conducted by MEGA5 software (3), from isolate 160-12 showed the highest nucleotide identity of 99.7% (100% amino acid identity) with eight pepper isolates of TSWV from Spain (FR693229, FR693231, FR693152-153, FR693078, FR693081, FR693089, and FR693092). To our knowledge, this is the first report on the occurrence of TSWV in Bosnia and Herzegovina. The presence of this harmful pathogen into a new area could have a serious threat to intensive and increasing production of ornamentals and numerous other TSWV susceptible species in Bosnia and Herzegovina. The discovery of TSWV on gloxinia should prompt more surveys, thorough inspections, and subsequent testing of other TSWV susceptible plants cultivated in Bosnia and Herzegovina. References: (1) Anonymous. OEPP/EPPO Bull. 34:271, 2004. (2) Daughtrey et al. Plant Dis. 81:1220, 1997. (3) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011. (4) A. Vucurovic et al. Eur. J. Plant Pathol. 133:935, 2012.

First Report of Alfalfa mosaic virus Infecting Lavandula × intermedia in Croatia.

Lavandin (Lavandula × intermedia Emeric ex Loiseleur) is cultivated on a large scale in some South European countries for the extraction of essential oils or as an ornamental plant for gardens and landscapes. In May of 2012, virus-like symptoms including bright yellow calico mosaic, leaf distortion, and growth reduction were observed on 15% of lavandin plants in a commercial nursery in Banovo Brdo locality, Baranja County, Republic of Croatia. Leaves from 15 symptomatic lavandin plants were collected and examined by double-antibody sandwich (DAS)-ELISA using commercial antisera (Bioreba AG, Reinach, Switzerland) against two viruses known to infect Lavandula spp.: Alfalfa mosaic virus (AMV) and Cucumber mosaic virus (CMV) (2,3). Commercial positive and negative controls and extracts from healthy lavandin leaves were included in each ELISA. Only AMV was detected serologically in all 15 tested samples. Five plants each of Chenopodium quinoa, C. amaranticolor, and Nicotiana benthamiana were mechanically inoculated with sap from an ELISA-positive sample (70-12) using 0.01 M phosphate buffer (pH 7). Local chlorotic spots accompanied by systemic mosaic on both Chenopodium species and bright yellow mosaic on N. benthamiana were observed 6 and 12 days post-inoculation, respectively. Test plants were assayed by DAS-ELISA and all inoculated plants of each species tested positive for AMV. The presence of AMV in all symptomatic lavandin plants was further confirmed by reverse transcription (RT)-PCR assay. Total nucleic acid was extracted using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) using AMV specific primer pair CP AMV1 (5'-TCCATCATGAGTTCTTCAC-3') and CP AMV2 (5'-AGGACTTCATACCTTGACC-3') (1). Total RNAs obtained from the Serbian AMV isolate from alfalfa (GenBank Accession No. FJ527748) and healthy L. × intermedia plant served as the positive and negative control, respectively. The 751-bp amplicons, covering the partial coat protein (CP) gene and 3'-UTR, were obtained from all 15 samples that were serologically positive to AMV as well as from positive control. No amplification product was observed when extract from healthy L. × intermedia plant was used as template in the RT-PCR assay. The RT-PCR product derived from isolate 70-12 was directly sequenced in both directions using the same primer pair as in RT-PCR and deposited in GenBank (JX996119). Multiple sequence alignment of the CP open reading frame was performed by MEGA5 software (4) and revealed that the isolate 70-12 showed the highest nucleotide identity of 99.4% (99.5% amino acid identity) with Serbian AMV isolate from tobacco (FJ527749). To our knowledge, this is the first report of AMV on L. × intermedia in Croatia. Because lavandin is an aromatic plant traditionally and widely grown in Croatia, the presence of AMV could be a limiting factor for its successful production. References: (1) M. M. Finetti-Sialer et al. J. Plant Pathol. 79:115, 1997. (2) T. Kobylko et al. Plant Dis. 92:978, 2008. (3) L. Martínez-Priego et al. Plant Dis. 88:908, 2004. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

First Report of Impatiens necrotic spot virus on Begonia in Bosnia and Herzegovina.

Impatiens necrotic spot virus (INSV) and Tomato spotted wilt virus (TSWV) are the most serious viral pathogens in the production of ornamental plants in Europe and North America (1). During a survey for the presence of tospoviruses in July 2012, potted begonia hybrids (Begonia × tuberhybrida Voss) exhibiting foliar chlorotic rings and zonal spots accompanied by leaf necrosis and distortion, were observed in a greenhouse in the vicinity of Banja Luka (Bosnia and Herzegovina). Leaf samples collected from 12 symptomatic plants were analyzed for the presence of INSV and TSWV by commercial double-antibody sandwich (DAS)-ELISA kits (Bioreba AG, Reinach, Switzerland). Commercial positive and negative controls and extracts from healthy begonia leaves were included in each ELISA. INSV was detected serologically in all 12 begonia samples and all tested samples were negative for TSWV. Five healthy plants of each Petunia × hybrida and Nicotiana benthamiana were mechanically inoculated with sap from an ELISA-positive sample (157-12) using chilled 0.01 M phosphate buffer (pH 7) containing 0.1% sodium sulphite. Local necrotic lesions on P. × hybrida and systemic chlorotic mottling on N. benthamiana were observed on all inoculated plants 4 and 10 days post-inoculation, respectively. For further confirmation of INSV infection, total RNAs were extracted from all ELISA-positive begonia plants as well as mechanically inoculated N. benthamiana plants with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and used as template in reverse transcription (RT)-PCR. RT-PCR was performed with the OneStep RT-PCR Kit (Qiagen) using primer pair INSV-589 and TOS-R15 (3), specific to the partial INSV nucleocapsid (N) gene. Total RNA obtained from Serbian INSV isolate from a begonia (GenBank Accession No. HQ724289) and RNA extracts from healthy begonia plants were used as positive and negative controls, respectively. All naturally and mechanically infected plants as well as the positive control yielded an amplicon of the expected size (589 bp), while no amplification products were obtained from the healthy controls. The RT-PCR product derived from the isolate 157-12 was sequenced directly after purification with QIAquick PCR Purification Kit (Qiagen) and submitted to GenBank (KC494869). Pairwise comparison of the 157-12 isolate N sequence with other homologous sequences available in GenBank, conducted using MEGA5 software (2), revealed that begonia isolate from Bosnia and Herzegovina showed the highest nucleotide identity of 99.7% (100% amino acid identity) with the Chinese INSV isolate (FN400772) originating from Oncidium sp. To our knowledge, this is the first report of INSV on begonia in Bosnia and Herzegovina. Begonias are very popular and widely grown ornamentals in Bosnia and Herzegovina and the presence of a new and devastating pathogen could represent a serious threat for its production. Since begonia is commonly grown together with numerous ornamental plants susceptible to INSV, further investigations are needed in order to prevent spread of this potentially harmful pathogen to new hosts in Bosnia and Herzegovina. References: (1) M. L. Daughtrey et al. Plant Dis. 81:1220, 1997. (2) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011. (3) H. Uga and S. Tsuda. Phytopathology 95:166, 2005.

First Report of Cucumber mosaic virus Infecting Peperomia tuisana in Serbia.

Peperomia tuisana C.DC. ex Pittier (family Piperaceae) is an attractive succulent grown as an ornamental. Despite its tropical origins, it can be successfully grown indoors in any climate. In March 2012, three samples of P. tuisana showing virus-like symptoms were collected from a commercial greenhouse in Zemun (District of Belgrade, Serbia) in which estimated disease incidence was 80%. Infected plants showed symptoms including necrotic ringspots and line patterns that enlarged and caused necrosis of leaves. A serious leaf drop led to growth reduction and even death of the plant. Leaves from three symptomatic P. tuisana plants were sampled and analyzed by double-antibody sandwich (DAS)-ELISA using commercial diagnostic kits (Bioreba AG, Reinach, Switzerland) against the most common viral pathogens of ornamentals: Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV), and Impatiens necrotic spot virus (INSV) (1,2). Commercial positive and negative controls were included in each ELISA. Serological analyses showed that all plants were positive for CMV and negative for TSWV and INSV. The ELISA-positive sample (isolate 1-12) was mechanically inoculated onto five plants each of three test species as well as of healthy young P. tuisana using 0.01 M phosphate buffer (pH 7). Chlorotic local lesions on Chenopodium quinoa and severe mosaic and leaf malformations were observed on all inoculated Nicotiana tabacum 'Samsun' and N. glutinosa. Also, the virus was successfully mechanically transmitted to P. tuisana that reacted with symptoms identical to those observed on the original host plants. All mechanically inoculated plants were positive for CMV in DAS-ELISA. For further confirmation of CMV infection, reverse transcription (RT)-PCR was performed on extracts made from symptomatic P. tuisana, N. tabacum 'Samsun,' and N. glutinosa leaf materials. Total RNAs were extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and RT-PCR was carried out using One-Step RT-PCR Kit (Qiagen). A CMV-specific primer pair, CMVCPfwd and CMVCPrev (3), which amplifies an 871-bp fragment of the entire coat protein (CP) gene and part of 3'- and 5'-UTRs, were used for both amplification and sequencing. Total RNAs obtained from the Serbian CMV isolate (HM065510) and healthy P. tuisana were used as positive and negative controls, respectively. A product of the correct predicted size was obtained in all naturally and mechanically infected plants, as well as positive control. No amplicon was recorded in the healthy control. The amplified product derived from isolate 1-12 was purified (QIAquick PCR Purification Kit, Qiagen), directly sequenced in both directions, deposited in GenBank (KC505441), and analyzed by MEGA5 software (4). Sequence comparison of the complete CP gene (657 nt) revealed that the Serbian isolate 1-12 shared the highest nucleotide identity of 99.1% (99.5% amino acid identity) with the Japanese isolate (AB006813). To our knowledge, this is the first report on the occurrence of CMV in P. tuisana in Serbia. This is also an important discovery since P. tuisana is commonly grown together with other ornamental hosts of CMV, and thus could represent a serious threat for future expansion of CMV in the greenhouse floriculture industry in Serbia. References: (1) M. L. Daughtrey et al. Plant Dis. 81:1220, 1997. (2) S. Flasinski et al. Plant Dis. 79:843, 1995. (3) K. Milojevic et al. Plant Dis. 96:1706, 2012. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

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

Brugmansia (Brugmansia spp.), also known as Angel's 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. Vucurovic et al. Eur. J. Plant Pathol. 133:935, 2012.

First Report of the Natural Infection of Robinia pseudoacacia with Alfalfa mosaic virus.

Robinia pseudoacacia L. (family Fabaceae), commonly known as black locust, is native to the southeastern United States, but has been widely planted and naturalized in temperate regions worldwide. In Europe it is often planted alongside streets and in parks, not only because of the dense canopy and impressive flower clusters in spring, but also because it tolerates air pollution well. In June 2012, several black locust trees exhibiting yellow leaf spots accompanied by mottling and leaf deformation were observed in a park in Backa Topola, North Backa District, Serbia. Numerous aphid colonies were found colonizing symptomatic trees. Leaves collected from nine symptomatic and 10 asymptomatic trees were tested for the presence of three common aphid-transmitted viruses, Alfalfa mosaic virus (AMV), Cucumber mosaic virus, and Potato virus Y, using double-antibody sandwich (DAS)-ELISA with commercial polyclonal antibody (Bioreba AG, Reinach, Switzerland). Commercial positive and negative controls and extracts from healthy black locust leaves were included in each assay. AMV was serologically detected in all symptomatic and also in four of the asymptomatic trees, while no other tested viruses were found. Sap from affected leaves of a ELISA-positive sample (373-12) was mechanically inoculated onto five plants each of Chenopodium quinoa and Nicotiana benthamiana using 0.01 M phosphate buffer (pH 7). Symptoms including local chlorotic leaf lesions followed by mosaic on C. quinoa and a bright yellow mosaic on N. benthamiana were observed on all inoculated plants 5 and 10 days post-inoculation, respectively. The identity of the virus was confirmed using reverse transcription (RT)-PCR analysis. Total RNAs from all naturally and mechanically infected plants were isolated using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RT-PCR was carried out using the One-Step RT-PCR Kit (Qiagen) with primer pair CP AMV1 and CP AMV2 specific to the partial CP gene and 3'-UTR of AMV RNA 3 (1). Total RNAs from Serbian AMV isolate from alfalfa (GenBank Accession No. FJ527748) and RNA extract from healthy leaves of R. pseudoacacia were used as positive and negative controls, respectively. All tested plants, as well as the positive control, yielded an amplicon of the correct predicted size (751 bp), while no amplicon was recorded in the healthy control. The amplified product of isolate 373-12 was purified with QIAquick PCR Purification Kit (Qiagen) and sequenced on ABI PRISM 3700 DNA analyzer (Macrogen, South Korea) in both directions (KC288155). Pairwise comparison of the 373-12 isolate CP sequence with those available in GenBank, conducted with MEGA5 software (4), revealed the maximum nucleotide identity of 99% (99% amino acid identity) with the soybean isolate (HQ185569) from Tennessee. AMV has a worldwide distribution and its natural host range includes over 150 plant species, including many herbaceous and several woody plants (2). To our knowledge, this is the first report of R. pseudoacacia as a natural host of AMV worldwide. This finding has potentially significant implications for the successful production of susceptible crops, considering that black locust could act as an important link in the epidemiology of AMV as it may serve as a virus reservoir (3). References: (1) M. M. Finetti-Sialer et al. J. Plant Pathol. 79:115, 1997. (2) R. Hull. Comparative Plant Virology. 2nd ed. Elsevier Academic Press, Burlington, MA, 2009. (3) E. E. Muller et al. Plant Dis. 96:506, 2012. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

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 Backa 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. Stankovic et al. Acta Virol. 55:337, 2011. (4) A. Vucurovic et al. Eur. J. Plant Pathol. 133:935, 2012.

Lamium maculatum is a Natural Host for Cucumber mosaic virus.

Lamium maculatum L. (spotted dead-nettle) is a flowering perennial ornamental that is commonly grown as a landscape plant for an effective ground cover. In June 2010, severe mosaic accompanied by reddish brown necrosis and leaf deformation was noticed on 80% of L. maculatum growing in shade under trees and shrubs in Sarajevo (Bosnia and Herzegovina). Leaves from 10 symptomatic L. maculatum plants were sampled and analyzed by double-antibody sandwich (DAS)-ELISA using commercial diagnostic kits (Bioreba AG, Reinach, Switzerland) against Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV), and Impatiens necrotic spot virus (INSV), the most important viral pathogens of ornamental plants (1,2). Commercial positive and negative controls and extracts from healthy L. maculatum leaves were included in each assay. All samples tested negative for TSWV and INSV and positive for CMV. The virus was mechanically transmitted to test plants and young virus-free plants of L. maculatum using 0.01 M phosphate buffer (pH 7). The virus caused chlorotic local lesions on Chenopodium quinoa, while systemic mosaic was observed on Capsicum annuum 'Rotund,' Nicotiana rustica, N. glutinosa, N. tabacum 'White Burley,' and Phaseolus vulgaris 'Top Crop.' The virus was transmitted mechanically to L. maculatum and induced symptoms resembling those observed on the source plants. Inoculated plants were assayed by DAS-ELISA and all five inoculated plants of each species tested positive for CMV. The presence of CMV in L. maculatum as well as mechanically infected N. glutinosa plants was further confirmed by RT-PCR. Total RNA from symptomatic leaves was isolated using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) following the manufacturer's instructions. The primer pair, CMVAu1u/CMVAu2d, that amplifies the entire coat protein (CP) gene and part of 3'- and 5'-UTRs was used for both amplification and sequencing (4). Total RNA obtained from the Serbian CMV isolate from pumpkin (GenBank Accession No. HM065510) and a healthy L. maculatum plant were used as positive and negative controls, respectively. All naturally and mechanically infected plants as well as the positive control yielded an amplicon of the expected size (850 bp). No amplicon was observed in the healthy control. The amplified product derived from isolate 3-Lam was purified (QIAquick PCR Purification Kit, Qiagen), directly sequenced in both directions and deposited in GenBank (JX436358). Sequence analysis of the CP open reading frame (657 nt), conducted with MEGA5 software, revealed that the isolate 3-Lam showed the highest nucleotide identity of 99.4% (99.1% amino acid identity) with CMV isolates from Serbia, Australia, and the USA (GQ340670, U22821, and U20668, respectively). To our knowledge, this is the first report of the natural occurrence of CMV on L. maculatum worldwide and it adds a new host to over 1,241 species (101 plant families) infected by this virus (3). This is also an important discovery for the ornamental industry since L. maculatum is commonly grown together with other ornamental hosts of CMV in nurseries and the urban environment as well as in natural ecosystems. References: (1) Y. K. Chen et al. Arch. Virol. 146:1631, 2001. (2) M. L. Daughtrey et al. Plant Dis. 81:1220, 1997. (3) M. Jacquemond. Adv. Virus Res. 84:439, 2012. (4) I. Stankovic et al. Acta Virol. 55:337, 2011.

First Report of Tomato spotted wilt virus Infecting Onion and Garlic in Serbia.

In June 2011, extensive bleaching and numerous small whitish spots on leaves were observed in an onion (Allium cepa) seed crop as well as chlorotic spots and streaks in the neighboring garlic (A. sativum) bulb crop in the Aleksandrovo locality (Central Banat District, Serbia). Affected plants occurred throughout the field and disease incidence was estimated at 60% in the onion and 40% in the garlic crop. A high population of Thrips tabaci that was found in both crops, and local necrotic spots on Petunia × hybrida mechanically inoculated with infected onion or garlic sap by a chilled 0.01 M phosphate buffer, pH 7.0, containing 0.1% sodium sulfite (1), suggested the presence of a Tospovirus. For these reasons, sampled symptomatic onion and garlic plants were tested for the presence of Tomato spotted wilt virus (TSWV) and Iris yellow spot virus (IYSV) using commercial double-antibody sandwich-ELISA diagnostic kits (Bioreba AG, Reinach, Switzerland). Commercial positive and negative controls and extracts from healthy onion and garlic tissue were included in each ELISA. Of the 18 onion and 10 garlic plants tested, 16 and 7 samples, respectively, were positive for TSWV, and all were negative for IYSV. The identity of TSWV was further confirmed by conventional reverse transcription (RT)-PCR analysis. Total RNAs were extracted with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) using TSWV-specific forward (5'-GGTTAAGCTCACTAAGAAARCA-3') and reverse primers (5'-TTTAACYCCRAACATTTCATAGA-3'), designed to amplify a 738-bp fragment of the nucleocapsid protein (N) gene. Total RNAs obtained from plants infected with a Serbian isolate of TSWV (GenBank Accession No. GQ373173) and healthy onion garlic plants were used as positive and negative controls, respectively. An amplicon of the expected size was produced from the 16 onion and 7 garlic ELISA-positive plants, but not from healthy controls. The amplified products derived from the two selected isolates, 114-11 from onion and 115-11 from garlic, were sequenced directly after purification with the QIAquick PCR Purification kit (Qiagen); the sequences obtained were allocated GenBank Accession Nos. JQ619234 and JQ619235, respectively. Sequence analysis of the partial N gene, conducted with MEGA5 software (4), revealed 99.9% nucleotide identity (100% amino acid identity) between the two Serbian Allium isolates. Serbian onion and garlic isolates showed the highest nucleotide identities of 100% and 99.9% with Serbian summer squash isolate (JF303081) and tobacco isolate from Montenegro (GU369729), respectively. Well-established in many European countries, TSWV has been reported as an important constraint to the production of tomato, pepper, tobacco, and ornamentals (2), but the information on TSWV naturally infecting Allium spp. is limited. The presence of TSWV on onion and garlic in Serbia revealed that its known host range has expanded in Europe. To our knowledge, other than Marchoux's unpublished data (3), there are no other reports of garlic as a natural host of TSWV. The TSWV presence on Allium spp. represents a serious threat for these crops in Serbia, considering that it is prevalent in other crops in the area and its vectors are widespread. References: (1) Anonymous. OEPP/EPPO Bull. 34:271, 2004. (2) H. R. Pappu et al. Virus Res. 141:219, 2009. (3) G. Parrella et al. J. Plant Pathol. 85:227, 2003. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

First Report of Oidium neolycopersici on Greenhouse Tomatoes in Serbia.

In September 2011, tomato (Solanum lycopersicum L. 'Big Beef') plants showing typical symptoms of powdery mildew were collected in a greenhouse in the vicinity of Padinska Skela (District of City of Belgrade) in Serbia. Numerous circular, white colonies of powdery mildew were observed predominantly on the adaxial surface of the leaves, the petioles, and the stems. The foliage of infected plants turned yellow and necrotic, which was followed by rapid defoliation. Disease incidence was estimated by counting plants with powdery mildew symptoms in a random batch of 100 plants in four replicates and estimated to be extremely high, approaching 90%. Tomato plants ('Novosadski Jabucar') were inoculated with conidia released from diseased tomato leaves positioned above the tomato leaves and maintained at 25°C with a 14-h photoperiod. Healthy tomato plants from the same lot, which were not exposed to the conidia shower, were used as negative control. The first white fungal colonies appeared on the leaves of the inoculated plants within 4 to 7 days after inoculation, while no fungal growth was observed in the control plants. To determine the morphological characteristics of the pathogen, surface mycelium was removed with small strips of clear adhesive tape and examined using light microscopy. Microscopic observation revealed mycelium with lobed appressoria and hyaline, ellipsoid-ovoid or doliform conidia (32.5 to 47.5 × 17.5 to 25 µm) with no distinct firosin bodies and which produced sub-terminal germ tubes. Conidia were produced on the unbranched, erect conidiophores (82.5 to 150 µm) consisting of a cylindrical foot-cell followed by one to three short cells. No chasmothecia were found. On the basis of morphological characteristics, the pathogen was identified as Oidium neolycopersici (4), which was confirmed by internal transcribed spacer (ITS) sequence analysis. Total DNA was extracted directly from the whitish spots of superficial mycelium on the leaves with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. PCR amplification and sequencing were performed with primers ITS1F and ITS4 (1). The nucleotide sequence of the representative isolate 809-11 (Accession No. JQ619840) shared 100% identity with 16 O. neolycopersici isolates deposited in GenBank from different parts of the world. Tomato powdery mildew caused by O. neolycopersici is present in many European (4) and other countries around the world (3) and is becoming economically very important as majority of the tomato cultivars have shown to be susceptible (2). To our knowledge, this is the first report of O. neolycopersici in Serbia. Because tomato is a very popular and widely grown vegetable in Serbia, the presence of a new and potentially harmful disease could endanger greenhouse as well as open field tomato production. References: (1) J. H. Cunnington et al. Australas. Plant Pathol. 32:421, 2003. (2) T. Jankovics et al. Phytopathology 98:529, 2008. (3) H. Jones et al. Mol. Plant Pathol. 2:303, 2001. (4) L. Kiss et al. Mycol. Res. 105:684, 2001.

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. Acimovic and N. Lacok. 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.