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

Plant Disease ◽  
2014 ◽  
Vol 98 (12) ◽  
pp. 1749-1749 ◽  
Author(s):  
V. Trkulja ◽  
J. Vasić ◽  
B. Vuković ◽  
I. Stanković ◽  
A. Vučurović ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Bosnia And Herzegovina ◽  
Citrullus Lanatus ◽  
Economic Losses ◽  
Watermelon Mosaic Virus ◽  
Post Inoculation ◽  
Rt Pcr ◽  
First Report ◽  
Leaf Deformation ◽  
Negative Controls

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 (Laktaši 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.

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

Plant Disease ◽  
2014 ◽  
Vol 98 (6) ◽  
pp. 858-858 ◽  
Author(s):  
V. Trkulja ◽  
D. Jošić Kovačić ◽  
J. Mihić Salapura ◽  
I. Stanković ◽  
A. Vučurović ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Bosnia And Herzegovina ◽  
Citrullus Lanatus ◽  
Zucchini Yellow Mosaic Virus ◽  
Yellow Mosaic Virus ◽  
Post Inoculation ◽  
Rt Pcr ◽  
Cp Gene ◽  
Leaf Deformation ◽  
Negative Controls

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.

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

Plant Disease ◽  
2014 ◽  
Vol 98 (4) ◽  
pp. 573-573 ◽  
Author(s):  
V. Trkulja ◽  
J. Stojčić ◽  
D. Kovačić ◽  
I. Stanković ◽  
A. Vučurović ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Bosnia And Herzegovina ◽  
Zucchini Yellow Mosaic Virus ◽  
Yellow Mosaic Virus ◽  
Watermelon Mosaic Virus ◽  
Post Inoculation ◽  
Rt Pcr ◽  
Sequence Comparisons ◽  
First Report ◽  
Negative Controls

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.

scholarly journals First Report of Cucumber mosaic virus on Melon in Bosnia and Herzegovina

Plant Disease ◽  
2013 ◽  
Vol 97 (8) ◽  
pp. 1124-1124 ◽  
Author(s):  
V. Trkulja ◽  
D. Kovačić ◽  
B. Ćurković ◽  
A. Vučurović, I. Stanković ◽  
A. Bulajić ◽  
...  
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Bosnia And Herzegovina ◽  
Cucumis Melo ◽  
Cucurbita Pepo ◽  
Yellow Mosaic Virus ◽  
Rt Pcr ◽  
First Report ◽  
Negative Controls ◽  
Das Elisa

During July 2012, field-grown melon plants (Cucumis melo L.) with symptoms of mosaic, chlorotic mottling, and vein banding as well as blistering and leaf malformation were observed in one field in the locality of Kladari (municipality of Doboj, Bosnia and Herzegovina). Disease incidence was estimated at 60%. A total of 20 symptomatic plants were collected and tested with double-antibody sandwich (DAS)-ELISA using commercial polyclonal antisera (Bioreba AG, Reinach, Switzerland) against four the most commonly reported melon viruses: Cucumber mosaic virus (CMV), Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV), and Papaya ringspot virus (PRSV) (1,3). Commercial positive and negative controls were included in each assay. Only CMV was detected serologically in all screened melon samples. Sap from an ELISA-positive sample (162-12) was mechanically inoculated to test plants using 0.01 M phosphate buffer (pH 7.0). The virus caused necrotic local lesions on Chenopodium amaranticolor 5 days after inoculation, while mild to severe mosaic was observed on Nicotiana rustica, N. glutinosa, N. tabacum ‘Samsun,’ Cucurbita pepo ‘Ezra F1,’ and Cucumis melo ‘Ananas’ 10 to 14 days post-inoculation. All five inoculated plants of each experimental host were DAS-ELISA positive for CMV. The presence of CMV in all naturally and mechanically infected plants was further verified by conventional reverse transcription (RT)-PCR. Total RNAs were extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and used as template in RT-PCR. RT-PCR was carried out with the One-Step RT-PCR Kit (Qiagen) using primer pair CMVCPfwd and CMVCPrev (4), amplifying the entire coat protein (CP) gene and part of 3′- and 5′-UTRs of CMV RNA 3. Total RNAs obtained from the Serbian CMV isolate from Cucurbita pepo ‘Olinka’ (GenBank Accession No. HM065510) and healthy melon leaves were used as positive and negative controls, respectively. An amplicon of the correct predicted size (871 bp) was obtained from all naturally and mechanically infected plants as well as from positive control, but not from healthy tissues. The amplified product derived from isolate 162-12 was purified with QIAquick PCR Purification Kit (Qiagen) and sequenced directly using the same primer pair as in RT-PCR (KC559757). Multiple sequence alignment of the 162-12 isolate CP sequence with those available in GenBank, conducted with MEGA5 software, revealed that melon isolate from Bosnia and Herzegovina showed the highest nucleotide identity of 99.7% (100% amino acid identity) with eight CMV isolates originating from various hosts from Serbia (GQ340670), Spain (AJ829770 and 76, AM183119), the United States (U20668, D10538), Australia (U22821), and France (X16386). Despite the fact that CMV is well established in majority of Mediterranean countries and represents an important threat for many agriculture crops, including pepper in Bosnia and Herzegovina (2), to our knowledge, this is the first report of CMV infecting melon in Bosnia and Herzegovina. Melon popularity as well as production value has been rising rapidly and the presence of CMV may have a drastic economic impact on production of this crop in Bosnia and Herzegovina. References: (1) E. E. Grafton-Cardwell et al. Plant Dis. 80:1092, 1996. (2) M. Jacquemond. Adv. Virus Res. 84:439, 2012. (3) M. Luis-Arteaga et al. Plant Dis. 82:979, 1998. (4) K. Milojević et al. Plant Dis. 96:1706, 2012.

scholarly journals First Report of Cucumber mosaic virus Infecting Watermelon in Serbia

Plant Disease ◽  
2012 ◽  
Vol 96 (11) ◽  
pp. 1706-1706 ◽  
Author(s):  
K. Milojević ◽  
I. Stanković ◽  
A. Vučurović ◽  
D. Ristić ◽  
D. Nikolić ◽  
...  
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Citrullus Lanatus ◽  
Mild Mosaic ◽  
Rt Pcr ◽  
Total Rna ◽  
First Report ◽  
Cp Gene ◽  
Plant Pathol ◽  
Negative Controls

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

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

Plant Disease ◽  
2013 ◽  
Vol 97 (6) ◽  
pp. 851-851 ◽  
Author(s):  
G. Delibašić ◽  
B. Tanović ◽  
J. Hrustic ◽  
I. Stanković ◽  
A. Bulajić ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Black Locust ◽  
Natural Infection ◽  
Robinia Pseudoacacia ◽  
Alfalfa Mosaic Virus ◽  
Natural Host ◽  
Post Inoculation ◽  
Rt Pcr ◽  
First Report ◽  
Negative Controls

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.

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

Plant Disease ◽  
2013 ◽  
Vol 97 (3) ◽  
pp. 430-430 ◽  
Author(s):  
V. Trkulja ◽  
J. Mihić Salapura ◽  
D. Kovačić ◽  
I. Stanković ◽  
A. Bulajić ◽  
...  
Keyword(s):  
Bosnia And Herzegovina ◽  
Iris Yellow Spot Virus ◽  
Yellow Spot ◽  
N Gene ◽  
Post Inoculation ◽  
Rt Pcr ◽  
Spot Virus ◽  
Susceptible Host ◽  
First Report ◽  
Negative Controls

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.

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

Plant Disease ◽  
2013 ◽  
Vol 97 (7) ◽  
pp. 1004-1004 ◽  
Author(s):  
V. Trkulja ◽  
J. Mihić Salapura ◽  
B. Ćurković ◽  
I. Stanković ◽  
A. Bulajić ◽  
...  
Keyword(s):  
Bosnia And Herzegovina ◽  
Ornamental Plants ◽  
N Gene ◽  
Impatiens Necrotic Spot Virus ◽  
Post Inoculation ◽  
Necrotic Spot ◽  
Rt Pcr ◽  
Spot Virus ◽  
First Report ◽  
Negative Controls

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.

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

Plant Disease ◽  
2014 ◽  
Vol 98 (10) ◽  
pp. 1449-1449 ◽  
Author(s):  
K. Milojević ◽  
I. Stanković ◽  
A. Vučurović ◽  
D. Nikolić ◽  
D. Ristić ◽  
...  
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
The United States ◽  
Post Inoculation ◽  
Rt Pcr ◽  
First Report ◽  
Cp Gene ◽  
Elisa Testing ◽  
Negative Controls ◽  
Das Elisa

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. Milojević et al. Plant Dis. 96:1706, 2012. (2) M. Samuitienė and M. Navalinskienė. 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.

scholarly journals First Report of Papaya ringspot virus and Zucchini yellow mosaic virus in Luohanguo (Siraitia grosvenorii) in China

Plant Disease ◽  
2005 ◽  
Vol 89 (5) ◽  
pp. 530-530 ◽  
Author(s):  
Y.-M. Liao ◽  
X.-J. Gan ◽  
B. Chen ◽  
J.-H. Cai
Keyword(s):  
Mosaic Virus ◽  
Zucchini Yellow Mosaic Virus ◽  
Viral Disease ◽  
Ringspot Virus ◽  
Yellow Mosaic Virus ◽  
Watermelon Mosaic Virus ◽  
Papaya Ringspot Virus ◽  
Rt Pcr ◽  
First Report ◽  
Siraitia Grosvenorii

Luohanguo, Siraitia grosvenorii (Swingle) C. Jeffrey, is a perennial cucurbitaceous plant that is an economically important medicinal and sweetener crop in Guangxi province, China. Surveys conducted during the summer to fall seasons of 2003-2004 in northern Guangxi showed symptoms typical of a viral disease, including leaf mottling, mosaic, vein clearing, curling, and shoestring-like distortion in the field. Mechanical inoculation of sap from leaves of symptomatic plants collected from the surveyed areas caused similar symptoms on tissue culture-derived healthy Luohanguo plants. Two sequences of 0.7 and 1.6 kb with 88 and 97% identity to Papaya ringspot virus (PRSV) and Zucchini yellow mosaic virus (ZYMV) were amplified using reverse transcription-polymerase chain reaction (RT-PCR) with purified flexuous viral particles or total RNA extracted from the symptomatic Luohanguo leaves as templates with conserved degenerate potyvirus primers (1). To confirm the results, primers specific for PRSV (PP1/PP2, genome coordinates 4064-4083/5087-5069, GenBank Accession No X97251) and ZYMV (ZP1/ZP2, genome coordinates 5540-5557/7937-7920, GenBank Accession No L31350) were used to perform RT-PCR from the same RNA templates. The expected 1.0- and 2.3-kb fragments were amplified and they were 90 and 95% identical to PRSV and ZYMV in sequence, respectively. Watermelon mosaic virus was not detected. To our knowledge, this is the first report of the occurrence of PRSV and ZYMV in Luohanguo. Reference: (1) A. Gibbs et al. J. Virol. Methods 63:9, 1997.

scholarly journals First Report of Cucumber mosaic virus Associated with Capsicum chinense var. Scotch Bonnet in Florida

Plant Disease ◽  
2014 ◽  
Vol 98 (7) ◽  
pp. 1016-1016 ◽  
Author(s):  
B. Babu ◽  
H. Dankers ◽  
M. L. Paret
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Yellow Mosaic Virus ◽  
Hot Pepper ◽  
Crystalline Inclusion ◽  
Post Inoculation ◽  
Capsicum Chinense ◽  
Rt Pcr ◽  
First Report ◽  
Pcr Assays

Scotch bonnet (Capsicum chinense) is a tropical hot pepper variety that is grown in South America, the Caribbean Islands, and in Florida, and is an important cash crop. In Florida, scotch bonnet is grown on ~100 acres annually. Virus-like leaf symptoms including mosaic and yellow mottling were observed on scotch bonnet plants in a field at Quincy, FL, with a disease incidence of ~5%. Two symptomatic and one non-symptomatic plant sample were collected from this field for identification of the causal agent associated with the symptoms. Viral inclusion assays (2) of the epidermal tissues of the symptomatic scotch bonnet samples using Azure A stain indicated the presence of spherical aggregates of crystalline inclusion bodies. Testing of the symptomatic samples using lateral flow immunoassays (Immunostrips, Agdia, Elkhart, IN) specific to Cucumber mosaic virus (CMV), Potato virus Y (PVY), Pepper mild mottle virus (PMMoV), Tobacco mosaic virus (TMV), Zucchini yellow mosaic virus (ZYMV), and Papaya ringspot virus (PRSV), showed a positive reaction only to CMV. The sap from an infected leaf sample ground in 0.01 M Sorensons phosphate buffer (pH 7.0) was used to mechanically inoculate one healthy scotch bonnet plant (tested negative for CMV with Immunostrip) at the 2- to 3-leaf stage. The inoculated plant developed mild mosaic and mottling symptoms 12 to 14 days post inoculation. The presence of CMV in the mechanically inoculated plant was further verified using CMV Immunostrips. Total RNA was extracted (RNeasy Plant Mini Kit, Qiagen, Valencia, CA) from the previously collected two symptomatic and one non-symptomatic scotch bonnet samples. The samples were subjected to reverse-transcription (RT)-PCR assays using SuperScript III One-Step RT-PCR System (Invitrogen, Life Technologies, Grand Island, NY), and using multiplex RT-PCR primer sets (1). The primers were designed to differentiate the CMV subgroup I and II, targeting the partial coat protein gene and the 3′UTR. The RT-PCR assays using the multiplex primers produced an amplicon of 590 bp, with the CMV subgroup I primers. The RT-PCR product was only amplified from the symptomatic leaf samples. The obtained amplicons were gel eluted, and directly sequenced bi-directionally (GenBank Accession Nos. KF805389 and KF805390). BLAST analysis of these sequences showed 97 to 98% nucleotide identities with the CMV isolates in the NCBI database. The isolates collected in Florida exhibited highest identity (98%) with the CMV isolate from tomato (DQ302718). These results revealed the association of CMV subgroup I with symptomatic scotch bonnet leaf samples. Although CMV has been reported from scotch bonnet, this is the first report of its occurrence in Florida. References: (1) S. Chen et al. Acta Biochim Biophys Sin. 43:465, 2011. (2) R. G. Christie and J. R. Edwardson. Plant Dis. 70:273, 1986.

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