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 Cucumber mosaic virus Infecting Peperomia tuisana in Serbia

Plant Disease ◽  
2013 ◽  
Vol 97 (7) ◽  
pp. 1004-1004 ◽  
Author(s):  
K. Milojević ◽  
I. Stanković ◽  
A. Vučurović ◽  
D. Ristić ◽  
D. Milošević ◽  
...  
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Disease Incidence ◽  
Impatiens Necrotic Spot Virus ◽  
Rt Pcr ◽  
Test Species ◽  
First Report ◽  
Cp Gene ◽  
Negative Controls ◽  
Das Elisa

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.

scholarly journals First Report of Zucchini yellow mosaic virus in Watermelon in Serbia

Plant Disease ◽  
2012 ◽  
Vol 96 (1) ◽  
pp. 149-149 ◽  
Author(s):  
A. Vučurović ◽  
A. Bulajić ◽  
I. Stanković ◽  
D. Ristić ◽  
D. Nikolić ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mosaic Virus ◽  
Sandwich Elisa ◽  
Zucchini Yellow Mosaic Virus ◽  
Yellow Mosaic Virus ◽  
Rt Pcr ◽  
Total Rna ◽  
First Report ◽  
Cp Gene ◽  
Negative Controls

During a survey of cucurbit viruses in the Gornji Tavankut locality (North Backa District), Serbia in June 2011, field-grown (a surface of 1.8 ha) watermelon plants (Citrullus lanatus [Thunb.] Matsum and Nakai) with mild mosaic symptoms were observed. Large numbers of Aphis gossypii were colonizing the crop. A total of 26 samples, six from plants exhibiting mosaic and 20 from asymptomatic plants, were analyzed by double-antibody sandwich-ELISA using polyclonal antisera virus (Bioreba AG, Reinach, Switzerland) against three cucurbit-infecting viruses known to infect Cucurbita pepo in Serbia: Zucchini yellow mosaic virus (ZYMV), Cucumber mosaic virus, and Watermelon mosaic virus (3). Commercial positive and negative controls were included in ELISA analysis. Only six symptomatic samples tested positive for ZYMV, but no other tested viruses were found. The virus was mechanically transmitted from a representative ELISA-positive watermelon sample (550-11) to five plants of C. pepo ‘Ezra F1’ and severe mosaic was noticed 10 days after inoculation. For further confirmation of ZYMV infection, total RNA from a naturally infected watermelon plant and symptomatic C. pepo ‘Ezra F1’ 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 primer pair ZY-2 and ZY-3 (2). Total RNA obtained from a Serbian isolate of ZYMV from pumpkin (GenBank Accession No. HM072432) and healthy watermelon plants were used as positive and negative controls, respectively. The expected sizes of the RT-PCR products (1,186 bp) were amplified from naturally and mechanically infected symptomatic samples, but not from healthy tissues. The amplified product that derived from isolate 550-11 was purified (QIAquick PCR Purification Kit, Qiagen), sequenced in both directions, deposited in GenBank (Accession No. JN561294), and subjected to sequence analysis using MEGA4 software. Sequence comparisons revealed a high nucleotide identity of 99.9 to 99.8% and 100 to 99.6% amino acid identity for the CP gene with Serbian ZYMV isolates from C. pepo (Accession Nos. JF308188, HM072431, and HM072432). The nucleotide and deduced amino acid sequences of the entire CP gene (837 nt) of the Serbian ZYMV isolate from watermelon shared 99.9 to 93.7% and 100 to 96.8% identity, respectively, with innumerous isolates of ZYMV deposited in the GenBank (e.g., Accession Nos. AJ420012–17 and FJ705262). To our knowledge, this is the first report of ZYMV spreading its host range to watermelon in Serbia. ZYMV infection has been responsible for severe epidemics on cucurbits throughout the world (1). The presence of ZYMV on watermelon could therefore represent a serious threat for this valuable crop in Serbia, especially considering that it is prevalent in other cucurbit crops in the country and the vectors are widespread. References: (1) H. Lecoq et al. Virus Res. 141:190, 2009. (2) K. G. Thomson et al. J. Virol. Methods 55:83, 1995. (3) A. Vučurović et al. Pestic. Phytomed. (Belgrade) 24:85, 2009.

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 Lamium maculatum is a Natural Host for Cucumber mosaic virus

Plant Disease ◽  
2013 ◽  
Vol 97 (1) ◽  
pp. 150-150 ◽  
Author(s):  
R. Bešta-Gajević ◽  
A. Jerković-Mujkić ◽  
S. Pilić ◽  
I. Stanković ◽  
A. Vučurović ◽  
...  
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Impatiens Necrotic Spot Virus ◽  
Natural Occurrence ◽  
Rt Pcr ◽  
Natural Ecosystems ◽  
New Host ◽  
Total Rna ◽  
Negative Controls ◽  
Das Elisa

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.

scholarly journals First report of costus stripe mosaic virus infecting Tradescantia spathacea plants in Brazil

Plant Disease ◽  
2021 ◽  
Author(s):  
Gabriel Madoglio Favara ◽  
Felipe Franco de Oliverira ◽  
Camila Geovana Ferro ◽  
Heron Delgado Kraide ◽  
Eike Yudi Nishimura Carmo ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Protein Gene ◽  
Infected Plant ◽  
Nucleotide Sequences ◽  
Mild Mosaic ◽  
Rt Pcr ◽  
Chenopodium Amaranticolor ◽  
Capsid Protein Gene ◽  
Total Rna ◽  
First Report

Tradescantia spathacea (family Commelinaceae) is cultivated worldwide as an ornamental (Golczyk et al., 2013) and as medicinal plant (Tan et al., 2020). In 2019, 90 of ~180 plants of T. spathacea, grown in two beds of 4 m2 and exhibiting leaf mosaic were found in an experimental area at ESALQ/USP (Piracicaba municipality, São Paulo state, Brazil). Potyvirus-like flexuous filamentous particles were observed by transmission electron microscopy in foliar extracts of two symptomatic plants stained with 1% uranyl acetate. Total RNA was extracted using the Purelink viral RNA/DNA kit (Thermo Fisher Scientific) from leaves of two symptomatic plants and separately subjected to a reverse transcription polymerase chain reaction (RT-PCR). The potyviruses degenerate pairs of primers CIFor/CIRev (Ha et al. 2008), which amplifies a fragment corresponding to part of the cylindrical inclusion protein gene, and WCIEN/PV1 (Maciel et al. 2011), which amplifies a fragment containing part of the capsid protein gene and the 3′ untranslated region, were used. The expected amplicons (~700bp) were obtained from both total RNA extracts. Two amplicons from one sample were purified using the Wizard SV Gel and PCR Clean-Up System kit (Promega) and directly sequenced in both directions at Macrogen Inc (Seoul, South Korea). The obtained nucleotide sequences (GenBank MW430005 and MW503934) shared 95.32% and 97.79% nucleotide identity, respectively, with the corresponding sequences of the Brazilian isolate of the potyvirus costus stripe mosaic virus (CoSMV, MK286375) (Alexandre et al. 2020). Extract from an infected plant of T. spathacea was mechanically inoculated in 10 healthy plants of T. spathacea and two plants each of the following species: Capsicum annuum, Chenopodium amaranticolor, Commelina benghalensis, Datura stramonium, Gomphrena globosa, Nicandra physaloides, Nicotiana tabacum cvs. Turkish and Samsun, Solanum lycopersicum, T. palida, and T. zebrina. All T. spathacea plants exhibited mosaic and severe leaf malformation. C. benghalensis plants developed mild mosaic, whereas infected T. zebrina plants were asymptomatic. The plants of other species were not infected. RT-PCR with specific CoSMV primers CoSMVHC-F and CoSMVHC-R (Alexandre et al. 2020) confirmed the infection. Nucleotide sequences of amplicons obtained from experimentally inoculated T. spathacea and T. zebrina (MW430007 and MW430008) shared 94.56% and 94.94% identity with the corresponding sequence of a Brazilian CoSMV isolate (MK286375). None of eight virus-free plants of T. spathacea inoculated with CoSMV using Aphis craccivora exhibited symptoms, nor was CoSMV detected by RT-PCR. Lack of CoSMV transmission by A. solanella, Myzus persicae, and Uroleucon sonchi was previously reported (Alexandre et al. 2020). T. spathacea plants are commonly propagated vegetatively, and by seeds. Virus-free seeds, if available, can provide an efficient and easy way to obtain healthy plants. Only three viruses were reported in plants of the genus Tradescantia: Commelina mosaic virus, tradescantia mild mosaic virus, and a not fully characterized potyvirus (Baker and Zettler, 1988; Ciuffo et al., 2006; Kitajima 2020). CoSMV was recently reported infecting Costus spiralis and C. comosus (Alexandre et al. 2020). As far as we know, this is the first report of CoSMV infecting T. spathacea plants.

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

Plant Disease ◽  
2013 ◽  
Vol 97 (6) ◽  
pp. 850-850 ◽  
Author(s):  
D. Nikolić ◽  
I. Stanković ◽  
A. Vučurović ◽  
D. Ristić ◽  
K. Milojević ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Tomato Spotted Wilt Virus ◽  
N Gene ◽  
Broad Host Range ◽  
Rt Pcr ◽  
First Report ◽  
Tomato Spotted Wilt ◽  
Plant Pathol ◽  
Negative Controls ◽  
Wilt Virus

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. Vučurović et al. Eur. J. Plant Pathol. 133:935, 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 in Taro Plants in China

Plant Disease ◽  
2014 ◽  
Vol 98 (4) ◽  
pp. 574-574 ◽  
Author(s):  
Y. F. Wang ◽  
G. P. Wang ◽  
L. P. Wang ◽  
N. Hong
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Central China ◽  
Post Inoculation ◽  
Rt Pcr ◽  
Cmv Infection ◽  
First Report ◽  
Cp Gene ◽  
Two Samples ◽  
Pcr Products

Taro (Colocasia esculenta L. Schott) is an important crop worldwide. In China, the growing area and productivity of taro increased greatly in recent years. During the 2010 to 2013 growing seasons (from May to July), the incidence of Cucumber mosaic virus (CMV) in taro was determined. Leaf samples from 91 taro plants, including 26 plants of cv. Hongyayu grown in Jiangxi Province in eastern China, 33 plants of cv. Eyu no.1 grown in Hubei Province in central China, and 32 plants of cv. Baiyu grown in Guangxi Province in southwest China were collected randomly and tested for the presence of CMV by reverse transcription (RT)-PCR. Some sampled plants of cv. Hongyayu and Eyu no.1 showed leaf chlorosis or chlorotic spots, and most of the plants of these three cultivars showed feather-like mosaic symptom on their leaves, which was confirmed to be associated with the infection of Dasheen mosaic virus (DsMV) in our previous studies (3). Total RNA was extracted from leaves using CTAB protocol reported by Li et al. (1). Primer set forward 5′-ATGGACAAATCTGAATCAACC-3′/reverse 5′-TAAGCTGGATGGACAACCCGT-3′ (4) was used for the amplification of a 777-bp fragment, which contains the complete capsid protein (CP) gene of 657 bp. PCR products of the expected size were identified from 11 taro samples, including two samples of Hongyayu, three Eyu no.1, and six Baiyu plants. The result did not show any specific association between the symptoms observed and CMV infection. The obtained PCR products were cloned individually into the vector pMD18-T (TaKaRa, Dalian, China). Three independent clones derived from each product were sequenced by Genscript Corp., Nanjing, China. Pairwise comparison of CP gene sequences (Accession No. of one representation CP sequence: KF564789) showed 99.7 to 99.8% nucleotide (nt) and 99.1 to 99.5% deduced amino acid (aa) sequence identity among themselves, and 92.0 to 94.3% and 76.5 to 77.7% nt identities with corresponding sequences of CMV isolates in subgroup I and subgroup II (2), respectively. The maximum likelihood phylogenetic trees of nt and aa sequences generated by Clustal X v1.8 revealed that all these CMV isolates from taro in China fell into subgroup I. To further confirm the CMV infection, leaf saps of CMV infected taro plants of cv. Eyu no.1 were mechanically inoculated onto Pinellia ternate and Cucumis sativus. Plants of P. ternate showed local chlorotic lesions on the inoculated leaves and downward curl of newly grown leaves, and C. sativus showed local chlorotic lesions on the inoculated leaves and crinkle of newly grown leaves at 10 to 15 days post inoculation. The RT-PCR detection confirmed the CMV infection in those inoculated plants, and that the plants of P. ternate were also positive to DsMV, further complementing the results obtained above. To our knowledge, this is the first report of CMV occurrence in taro plants grown in China. Our results indicated that taro plants were widely infected by CMV isolates in subgroup I. This study provides important information for further evaluating the viral sanitary status of taro germplasm and improving the certification program of taro propagation materials in China. References: (1) R. Li et al. J. Virol. Methods 154:48, 2008. (2) P. Palukaitis et al. Adv. Virus. Res. 62:241, 2003. (3) S. M. Shi et al. Acta Hortic. Sin. 39:509, 2012. (4) P. D. Xu et al. Chinese J. Virol. 15:164, 1999.

scholarly journals First Report of Clover yellow vein virus on White Clover (Trifolium repens) in South Korea

Plant Disease ◽  
2014 ◽  
Vol 98 (10) ◽  
pp. 1450-1450 ◽  
Author(s):  
C.-Y. Park ◽  
M.-A. Lee ◽  
M. Nam ◽  
E.-H. Park ◽  
Y. S. Bae ◽  
...  
Keyword(s):  
South Korea ◽  
Mosaic Virus ◽  
White Clover ◽  
Trifolium Repens ◽  
Rt Pcr ◽  
Total Rna ◽  
Chlorotic Spot ◽  
Cp Gene ◽  
Clover Yellow Vein Virus ◽  
Plant Pathol

White clover (Trifolium repens L.) is a herbaceous, perennial plant that has become one of the most widely distributed legumes in the world. It is extensively used in grass-legume pastures, but also has the potential to invade agricultural lands and natural ecosystems. White clover is a well-known natural host for Alfalfa mosaic virus (AMV), Clover yellow vein virus (ClYVV), Soybean dwarf virus (SbDV), Beet western virus (BWYV), Tomato spotted wilt virus (TSWV), Zucchini yellow mosaic virus (ZYMV), etc (1). In July 2013, during a survey to determine the presence of different viruses infecting weed plants in South Korea, three white clover leaf samples showing yellow mosaic symptoms were collected from Taean County, South Chungcheong Do Province, South Korea. In order to identify the infecting virus, total RNA from three leaf samples was extracted using the Tri-reagent (MRC Reagent, Inc., OH) as described by the manufacturer, and was applied to the large-scale oligonucleotide (LSON) chip (3), wherein probes specific to a ClYVV isolate produced a positive reaction. All three samples tested were positive for ClYVV. To confirm this result, ClYVV-specific primers were designed using the sequences of four ClYVV isolates from NCBI (GenBank Accession Nos. AF185959, AF203536, DQ333346, and NC003536). Total RNA was extracted from symptomatic white clover samples using Easy-Spin Total RNA Extraction Kit (iNtRon, Daejeon, Korea) and used as template for RT-PCR. The positive control RNA was used from ClYVV GM isolate (KF975894) and negative control RNA used symptomless white clover plants. The ClYVV coat protein (CP) gene was amplified by RT-PCR using the specific primer pairs ClYVV-CP-F / ClYVV-CP-R (5′-CAAGAGCAGCACGATGAG-3′ and 5′-CTCGCTCTATAAAGATCAGAT-3′). DNA fragments of the expected size (1,042 bp) were obtained from the white clover Korea isolate (AB930132), and the PCR product was cloned into a T&A cloning vector (RBC Bioscience, Taipei, Taiwan) and sequenced directly in both directions. BLAST analyses of the nucleotide sequence CP gene fragments revealed the highest identity with 98% with other ClYVV isolates (AF203536). To determine the experimental host range of the ClYVV Korea isolate, we inoculated five species (Chenopodium amaranticolor, C. quinoa, Nicotiana clevelandii, N. benthamiana, and Trifolium repens) in three families using this isolate. All test plants were mechanically inoculated with 0.1 M phosphate buffered saline (Takara, Tokyo, Japan). Each test plant was inoculated nine times and grown in a greenhouse maintained at 27 to 33°C. Necrotic local lesions were produced on inoculated leaves of C. amaranticolor, C. quinoa, and N. clevelandii 4 to 6 days post-inoculation. After 10 to 14 days, C. amaranticolor and C. quinoa showed systemic chlorotic spot symptoms, and N. clevelandii, N. benthamiana, and T. repens showed chlorotic spot, mild mosaic, and mosaic in the upper leaves, respectively. Up to now, in South Korea, ClYVV has been detected in gladiolus (Gladiolus gandavensis) (3) and soybean (Glycine max) (4). ClYVV can be easily transmitted by insect, aphid, or mechanical inoculation and has a host range including tobacco, soybean, etc. The presence of ClYVV could become an important threat to crop production in South Korea. To our knowledge, this is the first report of a ClYVV infection of the white clover plant in South Korea. References: (1) B. L. Denny and P. L. Guy. Australas. Plant Pathol. 38:270, 2009. (2) M. Nam et al. Plant Pathol. J. 30:51, 2014. (3) I. S. Park et al. Korean J. Plant Pathol. 14:74, 1998. (4) J. C. Shin et al. Plant Dis. 98:1283, 2014.

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.

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