First Report of Zucchini yellow mosaic virus in Watermelon in Serbia

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.

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First Report of Cucumber mosaic virus Infecting Watermelon in Serbia

Plant Disease ◽

10.1094/pdis-07-12-0631-pdn ◽

2012 ◽

Vol 96 (11) ◽

pp. 1706-1706 ◽

Cited By ~ 3

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.

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

Plant Disease ◽

10.1094/pdis-11-13-1156-pdn ◽

2014 ◽

Vol 98 (6) ◽

pp. 858-858 ◽

Cited By ~ 1

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.

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

Plant Disease ◽

10.1094/pdis-08-13-0834-pdn ◽

2014 ◽

Vol 98 (4) ◽

pp. 573-573 ◽

Cited By ~ 1

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.

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Heteroduplex Mobility and Sequence Analyses for Assessment of Variability of Zucchini yellow mosaic virus

Phytopathology ◽

10.1094/phyto.2000.90.3.228 ◽

2000 ◽

Vol 90 (3) ◽

pp. 228-235 ◽

Cited By ~ 15

Author(s):

Shih-Shun Lin ◽

Roger F. Hou ◽

Shyi-Dong Yeh

Keyword(s):

Amino Acid ◽

Mosaic Virus ◽

Sequence Data ◽

Zucchini Yellow Mosaic Virus ◽

The Other ◽

Yellow Mosaic Virus ◽

Cdna Clones ◽

Rt Pcr ◽

Genotype I ◽

Cp Gene

A heteroduplex mobility assay (HMA) was used to analyze the variability among five isolates of Zucchini yellow mosaic virus (ZYMV; TW-TC1, TW-CY2, TW-TN3, TW-TNML1, and TW-NT1) collected from cucurbit fields in different areas of Taiwan. A cDNA fragment of 760 bp covering the variable region of the N terminal half of the coat protein (CP) gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and subsequently subjected to HMA analysis for sequence variation. When TW-NT1 combined with any of the other Taiwan isolates, the heteroduplexes obtained migrated much more slowly than did the heteroduplexes obtained in combinations among the other four Taiwan isolates, indicating that TW-TC1, TW-CY2, TW-TN3, and TW-TNML1 share a high degree of sequence homology, while the TW-NT1 isolate is more distinct. The complete nucleotide sequences of the CP genes and the 3′ noncoding regions of the five isolates were determined from RT-PCR-derived cDNA clones. A phylogenetic tree derived from the actual sequences of the 760-bp fragments of the five Taiwan and another six ZYMV isolates from different geographic areas revealed four genotypes. TW-TNML1, TW-TC1, TWC-Y2, and TW-TN3 were in genotype I, while TW-NT1 and U.S. isolates were in genotype II. The Singapore and Reunion Island isolates were separated into genotypes III and IV, respectively. Comparison of the CP genes of the five Taiwan isolates indicated that they share 92.8 to 98.7% nucleotide identities and 96.4 to 99.3% amino acid identities. The amino acid positions 73, 102, 109, and 149 of the CP gene, where lysine, serine, arginine, and aspartic acid reside, respectively, were uniquely conserved for genotype I Taiwan isolates. Thus, results of HMA agreed well with those of phylogenetic analysis based on the sequence data of the five Taiwan ZYMV isolates. These five ZYMV isolates of known sequence can be used as reference strains for HMA to analyze the variability of ZYMV in Taiwan.

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

Plant Disease ◽

10.1094/pd-89-0530a ◽

2005 ◽

Vol 89 (5) ◽

pp. 530-530 ◽

Cited By ~ 4

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.

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

Plant Disease ◽

10.1094/pdis-02-13-0135-pdn ◽

2013 ◽

Vol 97 (8) ◽

pp. 1124-1124 ◽

Cited By ~ 1

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.

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First Report of Cucumber mosaic virus Infecting Peperomia tuisana in Serbia

Plant Disease ◽

10.1094/pdis-01-13-0089-pdn ◽

2013 ◽

Vol 97 (7) ◽

pp. 1004-1004 ◽

Cited By ~ 2

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.

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First Report of Pepper veinal mottle virus in Tomato and Pepper in Taiwan

Plant Disease ◽

10.1094/pdis-93-1-0107a ◽

2009 ◽

Vol 93 (1) ◽

pp. 107-107 ◽

Cited By ~ 9

Author(s):

Y. H. Cheng ◽

R. Y. Wang ◽

C. C. Chen ◽

C. A. Chang ◽

F.-J. Jan

Keyword(s):

Amino Acid ◽

Mosaic Virus ◽

Polyclonal Antibodies ◽

Amino Acid Identity ◽

Mottle Virus ◽

Rt Pcr ◽

Acid Identity ◽

First Report ◽

Cp Gene ◽

Pepper Veinal Mottle Virus

In May of 2006, samples from tomato plants (Solanum lycopersicum cv. Known-you 301) exhibiting necrotic symptoms on stems, petioles, and leaves were collected from Chiayi County, Taiwan. Double-antibody sandwich-ELISAs were performed using Cucumber mosaic virus, Tomato mosaic virus, Potato virus Y, Watermelon silver mottle virus, and Chilli veinal mottle virus (ChiVMV) polyclonal antibodies. Three of eight samples reacted with antibodies against ChiVMV but not with the others. Using the potyvirus degenerate primers (Hrp 5/Pot 1) (2), an expected 1.5-kb DNA fragment including the 3′-end of the NIb gene, the complete coat protein (CP) gene, and the 3′-nontranslatable region of the virus was amplified from total RNA isolated from these three samples by reverse transcription (RT)-PCR. A homology search in GenBank indicated that the new tomato-infecting virus in Taiwan belongs to Pepper veinal mottle virus (PVMV) since they shared >90% amino acid identity in the CP gene. A virus culture (Tom1) isolated from one of the diseased tomatoes was then established in Chenopodium quinoa and Nicotiana benthamiana and the CP gene was amplified and sequenced (GenBank Accession No. EU719647). Comparisons of the 807-nt CP gene with those of five PVMV isolates available in GenBank showed 81.5 to 93.1% nucleotide and 90.0 to 97.8% amino acid identity. Tom1 induced irregular necrotic lesions on stems, petioles, and leaves of tomato while inducing only mild mottle symptoms on pepper. Serological cross reaction between ChiVMV and PVMV has been observed previously (1,3) and also found in this study. To differentiate these two potyviruses by RT-PCR, primer pair CPVMVup/dw (5′-TATTC(T/C)TCAGTGTGG(A/T/C)T(T/C)CCACCAT and 5′-(T/C)C(A/T)C(A/T)(A/T/G)(A/T)AA(A/G)CCATAA(A/C)(A/C)ATA(A/G)T(T/C)T) was designed on the basis of the comparison of the CP gene and the 3′-nontranslatable region of the PVMV and ChiVMV. DNA fragments of 171 and 259 bp are expected to be amplified from ChiVMV and PVMV, respectively, by RT-PCR with primers CPVMVup/dw. In a field survey done in 2006, samples from diseased peppers (Capsicum annuum) that reacted with the polyclonal antibodies against ChiVMV were further identified by RT-PCR with primers CPVMVup/dw, indicating that both ChiVMV and PVMV infected pepper crops (Capsicum spp.) in Taiwan. A pepper isolate (Pep1) of PVMV was obtained from Nantou County through three times of single lesion passages on C. quinoa and then propagated on N. benthamiana. The CP gene of Pep1 was amplified and sequenced (GenBank Accession No. EU719646) and found to share 99.1% nucleotide and 100% amino acid identity with that of Tom1. Pep1 caused mild mottle symptoms on leaves of both tomato and pepper. To our knowledge, this is the first report of the presence of PVMV in Taiwan as well as in East Asia. References: (1) B. Moury et al. Phytopathology 95:227, 2005. (2) S. S. Pappu et al. Plant Dis. 82:1121, 1998. (3) W. S. Tsai et al. Plant Pathol. 58:408, 2008.

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First Report of Narcissus mosaic virus Infecting Crocus spp. Cultivars in the Netherlands

Plant Disease ◽

10.1094/pd-89-0342c ◽

2005 ◽

Vol 89 (3) ◽

pp. 342-342 ◽

Cited By ~ 9

Author(s):

R. Miglino ◽

A. Jodlowska ◽

A. R. van Schadewijk

Keyword(s):

The Netherlands ◽

Mosaic Virus ◽

Yellow Mosaic Virus ◽

Specific Antiserum ◽

Rt Pcr ◽

Pcr Assay ◽

Total Rna ◽

First Report ◽

Plant Research ◽

Narcissus Mosaic Virus

A survey to identify virus diseases affecting Crocus spp. in the Netherlands was conducted during April 2004. Crocus spp. (cvs. Flavus, Pick-wick, Remembrance, and Grand Maitre) with symptoms suggestive of virus infection (stunting, yellowing, necrosis, and flower color breaking) were collected from several fields in the Breezand and Lisse districts in northern and southern Netherlands, respectively. All samples were tested for the presence of six known crocus-infecting viruses (1,2) using enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) assays. The ELISA assay was performed with the following polyclonal and monoclonal antibodies: Iris severe mosaic virus (ISMV); Tobacco rattle virus (TRV) isolates F, Y, and J obtained from the Applied Plant Research Institute, Lisse, Netherlands; Arabis mosaic virus; Cucumber mosaic virus from the Plant Research International Institute, Wageningen, Netherlands; Iris yellow spot virus (IYSV) from the Virology Department at Wageningen University, Netherlands; and the potyvirus group-specific monoclonal antiserum from the DSMZ, Braunschweig, Germany. All samples that tested positive with a potyvirus antiserum were further tested for the presence of Bean yellow mosaic virus (BYMV) using a BYMV-specific antiserum. Serological results obtained indicated that BYMV, detected with the potyvirus antiserum and BYMV-specific antiserum, and ISMV were the most commonly encountered viruses. Tobacco necrosis virus (TNV) and TRV were only found occasionally, whereas IYSV, was not detected in any of the samples tested. To study the presence of viruses not yet reported, total RNA was extracted and tested with a RT-PCR assay with carlavirus, potexvirus, necrovirus (R. Miglino, unpublished), and potyvirus (3) genus-specific oligonucleotides. In accordance with the ELISA results, PCR amplicons were obtained with the potyvirus, TNV, and TRV primer sets. Furthermore, a 280-bp amplicon corresponding to the expected size was amplified in a RT-PCR assay performed on total RNA with a potexvirus genus-specific primer set. The reverse primer (5′-AGC ATG GCG CCA TCT TGT GAC TG-3′) was located upstream in the conserved viral replicaseencoding region at position 4254-4231 of Narcissus mosaic virus (NMV) RNA genome (Genbank Accession No. D13747) and the forward primer (5′-CTG AAG TCA CAA TGG GTG AAG AA-3′) was located downstream at position 3969–3992. Sequence homology using BLAST analysis of the cloned and sequenced PCR product showed 98% identity with NMV. Although the virus has a very narrow host range, the results of this study may have a significant impact on the crocus industry in the Netherlands. To our knowledge, this is the first report of NMV infecting crocus. References: (1) M. G. Bellardi and A. Pisi. Inf. Fitopatol. 37:33, 1987. (2) A. F. L. M. Derks. Crocus spp. Pages 260–264 in: Virus and Virus-like Diseases of Bulbs and Flower Crops. G. Loebenstein et al., eds. Wiley publishers, West Sussex, UK, 1995. (3) S. A. Langeveld et al. J. Gen. Virol. 72:1531, 1991.

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First Report of Papaya ringspot virus–Type W and Zucchini yellow mosaic virus Infecting Trichosanthes cucumerina in Brazil

Plant Disease ◽

10.1094/pdis-94-6-0789b ◽

2010 ◽

Vol 94 (6) ◽

pp. 789-789 ◽

Cited By ~ 4

Author(s):

A. S. Jadão ◽

J. E. Buriola ◽

J. A. M. Rezende

Keyword(s):

Amino Acid ◽

Mosaic Virus ◽

Virus Type ◽

Zucchini Yellow Mosaic Virus ◽

Amino Acid Sequences ◽

Ringspot Virus ◽

Yellow Mosaic Virus ◽

Papaya Ringspot Virus ◽

Mechanical Inoculation ◽

Cp Gene

Trichosanthes cucumerina L., known as snake gourd, is a cucurbitaceous plant that is probably native to and originally domesticated in India. It is cultivated in humid subtropical and tropical countries of Australia, Latin America, and Africa (2). Plants of this species exhibiting symptoms of mosaic and leaf malformation were found during November 2008 near an experimental field of the Departamento de Fitopatologia e Nematologia, Universidade de São Paulo, Piracicaba, State of São Paulo, Brazil. Electron microscopy examination of negatively stained extract of infected tissue showed the presence of filamentous potyvirus-like particles. Sap from these infected plants reacted in plate-trapped antigen (PTA)-ELISA with the antiserum against Papaya ringspot virus–type W (PRSV-W) or Zucchini yellow mosaic virus (ZYMV), but not with the antiserum against Cucumber mosaic virus (CMV) or Zucchini lethal chlorosis virus (ZLCV). PRSV-W and ZYMV were simultaneously transmitted by mechanical inoculation to four plants of Cucurbita pepo cv. Caserta and one plant of T. cucumerina, causing mosaic. In addition, PRSV-W and ZYMV isolates from our virus collection separately infected one plant of T. cucumerina after mechanical inoculation. Infections were confirmed by PTA-ELISA. Total RNA extracted from infected and healthy T. cucumerina was analyzed by reverse transcription (RT)-PCR using a primer pair specific to the coat protein (CP) gene of PRSV-W (4) or ZYMV (3). Fragments of 864 bp and 1,045 bp were amplified with each pair of primers, respectively. Nucleotide sequences directly obtained from purified PCR products were used for further identification of these potyviruses. The nucleotide and deduced amino acid sequences of part of the CP gene (792 nt) of PRSV-W (GenBank Accession No. GU586789) shared 99 and 98% identity, respectively, with that of the Brazilian isolate PRSV-W-C (GenBank Accession No. 4152). The nucleotide and deduced amino acid sequences of the entire CP gene (837 nt) of ZYMV (GenBank Accession No. 6790) shared 91 to 98% and 94 to 100% identity, respectively, with innumerous isolates of ZYMV deposited in the GenBank (e.g., Accession Nos. AB004640, D13914, AB004641, and AJ420019). Natural infection of T. cucumerina by PRSV-W was reported in Nepal (1). To our knowledge, this is the first report of T. cucumerina infected by PRSV-W and ZYMV in Brazil. References: (1) G. Dahal et al. Ann. Appl. Biol. 130:491, 1997. (2) R. W. Robinson and D. S. Decker-Walters. Cucurbits. CAB International, Wallingford, UK. 1997. (3) K. G. Thomson et al. J. Virol. Methods 55:83, 1995. (4) M. G. S. D. Vechia. Fitopatol. Bras. 28:678, 2003.

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