First Report of Schlumbergera virus X in Dragon Fruit (Hylocereus spp.) in Spain

Dragon fruit (Hylocereus undatus) is a high-value fruit crop, introduced about a decade ago in the mainland of Spain. In 2021, chlorotic spots were observed on young cladodes in a commercial dragon fruit orchard in the province of Seville (southern Spain). Sap extracts from 4 symptomatic cladodes were used to mechanically inoculate indicator plants: no symptoms were produced in Datura stramonium plants, but Chenopodium amaranticolor reacted with chlorotic local lesions and prickly pear plants (Opuntia ficus-indica) showed irregular yellow ringspot symptoms on young cladodes at 30 days post inoculation. Total RNA was extracted from all 4 symptomatic cladodes as previously described (Pallas et al. 1987). Reverse transcription (RT)-PCR, which was carried out with M-MLV-RT and Go Taq Pol (Promega Biotech Ibérica, SL, Madrid, Spain) and tobamovirus primers (Dovas et al. 2004), failed to produce any amplicons. Electrophoretic analysis of dsRNA, extracted from symptomatic cladodes, yielded a banding pattern similar to the one reported for potexviruses (Valverde et al. 1986). Primers specific for Cactus virus X (Kim et al., 2016) failed to produce amplicons, whereas potexvirus group primers (Potex F5/Potex R2) (van der Vlugt and Berendsen 2002), amplified an expected 584-bp amplicon from RNA extracts of all 4 field-collected samples. The RT-PCR products from the four samples were Sanger-sequenced. All showed identical sequence results (GenBank Accession MZ614940) with a predicted amino acid identity of 99% with the corresponding RNA-dependent RNA polymerase amino acid sequence of Schlumbergera virus X (SchVX) (GenBank Accession No. ACD99908). SchVX-specific primers (431s, 5‘-TTTGAGGAGTTCGTCAGCAAGA-3‘ and 431As, 5‘-TCAAGAGCCCATTGAGAGAGTG-3‘) that were designed based on the new sequence, amplified the expected amplicon of around 430 nucleotides from the total RNA extracts of the four samples. The amplicons were Sanger-sequenced and the expected nucleotide sequence was obtained. This pair of primers were used in RT-PCR tests on subsequent surveys in 2 commercial dragon fruit greenhouses from the province of Seville, and in 1 experimental greenhouse in the province of Almeria. All samples from 25 symptomatic plants of H. undatus, H. hybridum, H. costaricensis, and H. purpusii in Seville and from 1 symptomatic H. undatus plant from Almeria tested positive for SchVX, while 15 asymptomatic plants tested negative. The results obtained in this investigation support that SchVX is present in the cladodes of dragon fruit plants expressing the symptoms. SchVX has been reported previously from H. undatus from Brasil (Duarte et al. 2008) and from prickly pear in Mexico (De La Torre-Almaráz et al. 2016), and to our knowledge, this is the first report of the virus in Spain. These findings suggest that SchVX has been introduced in dragon fruit farms from Spain and propagation of this emerging crop through planting of cuttings should include testing for this virus in order to prevent further spread.

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First Report of Bidens mottle virus Infecting Calendula in Taiwan

Plant Disease ◽

10.1094/pdis-10-10-0753 ◽

2011 ◽

Vol 95 (3) ◽

pp. 362-362 ◽

Cited By ~ 3

Author(s):

C.-H. Huang ◽

F.-J. Jan

Keyword(s):

Amino Acid ◽

Inclusion Bodies ◽

Amino Acid Identity ◽

Mottle Virus ◽

Rt Pcr ◽

Degenerate Primers ◽

Acid Identity ◽

First Report ◽

Cp Gene ◽

Dna Fragment

In March of 2010, calendula (Calendula officinalis L.), a perennial herb known as the pot marigold, showing chlorotic spots on leaves, chlorosis, and stunting were collected from Puli Township, Nantou County, Taiwan. The disorder occurred in more than 50% of the calendula plants in the field. A virus culture isolated from one of the symptomatic calendulas was established in Chenopodium quinoa through triple single-lesion isolation and designated as TwCa1. With transmission electron microscopy (TEM), negatively stained flexuous filamentous virions approximately 12 × 720 nm were observed in the crude sap of TwCa1-infected C. quinoa leaves and pinwheel inclusion bodies were found in the infected cells. On the basis of the sizes of the viral particles and inclusion bodies, isolate TwCa1 was a suspected potyvirus. By reverse transcription (RT)-PCR and potyvirus degenerate primers (Hrp5/Pot1) (1,2), a 0.65-kb DNA fragment, which included the 3′-end of the NIb gene and the 5′-end of coat protein (CP) gene of the virus, was amplified from total RNA isolated from TwCa1-infected plants. The amplified DNA fragment was cloned and sequenced. A homology search indicated that the new calendula-infecting virus in Taiwan might belong to Bidens mottle virus (BiMoV) because its partial genomic sequence shared 94.9 to 97.3% nucleotide and 96.6 to 98.1% amino acid identity with 11 BiMoV isolates available in NCBI GenBank. Primer pairs Hrp5/oligo d(T) were used to amplify the 3′-end genome of BioMV TwCa1 including the 3′-end of the NIb gene, the full-length CP gene, and the 3′-nontranslatable region of the virus. The 807-nt CP gene of TwCa1 (Accession No. HQ117871) shared 97.3 to 98.6% nucleotide and 98.5 to 98.9% amino acid identity with those of 11 BiMoV isolates available in GenBank. Results from TEM observations and CP gene sequence analysis indicated that TwCa1 is an isolate of BiMoV. BiMoV was later detected by RT-PCR in eight symptomatic calendulas collected from the same field. To our knowledge, this is the first report of BiMoV infecting calendula in Taiwan. This newly identified calendula-infecting BiMoV could have a direct impact on the economically important vegetable and floral industry in Taiwan. References: (1) C. C. Chen et al. Bot. Stud. 947:369, 2006. (2) D. Colinet and J. Kummert. J. Virol. Methods 45:149, 1993.

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

Plant Disease ◽

10.1094/pdis-02-21-0342-pdn ◽

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.

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First Report of Zucchini yellow mosaic virus in Watermelon in Serbia

Plant Disease ◽

10.1094/pdis-08-11-0664 ◽

2012 ◽

Vol 96 (1) ◽

pp. 149-149 ◽

Cited By ~ 1

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.

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First Report of Passiflora chlorosis virus in Bituminaria bituminosa in Europe

Plant Disease ◽

10.1094/pdis-93-2-0196a ◽

2009 ◽

Vol 93 (2) ◽

pp. 196-196 ◽

Cited By ~ 1

Author(s):

L. Cardin ◽

B. Moury

Keyword(s):

Amino Acid ◽

Green Peach Aphid ◽

Amino Acid Sequences ◽

Cdna Clones ◽

Coding Region ◽

Rt Pcr ◽

Leaf Extracts ◽

Chenopodium Amaranticolor ◽

First Report ◽

Bituminaria Bituminosa

Bituminaria bituminosa (L.) Stirton (pitch trefoil) is a perennial legume endemic to the Mediterranean Basin used as forage in arid areas and for stabilization of degraded soils. Mosaic and chlorotic ringspot symptoms have been observed in leaves of B. bituminosa in the Provence-Alpes-Côte d'Azur and Rhône-Alpes regions (France), Liguria (Italy), and Spain since 1975. In crude leaf extracts from more than 50 samples of diverse geographical origins, flexuous particles 680 to 720 nm long and 12 nm wide and pinwheel-like inclusions have been observed with the electron microscope, suggesting infection with a member of the family Potyviridae. The presence of a virus was confirmed by the use of potyvirus-polyvalent ELISA reagents (Potyvirus group test; Agdia, Elkhart, IN) and by the amplification of a DNA fragment of the expected size (≈1,650 bp) with extracts of isolates from different locations using reverse transcription (RT)-PCR with primers specific to members of the Potyviridae (3) corresponding to the 3′ end of the virus genome. The amplified fragment of an isolate from Coaraze (Alpes Maritimes Department, France) was cloned and two cDNA clones corresponding to this amplicon were sequenced (GenBank Accession Nos. EU334546 and EU334547). These two sequences facilitated development of new primers (5′-AAARGCRCCCTATATAGCAG-3′ and 5′-TATAAAGGTAACGCTAGGTGG-3′) to specifically amplify and sequence the coat protein (CP)-coding region of isolates of the virus from five additional French locations. The amino acid sequences of the CP amplicon were more than 96% identical among the French isolates. Comparison with other virus sequences with the BLASTn program revealed that these isolates belonged to the same species as the potyvirus Passiflora chlorosis virus (2), with 89 to 90% and 95 to 97% identity at the nucleotide and amino acid levels, respectively, for the CP-coding region (1). The host range of the virus was evaluated by manual inoculation with the Coaraze isolate and was found to be very narrow. No symptoms and no infections were obtained in Capsella bursa-pastoris, Capsicum annuum, Claytonia perfoliata, Cucumis melo, Cucumis sativus, Cucurbita pepo, Datura stramonium, Gomphrena globosa, Medicago sativa, Nicotiana benthamiana, N. glutinosa, N. tabacum, Ocimum basilicum, Petunia hybrida, Phaseolus mungo, Physalis peruviana, Pisum sativum, Psoralea glandulosa, Ranunculus sardous, Salvia splendens, Solanum lycopersicum, Trifolium repens, Vicia faba, Vigna unguiculata, or Zinnia elegans. Necrotic local lesions were observed in Chenopodium amaranticolor, C. quinoa, and in all eight cultivars of Phaseolus vulgaris tested. The virus was transmitted either manually or by the green peach aphid (Myzus persicae) to healthy B. bituminosa seedlings. Symptoms appeared in 10 to 15 weeks, and the virus was detected in the symptomatic plants by RT-PCR. To our knowledge, this is the first report of a virus infecting B. bituminosa. References: (1) M. J. Adams et al. Arch. Virol. 150:459, 2005. (2) C. A. Baker and L. Jones. Plant Dis. 91:227, 2007. (3) A. Gibbs and A. M. Mackenzie. J. Virol. Methods 63:9, 1997.

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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 Saguaro Cactus Virus Infecting Gymnocalycium mihanovichii in South Korea

Plant Disease ◽

10.1094/pdis-04-21-0770-pdn ◽

2021 ◽

Author(s):

Mi Sang Lim ◽

Byoung-Eun Min ◽

Sun Hee Choi

Keyword(s):

Amino Acid ◽

South Korea ◽

Rna Extraction ◽

Amino Acid Sequences ◽

Rt Pcr ◽

Systemic Necrosis ◽

Genome Database ◽

Total Rna ◽

First Report ◽

Saguaro Cactus Virus

Saguaro cactus virus (SgCV, genus Carmovirus, family Tombusviridae) was first isolated from an asymptomatic giant saguaro cactus (Carnegiea gigantea) in Arizona, USA (Milbrath and Nelson, 1972). In November 2017, 30 asymptomatic grafted cactus plants (Gymnocalycium mihanovichii grafted onto Hylocereus trigonus) were randomly collected from a commercial market in Gyeonggi Province, South Korea. Total RNA was extracted from both the scions and rootstocks of the plants using an RNeasy Plant Mini Kit (Qiagen, Germany) then subjected to reverse transcription polymerase chain reaction (RT-PCR) using RevertAid reverse transcriptase (Thermo Scientific, USA), TaKaRa Taq (TaKaRa, Japan), and SgCV-CP primers (forward, 5′- ATGGACGCTAAGTATGCG-3′; reverse, 5′- TCAGAGCCTAGCAACATA-3′). A validated SgCV stock (PV 0734, DSMZ, Germany) was used as an RT-PCR positive control. Out of 30 samples each of the rootstocks and scions, 21 and 8 produced, respectively, an amplicon at the expected size of 1,035 bp. The amplicons from three samples were cloned into a pGEM-T easy vector (Promega, USA), and three clones of each sample were sequenced (Macrogen, South Korea). The amplicons shared 100 % sequence identity with each other. BLASTn analysis showed that the sequence shared the highest identity at 66.3% with SgCV isolate Arizona (GenBank U72332). For bioassay of the virus, sap from infected G. mihanovichii was mechanically inoculated on four indicator plant species. The virus induced local lesions in Chenopodium amaranticolor, C. quinoa, and Gomphrena globosa, and systemic necrosis including growth reduction in C. capitatum. These results are consistent with those reported on SgCV by Milbrath and Nelson (1972). For determination of the exact species of the virus, non-inoculated leaves of C. capitatum were harvested 21 days after mechanical inoculation and subjected to total RNA extraction using the RNeasy Plant Mini Kit (Qiagen). A cDNA library was prepared using TruSeq RNA sample preparation v2, and sequenced on a NovaSeq 6000 system sequencer (Macrogen, South Korea). A total of 137,393,766 raw reads were quality-trimmed, and assembled into 120,408 contigs with sizes ranging from 201 to 15,898 nt using the Trinity program (r20140717). The assembled contigs were screened against the NCBI viral genome database using BLASTn, and a single contig of 3,858 nt matched the SgCV (acc. number U72332, coverage 88%, identity 70.3%). The sequence was deposited in GenBank (SgCV-gm, MW590184) and contained five open reading frames (ORFs), which is consistent with those of SgCV reported by Weng and Xiong (1997). Using DNAMAN software (Lynnon Biosoft, Canada) the deduced amino acid sequences encoded by the ORFs were determined and their homology with respective ORF proteins of various carmoviruses was subsequently compared (Table S1). The deduced protein sequences shared the highest identity of 68.2 to 81% with those of the SgCV isolate Arizona. King et al. (2012) suggested respective artificial host range reactions and percentage of coat protein and polymerase amino acid sequence identities of less than 52% and 57% as criteria for species demarcation in Carmovirus. These features suggest that SgCV-gm should possibly be designated a new SgCV isolate. To the best of our knowledge, this is the first report of SgCV naturally infecting G. mihanovichii in South Korea. Further research is needed to gain more in-depth insight into the biological and pathological properties of this virus.

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First Report of Grapevine leafroll-associated virus 5 in Spain

Plant Disease ◽

10.1094/pdis-07-10-0508 ◽

2010 ◽

Vol 94 (12) ◽

pp. 1507-1507 ◽

Cited By ~ 4

Author(s):

C. V. Padilla ◽

E. Cretazzo ◽

I. Hita ◽

N. López ◽

V. Padilla ◽

...

Keyword(s):

United States ◽

Amino Acid ◽

The United States ◽

Amino Acid Identity ◽

Wine Industry ◽

Rt Pcr ◽

Acid Identity ◽

First Report ◽

Virus Indexing ◽

Homologous Genes

Grapevine leafroll-associated viruses (GLRaVs) cause significant reductions in yield and quality in the wine industry worldwide. At least nine different GLRaVs have been found in different regions of the world. In the process of virus indexing of candidate grapevine clones for certification, which includes grafting of scions onto rootstocks, we observed strong leafroll symptoms 1 year after grafting with one vine of cv. Estaladina in Castilla y León, Spain and one vine of cv. Tempranillo in La Rioja, Spain, collected in 2008 and 2007, respectively. Both vines tested positive by real-time reverse transcription (RT)-PCR with TaqMan probes specific for Grapevine leafroll-associated virus 5 and double-antibody sandwich (DAS)-ELISA with a mix of monoclonal antibodies that recognizes GLRaV-4, 5, 6, 7, and 9 (Bioreba, Reinach, Switzerland). RNA extracts of both GLRaV-5 positive vines were analyzed by conventional RT-PCR with a pair of consensus degenerated primers derived from GLRaV-5 hsp70 sequences available in GenBank: LR5HYF (5′-TGGGATGAAYAARTTCAATGC-3′) and LR5HYR (5′-TGAAATTCCTCATRTARGAGC-3′) that amplified a 250-bp fragment. Amplicons were cloned and the comparison of the amino acid sequences (Estaladina isolate, Est110: Accession No. HM208622; Tempranillo isolate, Tem020: Accession No. HM208618) showed in the case of the Est110 isolate, 100 and 82.6% identity, respectively, with the homologous genes of one GLRaV-5 isolate from the United States (AF233934 [3]) and Argentina (EU815935 [2]). For isolate Tem020, the hsp70 gene showed 97.1 and 81.2% amino acid identity with the homologous hsp70 genes of the United States and Argentina isolates. The coat protein (cp) genes of both isolates were also amplified and cloned using the specific GLRaV-5 primers, LR53413 (5′-CGTGATACAAGGTAGGACAACCGT-3′) and LR53843 (5′-CTTGCACTATCGCTGCCGTGAAT-3′), designed according to the sequence of AF233934. Fragments were of the expected size (430 bp) and the nucleotide sequences were obtained (Est110: Accession No. HM363522; Tem020: Accession No. HM363523) and used for pairwise nucleotide comparisons. The Est110 isolate showed 96.7 and 97.5% amino acid identity with the isolates from the United States and Argentina, respectively, while the Tem020 isolate showed 94.8 and 95.6% identity, respectively. Amino acid identity of Est110 and Tem020 cp genes was 100% when compared with the homologous genes of isolates AF233934 and EU815935. To our knowledge this is the first report of GRLaV-5 in Spain. Since 2008, we have detected eight additional vines positive for this virus in 200 clones analyzed for certification, suggesting that the incidence of GLRaV-5 in Spain could be widespread. This research indicates that virus indexing for GLRaV should be included in certification schemes for grapevine candidate clones (1) in Spain. References: (1) Anonymous. OEPP/EPPO Bull. 38:422, 2008. (2) S. Gomez Talquenca et al. Virus Genes 38:184, 2009. (3) F. Osman et al. J. Virol. Methods 141:22, 2007.

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First Report of Cucumber vein yellowing virus on Cucumber in Lebanon

Plant Disease ◽

10.1094/pdis-05-13-0471-pdn ◽

2013 ◽

Vol 97 (11) ◽

pp. 1516-1516 ◽

Cited By ~ 1

Author(s):

P. E. Abrahamian ◽

H. Sobh ◽

R. Seblani ◽

J. Samsatly ◽

M. Jawhari ◽

...

Keyword(s):

Amino Acid ◽

Early Spring ◽

Post Inoculation ◽

Northern Coast ◽

Southern Coast ◽

Rt Pcr ◽

Economic Implications ◽

First Report ◽

Access Period ◽

Yellowing Virus

In a 2-year (2008 to 2009) wide-scale survey of viruses infecting cucurbits, a limited number of greenhouse-grown cucumber (Cucumis sativus) plants showed vein-yellowing symptoms. Greenhouses were infested with whiteflies and infection with Cucumber vein yellowing virus (CVYV) was suspected. CVYV is widely distributed in southern Europe in both open field and protected cucurbit crops (2). Total RNA was extracted from seven plants with vein yellowing symptoms using TRI Reagent (Sigma-Aldrich, St Louis, MO). RT-PCR tests using CVYV-specific primers (CV+/CV–) targeting the coat protein of CVYV (2) gave amplicons of the expected size from seven plants. The sequence of one representative isolate, CVYV-LB3 (GenBank Accession No. JF289167), showed 97, 95.6, and 95.2% pairwise nucleotide identity with isolates from Tunisia (EF538680), Israel (AF233429), and Jordan (JF460793), respectively. In 2012, CVYV like symptoms were not observed in greenhouses in the same areas. In early spring 2013, a total of 16 leaf samples with vein-yellowing symptoms were collected from the northern coastal areas (Jbeil, Amshit, Tabarja) and 11 samples showing only yellowing on older leaves from the southern coast (Jiyeh). CVYV was detected in all samples from the northern coast and in four samples from the southern coast. Four isolates from the North and two from the South were sequenced (KC990497 to KC990502) and showed high sequence variation. The pairwise nucleotide and amino acid identities between these isolates ranged from 95.1 to 100% and 98.5 to 100%, respectively. Pairwise nucleotide and amino acid identities of the isolates with CVYV-LB3 showed variable homology between 93.7 to 98.2% and 94.6 to 96.6%, respectively. The inter-population structure of CVYV in Lebanon showed high variability as compared to the homogenous Spanish population (4). For transmission tests, non-viruliferous whiteflies (Bemisia tabaci) were exposed for an 18-h acquisition access period on vein-yellowed leaves followed by a 24-h inoculation access period to healthy cucumber plants (5-leaf stage). In addition, leaves with vein-yellowing symptoms were ground in 0.1 M phosphate buffer (pH 7.0) and sap-inoculated on carborundum-dusted cucumber (cv. Delta) and squash (Cucurbita pepo cv. FarajF1) plants. Vein yellowing symptoms developed 9 to 11 days post-inoculation on all whitefly inoculated plants, while symptoms were delayed till 3 weeks post inoculation on seven out of eight sap-inoculated plants. All symptomatic plants were positive for CVYV by RT-PCR. Furthermore, surveyed plants were also tested for Cucurbit chlorotic yellows virus (CCYV) and Cucurbit yellow stunting disorder virus (CYSDV), two criniviruses reported previously in Lebanon, by RT-PCR (1). Double or triple infection of CCYV and CYSDV occurred in 18 out of 20 of the CVYV-infected plants. During the past 5 years, a limited number of cucumber plants showed CVYV symptoms. This indicates that CVYV occurrence is sporadic. However, its occurrence in mixed infection with criniviruses may have damaging economic implications to cucurbit production (3). To our knowledge, this is the first report of CVYV on cucurbits in Lebanon and its occurrence in co-infection with CCYV. References: (1) P. E. Abrahamian et al. Plant Dis. 96: 1704, 2012. (2) I. M. Cuadrado et al. Plant Dis. 85:336, 2001. (3) F. M. Gil-Salas et al. Plant Pathol. 61:468, 2011. (4) D. Janssen et al. Virus Genes 34:367, 2007.

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

Plant Disease ◽

10.1094/pdis-08-12-0777-pdn ◽

2013 ◽

Vol 97 (3) ◽

pp. 429-429 ◽

Cited By ~ 2

Author(s):

V. Trkulja ◽

J. Mihić Salapura ◽

B. Ćurković ◽

I. Stanković ◽

A. Bulajić ◽

...

Keyword(s):

Bosnia And Herzegovina ◽

Tomato Spotted Wilt Virus ◽

Amino Acid Identity ◽

N Gene ◽

Impatiens Necrotic Spot Virus ◽

Post Inoculation ◽

Rt Pcr ◽

First Report ◽

Tomato Spotted Wilt ◽

Wilt Virus

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

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First Report of Capsicum chlorosis virus Infecting Tomato in Taiwan

Plant Disease ◽

10.1094/pdis-04-10-0275 ◽

2010 ◽

Vol 94 (10) ◽

pp. 1263-1263 ◽

Cited By ~ 7

Author(s):

C.-H. Huang ◽

Y.-X. Zheng ◽

Y.-H. Cheng ◽

W.-S. Lee ◽

F.-J. Jan

Keyword(s):

Amino Acid ◽

Amino Acid Identity ◽

N Gene ◽

Rt Pcr ◽

Degenerate Primers ◽

Tomato Plants ◽

Acid Identity ◽

First Report ◽

Conserved Region ◽

Capsicum Chlorosis Virus

In December 2009, two samples from tomato plants (Solanum lycopersicum cv. Known-you 301) showing symptoms of chlorosis and necrosis on leaves were collected from two different fields that exhibited 5% disease incidence in Wufeng Township, Taichung County. Reverse transcription (RT)-PCR was applied to detect the presence of potential viruses in collected samples using three degenerate primers (3), gL3637/gL4435c for tospoviruses, Tob-Uni1/Tob-Uni2 for tobamoviruses, and Hrp5/Pot1 for potyviruses, and one specific primer, FJJ2001-7/FJJ2001-8, for the coat protein gene of Cucumber mosaic virus (3). An 816-nt DNA fragment was amplified from each of these two field samples by RT-PCR with the tospovirus degenerate primers, gL3637/gL4435c, designed from the conserved region of L RNA. One of the amplified fragments was cloned and sequenced. A homology search indicated that the new tomato-infecting virus in Taiwan might belong to Capsicum chlorosis virus (CaCV) since the partial L RNA shared more than 87% nucleotide and 99.6% amino acid identity with two CaCV isolates from Thailand (GenBank Accession Nos. DQ256124 and NC_008302). A virus culture isolated from the symptomatic tomato was established in Chenopodium quinoa through triple single-lesion isolation and designated as TwTom1. The partial L RNA and full-length nucleocapsid (N) gene of TwTom1 were obtained by RT-PCR with primer pairs gL3637/gL4435c and FJJ 2010-2 (5′-TTAAAT(C/T)ACAC(C/T)TCTATAGA)/N3534c (1), respectively. The 816-nt L RNA conserved region of TwTom1 (Accession No. HM021140) also shared 87% nucleotide and 99.6% amino acid identity with those of the above mentioned two CaCV isolates available in GenBank. The 828-nt N gene of TwTom1 (Accession No. HM021139) shared 85 to 98.1% nucleotide and 92 to 100% amino acid identity with those of 26 CaCV isolates available in GenBank. TwTom1 shared the highest N gene nucleotide and amino acid identity, 98.1 and 100%, respectively, with a gloxinia isolate (Accession No. AY312061). Sequence analysis results indicated that TwTom1 is an isolate of CaCV. The TwTom1 isolate was back inoculated onto three tomato (cv. Known-you 301) plants for pathogenicity test. The inoculated tomato plants showed symptoms of chlorosis at 13 days postinoculation (dpi) and symptoms of chlorosis plus necrosis on leaves at 20 dpi, which were similar to that observed in the field. A protein band measuring approximately 30 kDa in the crude sap of the TwTom1-infected tomato was observed in western blotting using the antiserum against the N protein of CaCV. In addition, CaCV was later detected by RT-PCR in two symptomatic tomato samples collected from another field. CaCV was first found in Australia, then Thailand, Taiwan, China, and India (2). Although CaCV was found to infect several species of ornamental crops in Taiwan, to our knowledge, this is the first report of CaCV that could naturally infect tomato, a nonornamental plant in Taiwan. References: (1) Y. H. Lin et al. Phytopathology 95:1482, 2005. (2) H. R. Pappu et al. Virus Res. 141:219, 2009. (3) Y.-X. Zheng et al. Plant Dis. 94:920, 2010.

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