scholarly journals First report of Cucurbit chlorotic yellows virus infecting Cucumis melo (muskmelon and oriental melon) in Korea

Plant Disease ◽  
2021 ◽  
Author(s):  
In Sook Cho ◽  
Tae-Bok Kim ◽  
Ju-Yeon Yoon ◽  
Bong Nam Chung ◽  
John Hammond ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Cucumis Melo ◽  
Korean Isolate ◽  
Rt Pcr ◽  
Genome Database ◽  
Total Rna ◽  
First Report ◽  
Oriental Melon ◽  
Cucumis Melo L ◽  
Cucurbit Chlorotic Yellows Virus

In December 2018, virus-like symptoms (yellowing, vein clearing) were observed on 2% of muskmelon (Cucumis melo L.) plants in plastic houses on a farm in Gyeongsang province, Korea Total RNA from two symptomatic and two asymptomatic plants was extracted using RNeasy Plant Mini Kit (Qiagen, Germany) for high throughput sequencing (HTS). After pre-processing and Ribo-Zero rRNA removal, a cDNA library was prepared (Illumina TruSeq Stranded Total RNA kit) and sequenced (Illumina NovaSeq 6000 system: Macrogen Inc. Korea). De novo assembly of 88,222,684 HTS reads with Trinity software (r20140717) yielded 146,269 contigs of 201-28,442 bp, which were screened against the NCBI viral genome database by BLASTn. Contigs from cucumber mosaic virus (CMV), melon necrotic spot virus (MNSV), tobacco mosaic virus (TMV) and watermelon mosaic virus (WMV) were identified, all previously reported in Korea. Two contigs (8,539 and 8,040 bp) with 99.9% sequence identity to distinct cucurbit chlorotic yellows virus (CCYV) isolates (JN641883, RNA1, Taiwan; MH819191, RNA2, China) were also identified. The ten sequences most closely related to each RNA of the Korean isolate (≥99% coverage, ≥99.6% nt identity) were from Japan, China, Taiwan, or Israel. CCYV presence was confirmed by reverse transcription-PCR (RT-PCR) using newly designed specific primers, RdRp-F/RdRp-R (5’-ACCGAACACTTGGCTATCCAA-3’/5’-CTTAATGCCGCGTATGAACTCA-3’) span style="font-family:'Times New Roman'; letter-spacing:-0.5pt">and HSP-F/HSP-R (5’-TGAACGACACTGAGTTCATTCCTA-3’/5’-CGCCAAGATCGTACATGAGGAA-3’), against RNA dependent RNA polymerase (RdRp; RNA1) and the heat shock protein 70 homolog (HSP70h; RNA2). Symptomatic samples yielded products of expected sizes (RdRp,450 bp; HSP70h, 510 bp) while asymptomatic samples did not. The amplicons were cloned, and two clones of each were sequenced (BIONEER, Korea; GenBank acc. nos. LC592226 and LC592227) showing 100% and 99.2% nt identity with RdRp and HSP70h genes of Chinese CCYV isolate SD (MH819190 and MH819191, respectively) and other Asian isolates. Primers specific for CMV, WMV, beet pseudo-yellows virus (BPYV) (Okuda et al., 2007), TMV (Kim et al., 2018), MNSV (F/R, 5ʹ-ATCTCGCATTTGGCATTACTC-3ʹ/5ʹ-ATTTGTAGAGATGCCAACGTA-3ʹ), cucurbit yellow stunting disorder virus (CYSDV; Zeng et al., 2011) and cucurbit aphid-borne yellows virus (CABYV; F/R, 5ʹ-CGGTCTATTGTCTGCAGTACCA-3ʹ/5ʹ- GTAGAGGATCTTGAATTGGTCCTCA-3ʹ) were also used. None of these viruses were detected in the symptomatic samples, but both asymptomatic plants were positive for CMV and WMV, and one also for MNSV. In June and September 2020, muskmelon and oriental melon (Cucumis melo L. var. makuwa) plants with yellowing disease (incidence 80-90%) and whiteflies were observed in all investigated plastic houses of one muskmelon and one oriental melon farm in Gyeonggi and Jeolla provinces. Symptomatic samples (14 muskmelon; 6 oriental melon) were collected and RT-PCR tested as above; 19/20 samples were positive for CCYV, but none for the other viruses. The oriental melon sequence (LC592895, LC592230) showed 99.7% and 100% nt identity with the RdRp and HSP70h genes of Chinese isolate SD, respectively. CCYV was first reported in Japan (Okuda et al., 2010), Taiwan, and China (Huang et al., 2010; Gu et al., 2011); to our knowledge, this is the first report of CCYV infecting muskmelon and oriental melon in Korea. Whitefly-transmitted CCYV could present a serious threat of yield losses to cucurbit crops in Korea, requiring control of vector populations to prevent spread of CCYV.

scholarly journals First Report of Bean common mosaic virus in Cudrania tricuspidata in Korea

Plant Disease ◽  
2015 ◽  
Vol 99 (2) ◽  
pp. 292-292 ◽  
Author(s):  
J.-K. Seo ◽  
M. Kang ◽  
O. J. Shin ◽  
H.-R. Kwak ◽  
M.-K. Kim ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Large Scale ◽  
De Novo ◽  
Bean Common Mosaic Virus ◽  
Deciduous Tree ◽  
Root Bark ◽  
Genome Database ◽  
Cudrania Tricuspidata ◽  
Total Rna ◽  
First Report

Cudrania tricuspidata (Moraceae) is a deciduous tree widely distributed in East Asia, including China, Korea, and Japan. It produces delicious fruit, and its cortex and root bark have been used as a traditional medicine to treat neuritis and inflammation. As C. tricuspidata has become known as a functional food, its cultivation area and production gradually have increased in Korea. However, information of viral disease in C. tricuspidata is very limited. In September 2012, open-field-grown C. tricuspidata trees showing virus-like symptoms of mosaic, yellowing, and distortion on the leaves were found in Naju, Korea. The fruit production in the diseased trees decreased to 20 to 40% of that in healthy trees. To identify causal agent(s), total RNA was isolated from the symptomatic leaves and used to generate a transcriptome library using the TruSeq Stranded Total RNA with Ribo-Zero Plant kit (Illumina, San Diego, CA) according to the manufacturer's instruction. The transcriptome library was analyzed by next-generation sequencing (NGS) using an Illumina HiSeq2000 sequencer. NGS reads were quality filtered and de novo assembled by the Trinity pipeline, and the assembled contigs were analyzed against the viral reference genome database in Genbank by BLASTn and BLASTx searches (3). The entire NGS procedure was perofrmed by Macrogen Inc. (Seoul, South Korea). Among the analyzed contigs, one large contig (10,043 bp) was of viral origin. Nucleotide blast searches showed that the contig has a maximum identity of 89% (with 100% coverage) to the isolate MS1 (Genbank Accession No. EU761198) of Bean common mosaic virus (BCMV), which was isolated from Macroptilium atropurpureum in Australia. The presence of BCMV was confirmed by a commercially available double-antibody sandwich (DAS)-ELISA kit (Agdia, Elkhart, IN). To confirm the BCMV sequence obtained by NGS, two large fragments covering the entire BCMV genome were amplified by reverse transcription-polymerase chain reaction (RT-PCR) using two sets of specific primers (5′-AAAATAAAACAACTCATAAAGACAAC-3′ and 5′-AGACTGTGTCCCAGAGCATTTC-3′ to amplify the 5′ half of the BCMV genome; 5′-GCATCCTGAGATTCACAGAATTC-3′ and 5′-GGAACAACAAACATTGCCGTAG-3′ to amplify the 3′ half of the BCMV genome) and sequenced. To obtain the complete genome sequence, the 5′ and 3′ terminal sequences were analyzed by the 5′ and 3′ rapid amplification of cDNA ends (RACE) method as described previously (1). The assembled full-length sequence of BCMV isolated from C. tricuspidata was 10,051 nucleotides in length without a poly(A) tail. It was deposited in Genbank under the accession number KM076650. BCMV, a member of the genus Potyvirus, is one of the most common viruses naturally infecting legumes, including Phaseolus vulgaris (2). In general, BCMV is known to have a restricted host range outside legume species (2). Therefore, the identification of BCMV from C. tricuspidata in this report is very exceptional. Because BCMV is easily transmitted by various aphids like other potyviruses, a large-scale survey may be required for exact investigation of the BCMV incidence in C. tricuspidata to prevent rapid spread of the virus. To the best of our knowledge, this is the first report of BCMV in C. tricuspidata. References: (1) H.-R. Kwak et al. Plant Pathol. J. 29:274, 2013. (2) M. Saiz et al. Virus Res. 31:39, 1994. (3) S.-E. Schelhorn et al. PLoS Comput. Biol. 9:e1003228, 2013.

scholarly journals First report of cucurbit chlorotic yellows virus infecting cucumber in South Korea

Plant Disease ◽  
2021 ◽  
Author(s):  
Hae-Ryun Kwak ◽  
Hui-Seong Byun ◽  
Hong-Soo Choi ◽  
Jong-Woo Han ◽  
Chang-Seok Kim ◽  
...  
Keyword(s):  
Coat Protein ◽  
South Korea ◽  
East Asia ◽  
Rna Extraction ◽  
Rt Pcr ◽  
Specific Primers ◽  
Total Rna ◽  
First Report ◽  
Chlorotic Mottle ◽  
Cucurbit Chlorotic Yellows Virus

In October 2018, cucumber plants showing yellowing and chlorotic mottle symptoms were observed in a greenhouse in Chungbuk, South Korea. The observed symptoms were similar to those caused by cucurbit aphid-borne yellows virus (CABYV), which has been detected on cucumber plants in the region since it was reported on melon in Korea in 2015 (Lee et al 2015). To identify the potential agents causing these symptoms, 28 samples from symptomatic leaves and fruit of cucumber plants were subjected to total RNA extraction using the Plant RNA Prep Kit (Biocubesystem, Korea). Reverse transcription polymerase chain (RT-PCR) was performed on total RNA using CABYV specific primers and protocols (Kwak et al. 2018). CABYV was detected in 17 of the 28 samples, while 11 symptomatic samples tested negative. In order to identify the cause of the symptoms, RT-PCR was performed using cucurbit chlorotic yellows virus (CCYV) and cucurbit yellow stunting disorder virus (CYSDV) specific primers (Wintermantel et al. 2019). Eight of the 28 samples were positive using the CCYV specific primers while seven samples were infected with only CCYV and one contained a mixed infection of CABYV with CCYV. None of the samples tested positive for CYSDV. The expected 373 nt amplicons of CCYV were bi-directionally sequenced, and BLASTn analysis showed that the nucleotide sequences shared 98 to 100% identity with CCYV isolates from East Asia, including NC0180174 from Japan. Two pairs of primers for amplification of the complete coat protein and RNA-dependent RNA polymerase (RdRp) genes (Wintermantel et al., 2019) were used to amplify the 753bp coat protein and 1517bp RdRp genes, respectively. Amplicons of the expected sizes were obtained from a CCYV single infection and ligated into the pGEM T- Easy vector (Promega, WI, USA). Three clones from each amplicon were sequenced and aligned using Geneious Prime and found to have identical sequences (Genbank accession nos. MW033300, MW033301). The CP and RdRp sequences demonstrated 99% nucleotide and 100% amino acid identity with the respective genes and proteins of the CCYV isolates from Japan. This study documents the first report of CCYV in Korea. Since CCYV was first detected on melon in Japan, it has been reported in many other countries including those in East Asia, the Middle East, Southern Europe, North Africa, and recently in North America. CCYV has the potential to become a serious threat to production of cucurbit crops in Korea, particularly due to the increasing prevalence of the whitefly, Bemisia tabaci, in greenhouse production systems. It will be important to continue monitoring for CCYV and determine potential alternate hosts in the region to manage and prevent further spread of CCYV in Korea.

scholarly journals First Report of Cucurbit chlorotic yellows virus Infecting Cucurbits in Taiwan

Plant Disease ◽  
2010 ◽  
Vol 94 (9) ◽  
pp. 1168-1168 ◽  
Author(s):  
L.-H. Huang ◽  
H.-H. Tseng ◽  
J.-T. Li ◽  
T.-C. Chen
Keyword(s):  
Citrullus Lanatus ◽  
Bottle Gourd ◽  
Insect Vector ◽  
Silverleaf Whitefly ◽  
Rt Pcr ◽  
Specific Primers ◽  
Total Rna ◽  
First Report ◽  
Dna Fragment ◽  
Cucurbit Chlorotic Yellows Virus

In April 2009, chlorosis, yellows, and bleaching accompanied with green veins and brittleness on the lower leaves of cantaloupe (Cucumis melo L.) were observed in Lunbei Township, Yunlin County, Taiwan. The same symptoms were also found on cucumber (Cucumis sativus L.), pumpkin (Cucurbita moschata Duchesne), watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai), bottle gourd (Lagenaria siceraria (Molina) Standl.), and oriental pickling melon planted in other areas of Yunlin and Changhua counties in central Taiwan. Large populations of whiteflies were observed in association with the diseased cucurbit crops, and they were further identified as silverleaf whitefly (Bemisia argentifolii Bellows & Perring) by PCR with specific primers BaBF (5′-CCACTATAATTATTGCTGTTCCCACA-3′) and l2-N-3014R (5′-TCCAATGCACTAATCTGCCATATTA-3′) (3). In June 2009, samples from symptomatic cantaloupe were collected for virus diagnosis. Flexuous filamentous virions of 700 to 900 nm were observed in crude sap of the symptomatic cantaloupe tissues with transmission electron microscopy. On the basis of the suspected insect vector, symptomology, and virus morphology, a Crinivirus species was suspected as the causal agent. A nested reverse transcription (RT)-PCR assay with degenerate deoxyinosine-containing primers developed for detection of Closterovirus and Crinivirus (1) was conducted. Total RNAs extracted from 16 symptomatic cantaloupe samples with a Plant Total RNA Miniprep Purification Kit (Hopegen, Taichung, Taiwan) were analyzed, and a 0.5-kb DNA fragment was amplified from eight of them. The PCR products were sequenced and the sequences were identical among samples. A comparison of the submitted sequence (Accession No. HM120250) with those in GenBank showed that the sequence was identical to the Hsp70h sequences of Cucurbit chlorotic yellows virus (CCYV) isolates from Japan (Accession No. AB523789) (4) and China (Accession Nos. GU721105, GU721108, and GU721110). To identify CCYV infection in the field, the specific primers, Crini-hsp70-f (5′-GCCATAACCATTACGGGAGA-3′) and Crini-hsp70-r (5′-CGCAGTGAAAAACCCAAACT-3′), that amplify a 389-bp DNA fragment corresponding to the nucleotide 1,324 to 1,712 of RNA2 of the original CCYV Japan isolate (Accession No. AB523789) were designed for detection of CCYV. In RT-PCR analyses, CCYV was identified in cantaloupe (305 of 599 samples), watermelon (27 of 93 samples), cucumber (all 15 samples), melon (82 of 92 samples), pumpkin (8 of 10 samples), and bottle gourd (10 of 17 samples) showing chlorosis and yellowing. The 389-bp DNA fragment was also amplified by RT-PCR with the primer pair Crini-hsp70-f/Crini-hsp70-r from total RNA extracts of 29 of 116 silverleaf whitefly individuals collected from the diseased cantaloupe fields in Lunbei Township from August to October, 2009. CCYV is a newly characterized Crinivirus species, first discovered in Japan in 2004 (2) and also found in China in 2009. To our knowledge, this is the first report that CCYV is emerging as a threat to cucurbit productions in Taiwan. References: (1) C. I. Dovas and N. I. Katis. J. Virol. Methods 109:217, 2003. (2) Y. Gyoutoku et al. Jpn. J. Phytopathol. 75:109, 2009. (3) C. C. Ko et al. J. Appl. Entomol. 131:542, 2007. (4) M. Okuda et al. Phytopathology 100:560, 2010.

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 Cucumber mosaic virus on Melon in Bosnia and Herzegovina

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

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

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

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

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

scholarly journals First Report of 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 Tomato spotted wilt virus in Brugmansia suaveolens in Korea

Plant Disease ◽  
2014 ◽  
Vol 98 (9) ◽  
pp. 1283-1283
Author(s):  
S.-K. Choi ◽  
I.-S. Cho ◽  
G.-S. Choi ◽  
J.-Y. Yoon
Keyword(s):  
Mosaic Virus ◽  
Tomato Spotted Wilt Virus ◽  
Korean Isolate ◽  
Rt Pcr ◽  
First Report ◽  
Tomato Spotted Wilt ◽  
Pcr Product ◽  
Brugmansia Suaveolens ◽  
Plant Pathol ◽  
Wilt Virus

Brugmansia suaveolens, also known as angel's trumpet, is a semi-woody shrub or a small tree. Because flowers of B. suaveolens are remarkably beautiful and sweetly fragrant, B. suaveolens is grown as ornamentals outdoors year-round in the tropics and subtropics, and as potted plants in temperate regions (1). In February 2013, virus-like symptoms including mosaic symptoms followed by distortion of leaves were observed in a potted B. suaveolens in a nursery in Chung-Nam Province, Korea. Symptomatic leaves were analyzed for the presence of several ornamental viruses including Cucumber mosaic virus (CMV), Tobacco mosaic virus (TMV), Tomato bush stunt virus (TBSV), and Tomato spotted wilt virus (TSWV) by immune-strip diagnostic kits that were developed by our laboratory. Positive controls and extract from healthy leaves of B. suaveolens as a negative control were included in each immune-strip assay. TSWV was detected serologically from the naturally infected B. suaveolens, but CMV, TBSV, and TMV were not detected from the B. suaveolens. The presence of TSWV (named TSWV-AT1) was confirmed by commercially available double-antibody sandwich (DAS)-ELISA kits (Agdia, Elkhart, IN). TSWV-AT1 was mechanically transmitted from the ELISA-positive B. suaveolens to Capsicum annuum and Nicotiana glutinosa, respectively. Inoculated C. annuum showed chlorotic rings in the inoculated leaves and inoculated N. glutinosa produced mosaic and systemic necrosis in the inoculated leaves after 7 days inoculation, respectively, which were consistent with symptoms caused by TSWV (2). To confirm further TSWV-AT1 infection, reverse transcription (RT)-PCR was performed using the One-Step RT-PCR (Invitrogen, Carlsbad, CA) with TSWV-specific primers, TSWV-NCP-For and TSWV-NCP-Rev (3), designed to amplify a 777-bp cDNA of the nucleocapsid protein (NCP) gene. Total RNAs from naturally infected B. suaveolens, symptomatic C. annuum, and N. glutinosa were extracted using RNeasy Plant Mini Kit (Qiagen, Valencia, CA). Total RNAs obtained from a Korean isolate of TSWV (Accession No. JF730744) and healthy B. suaveolens were used as positive and negative controls, respectively. The expected size of the RT-PCR product was amplified from symptomatic B. suaveolens, C. annuum, and N. glutinosa but not from healthy leaves of B. suaveolens. The amplified RT-PCR product from TSWV-AT1 was directly sequenced using BigDye Termination kit (Applied Biosystems, Foster City, CA). Multiple alignment of the TSWV-AT1 NCP sequence (AB910533) with NCP sequences of other TSWV isolates using MEGA5 software (4) revealed 99.0% aa identity with an Korean TSWV isolate (AEB33895) originating from tomato. These results provide additional confirmation of TSWV-AT1 infection. It is known that high-value ornamentals may act also as reservoirs for TSWV that can infect other ornamentals and cultivated crops, because TSWV has a very broad host range (2). Elaborate inspections for TSWV and other viruses are necessary for production of healthy B. suaveolens, since the popularity and economic importance of this ornamental plant is increasing. To our knowledge, this is the first report of TSWV in B. suaveolens in Korea. References: (1) Anonymous. OEPP/EPPO Bull. 34:271, 2004. (2) G. Parrella et al. J. Plant Pathol. 85:227, 2003. (3) B.-N. Chung et al. Plant Pathol. J. 28:87, 2012. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

scholarly journals First Report of Saguaro Cactus Virus Infecting Gymnocalycium mihanovichii in South Korea

Plant Disease ◽  
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.

scholarly journals First Report of Tomato Brown Rugose Fruit Virus on Tomato in Syria

Plant Disease ◽  
2021 ◽  
Author(s):  
Ziad M Hasan ◽  
Nidà Mohammed Salem ◽  
Imad D. Ismail ◽  
Insaf Akel ◽  
Ahmad Y Ahmad
Keyword(s):  
Mosaic Virus ◽  
Rna Extraction ◽  
Coastal Region ◽  
Rt Pcr ◽  
Specific Primers ◽  
Total Rna ◽  
First Report ◽  
Species Specific ◽  
Das Elisa ◽  
Total Rna Extraction

Tomato (Solanum lycopersicum L.) is an important vegetable crop worldwide. In spring and autumn 2017, virus-like symptoms were observed on greenhouse grown tomato plants in the east of Akkar plain (south of coastal region, Tartous governorate, Syria). These symptoms were: mild to severe mosaic on the apical leaves, brown necrosis on sepals, receptacle and flower’s cluster carrier, and severe symptoms of brown rugose and discoloration on fruit. During next growing seasons, disease spread was observed in most of Syrian coastal region with disease incidence ranged from 40% to 70% by 2020. Tomato brown rugose fruit virus (ToBRFV) was suspected as a main causal agent of the disease, especially since its first report in Jordan, a neighboring country (Salem et al. 2016), Palestine (Alkowni et al. 2019), Turkey (Fidan et al. 2019), Germany (Menzel et al. 2019), Italy (Panno et al. 2019), America (Camacho-Beltrán et al. 2019), Egypt (Amer and Mahmoud, 2020), and recently in Spain (Alfaro-Fernandez et al. 2021). In November and December 2020, seventy-one leaf samples from symptomatic plants (59 from Tartous and 12 from Lattakia governorates) and seven from asymptomatic ones (5 from Tartous and 2 from Lattakia) were collected and tested for the presence of ToBRFV by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), using ToBRFV-commercial kit (LOEWE® Biochemia, Germany) following the manufacturer’s instructions. Results showed, forty-three of symptomatic samples reacted positively (38 in Tartous and 5 in Lattakia) and none of asymptomatic ones. On the other hand, sap mechanical inoculation of 10 tomato cv. Mandaloun F1 (Enza Zaden, the Netherlands) plants using a positive tomato isolate gave systemic mosaic symptoms in all plants identical to those observed in the original plants in the field, after 13 days of inoculation, and necrotic local lesions on 10 plants of Nicotiana tabacum after 5 days, indicating the presence of a tobamovirus in general. ToBRFV infection was confirmed in all mechanically-inoculated plants by DAS-ELISA. Further tests were necessary to investigate ToBRFV presence, because of its serological relationships with another tobamoviruses. Six representative symptomatic samples (ELISA-positive) and two asymptomatic (ELISA-negative) samples were subjected to total RNA extraction using the SV-Total RNA Extraction kit (Promega, U.S.A.) following the manufacturer’s instructions. The samples were tested by two-step reverse transcription-polymerase chain reaction (RT-PCR) using species-specific primers and protocols for most common tomato-infecting viruses, including: tomato chlorosis virus and tomato infectious chlorosis virus (Dovas et al. 2002), pepino mosaic virus (PepMV) and tomato torrado virus (Wieczorek et al. 2013), alfalfa mosaic virus (Parrella et al. 2000), tomato spotted wilt virus (Salem et al. 2012) and a pair of primers: ToBRFV-F2 (5’-CATATCTCTCGACACCAGTAAAAGGACCCG-3’) and ToBRFV-R2 (5’-TCCGAGTATAGGAAGACTCTGGTTGGTC-3’) targeting a region of the RNA dependent RNA polymerase (RdRp), of the ToBRFV genome (KT383474; Salem et al. 2016). First-strand cDNA synthesis was carried out using Moloney murine leukemia virus reverse transcriptase (M-MLV RT; Promega) and random primer according to the manufacturer's protocol, then followed by PCR with the seven species-specific primers. Only ToBRFV was detected among all tested viruses in symptomatic samples (ELISA-positive), and none of the tested viruses was detected in the asymptomatic plants. To confirm the presence of ToBRFV, two selected RdRp-specific PCR amplicons (872 bp) were purified and ligated into pGEM T-Easy Vector (Promega), and three clones were sequenced (GenBank accession nos. MZ447794 to 96). BLASTn analysis showed that the nucleotide sequences are 99.77-100% identical and shared around 99% identity to RdRp of ToBRFV isolate (MT118666) from Turkey available in the GenBank. Accordingly, the presence of ToBRFV was confirmed by bioassays on indicator plants, DAS-ELISA, RT-PCR, and further sequencing. To our knowledge, this is the first report of ToBRFV infecting tomato in Syria, and this requires special emphasis for further investigations because of the virus severity, easy transmission ability and absent of commercial resistance varieties till now.

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