First report of watermelon crinkle leaf-associated virus 1 (WCLaV-1) and WCLaV-2 in watermelon (Citrullus lanatus L.) plants co-infected with Cucurbit chlorotic yellows virus in Florida

Watermelon (Citrullus lanatus L.) and other cucurbits are major crops in Florida. During the 2020 and 2021 seasons, watermelon plants with foliar virus-like symptoms of yellow mottling and chlorosis, mild leaf wrinkling and thickened leaves were observed in commercial fields (40 to 150 ha) in five counties (Desoto, Glades, Osceola, Seminole, and Charlotte) at >50% field incidence. Initial screening of 13 field-collected samples (2 to 4/County) for potyviruses with the Agdia POTY Immunostrip (Agdia, Inc. Elkhart, IN) were negative. Total nucleic acid extracts from each sample (RNeasy Plant Mini Kit, Qiagen, Germantown, MD) were used in one-step RT-PCR (Qiagen OneStep RT-PCR kit) with species-specific primer targeting squash vein yellowing virus (SqVYV), papaya ringspot virus-W (PRSV-W) (Adkins et al., 2008), cucurbit yellow stunting disorder virus (CYSDV) (Polston et al. 2008), cucurbit chlorotic yellows virus (CCYV) (Hernandez et al., 2021a), watermelon crinkle leaf-associated virus 1 (WCLaV-1), and WCLaV-2 (Hernandez et al., 2021b). The samples were also tested for cucurbit leaf crumple virus (CuLCrV) as per Hagen et al. (2008). All 13 samples were negative for SqVYV, PRSV-W, CuLCrV, and CYSDV, but 7 samples (53.8%) from 4 counties tested positive for CCYV, 12 (92.3%) from 5 counties were positive for WCLaV-1, and WCLaV-2 was detected in 8 samples (61.5%) from 5 counties. Three samples were singly infected with WCLaV-1 while the remaining 10 were mixed infected with different combination of 2 or 3 viruses. Notably, symptoms on all 13 plants were visually indistinguishable. To verify the results, two randomly chosen gene-specific fragments per virus, obtained with primers CCYV-v1330/c2369, CCYV-v4881/c5736, WCLaV-1vRP/1cRP, WCLaV-1vMP/1cMP, WCLaV-2vRP/2cRP, and WLaV 2vMP/2cMP, were excised from the gel, cloned, and Sanger-sequenced as described (Hernandez et al., 2021a, 2021b). In pairwise comparisons, the ~1 kb partial ORF1a (GenBank accession nos. MZ325846 to MZ325847) and 753 bp complete coat protein cistron (MZ325848 to MZ325849) of CCYV from Florida shared 98.9-99.5%/98.4-99.6% nucleotide (nt)/amino acid (aa) and 99.4-99.8%/99.6-100% nt/aa identities, respectively with the corresponding sequences of global CCYV isolates. The partial RNA1 (MZ325850 to MZ325851) and RNA2 (MZ325852 to MZ325853) sequences of WCLaV-1 from Florida shared 99.2-99.8%/100% nt/aa and 98.9-100%/99.3-100% nt/aa identities, respectively with the corresponding global sequences of WCLaV-1 isolates. Lastly, the partial RNA1 (MZ325854 to MZ325857) and RNA2 (MZ325858 to MZ325861) sequences of WCLaV-2 from Florida shared 96.4-99.8%/97-100% nt/aa and 96.5-100%/95.9-100% nt/aa identities, respectively with the corresponding global sequences of WCLaV-2 isolates. This is the first report of WCLaV-1 and WCLaV-2 from Florida and the first documentation of the occurrence of CCYV in South Florida. CCYV has been reported previously from California (Wintermantel et al. 2019), Georgia (Kavalappara et al. 2021), and recently from North Florida (M. Paret, pers. comm) but WCLaV-1 and WCLaV-2 have only been reported from Texas (Hernandez et al., 2021b), after their discovery in China (Xin et al. 2017). The results indicate further expansion of the geographical range of these cucurbit-infecting viruses, although their longer but undetected presence in Florida is plausible due to the resemblance of their associated symptoms with those attributed to known viruses. References Adkins, et al., 2008. Plant Dis. 92:1119-1123. https://apsjournals.apsnet.org/doi/10.1094/PDIS-92-7-1119. Hagen, et al. 2008. Plant Dis. 92:781-793. https://apsjournals.apsnet.org/doi/pdfplus/10.1094/PDIS-92-5-0781. Hernandez, et al. 2021a. Plant Dis. https://doi.org/10.1094/PDIS-02-21-0378-PDN. Hernandez, et al., 2021b. Plant Dis. https://doi.org/10.1094/PDIS-02-21-0249-PDN Kavalappara, et al., 2021. Plant Dis. https://doi.org/10.1094/PDIS-11-20-2429-PDN. Polston, et al. 2008. Plant Dis. 92(8):1251. https://apsjournals.apsnet.org/doi/10.1094/PDIS-92-8-1251B. Wintermantel, et al., 2019. Plant Dis. 103(4):778. https://doi.org/10.1094/PDIS-08-18-1390-PDN. Xin, et al., 2017. Front. Microbiol. 8:1514, doi: 10.3389/fmicb.2017.01514.

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First Report of Cucurbit chlorotic yellows virus Infecting Cucurbits in Taiwan

Plant Disease ◽

10.1094/pdis-94-9-1168b ◽

2010 ◽

Vol 94 (9) ◽

pp. 1168-1168 ◽

Cited By ~ 33

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.

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First Report of Cucurbit chlorotic yellows virus in Cucumber, Melon, and Watermelon in Greece

Plant Disease ◽

10.1094/pdis-03-14-0311-pdn ◽

2014 ◽

Vol 98 (10) ◽

pp. 1446-1446 ◽

Cited By ~ 23

Author(s):

C. Orfanidou ◽

V. I. Maliogka ◽

N. I. Katis

Keyword(s):

Citrullus Lanatus ◽

Disease Incidence ◽

Access Time ◽

Cucumber Plant ◽

Post Inoculation ◽

Rt Pcr ◽

First Report ◽

Yellowing Disease ◽

Beet Pseudo Yellows Virus ◽

Cucurbit Chlorotic Yellows Virus

In 2011, an outbreak of a yellowing disease causing chlorosis and Interveinal chlorotic spots on lower leaves was observed in cucumber (Cucumis sativus) and melon (C. melo) plants in two greenhouses on the island of Rhodes, Greece. Similar symptoms were observed in 2012 in open field watermelon (Citrullus lanatus) plants in Rhodes and in November 2013 in a cucumber greenhouse in Tympaki, Crete. Disease incidence ranged from 10 to 40%. The observed symptoms were similar to those caused by whitefly transmitted criniviruses (family Closteroviridae) Cucurbit yellow stunting disorder virus (CYSDV) and Beet pseudo-yellows virus (BPYV), as well as Cucurbit chlorotic yellows virus (CCYV), a recently described crinivirus that infects cucurbits in Japan (4) and by the aphid transmitted polerovirus (family Luteoviridae) Cucurbit aphid-borne yellows virus (CABYV). Dense populations of whiteflies were present in all the affected crops. Leaf samples from cucumber (10 from Rhodes and 10 from Crete), melon (10), and watermelon (10) were collected and tested for the presence of the above viruses. Total RNA was extracted from the samples (2) and detection of BPYV, CYSDV, and CABYV was done as previously described (1,3) whereas detection of CCYV was conducted by herein developed two-step RT-PCR assays. Two new pairs of primers, ‘CC-HSP-up’ (5′-GAAGAGATGGGTTGGTGTAGATAAA-3′)/‘CC-HSP-do’ (5′-CACACCGATTTCATAAACATCCTTT-3′) and ‘CC-RdRp-up’ (5′-CCTAATATTGGAGCTTATGAGTACA-3′)/‘CC-RdRp-do’ (5′-CATACACTTTAAACACAACCCC-3′) were designed based on GenBank deposited sequences of CCYV for the amplification of two regions partially covering the heat shock protein 70 homologue (HSP70h) (226 bp) and the RNA dependent RNA polymerase (RdRp) genes (709 bp). Interestingly, CCYV was detected in all samples tested, while CYSDV was detected in 18 cucumbers (10 from Rhodes and 8 from Crete), 1 melon, and 3 watermelon plants. Neither BPYV nor CABYV were detected. In order to verify the presence of CCYV, the partial HSP70h and RdRp regions of a cucumber isolate from Crete were directly sequenced using the primers ‘CC-HSP-up’/‘CC-HSP-do’ and ‘CC-RdRp-up’/‘CC-RdRp-do’. BLAST analysis of the obtained sequences (HG939521 and 22) showed 99% and 100% identities with the HSP70h and RdRp of cucumber CCYV isolates from Lebanon, respectively (KC990511 and 22). Also, the partial HSP70h sequence of a watermelon CCYV isolate from Rhodes showed 99% identity with the cucumber isolate from Crete. Whitefly transmission of CCYV was also carried out by using an infected cucumber from Crete as virus source. Four groups of 30 whitefly adults of Bemisia tabaci biotype Q were given an acquisition and inoculation access time of 48 and 72 h, respectively. Each whitefly group was transferred to a healthy cucumber plant (hybrid Galeon). Two weeks post inoculation, the plants, which have already been showing mild interveinal chlorosis, were tested for virus presence by RT-PCR. CCYV was successfully transmitted in three of four inoculated cucumbers, which was further confirmed by sequencing. In Greece, cucurbit yellowing disease has occurred since the 1990s, with CYSDV, BPYV, and CABYV as causal agents. To our knowledge, this is the first report of CCYV infecting cucurbits in Greece; therefore, our finding supports the notion that the virus is spreading in the Mediterranean basin and is an important pathogen in cucurbit crops. References: (1) I. N. Boubourakas et al. Plant Pathol. 55:276, 2006. (2) E. Chatzinasiou et al. J. Virol. Methods 169:305, 2010. (3) L. Lotos et al. J. Virol. Methods 198:1, 2014. (4) M. Okuda et al. Phytopathology 100:560, 2010.

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First Report of Cucurbit Chlorotic Yellows Virus affecting Watermelon in USA

Plant Disease ◽

10.1094/pdis-03-21-0639-pdn ◽

2021 ◽

Author(s):

A. Abdul Kader Jailani ◽

Fanny Iriarte ◽

Robert Hochmuth ◽

Sylvia M. Willis ◽

Mark W. Warren ◽

...

Keyword(s):

Mixed Infection ◽

Citrullus Lanatus ◽

Disease Incidence ◽

Pcr Amplification ◽

Accession Number ◽

Rt Pcr ◽

Imperial Valley ◽

First Report ◽

The U.S ◽

Cucurbit Chlorotic Yellows Virus

Watermelon (Citrullus lanatus) is a high nutrient crop, high in vitamins and very popular in the U.S and globally. The crop was harvested from 101,800 acres with a value of $560 million in the U.S (USDA-NASS, 2020). California, Florida, Georgia and Texas are the four-leading watermelon-producing states in the U.S. During the fall season of 2020, plants in two North Florida watermelon fields, one in Levy County (~20 acres) and one in Suwannee County (~80 acres) with varieties Talca and Troubadour, respectively, exhibited viral-like symptoms. The fields had 100% disease incidence that led to fruit quality issues and yield losses of 80% and above. Symptoms observed in the watermelon samples included leaf crumpling, yellowing and curling, and vein yellowing similar to that of single/and or mixed infection of cucurbit leaf crumple virus (CuLCrV; genus: Begomovirus, family: Geminiviridae), cucurbit yellow stunting disorder virus (CYSDV; genus: Crinivirus, family: Closteroviridae) and squash vein yellowing virus (SqVYV; genus: Ipomovirus, family: Potyviridae), although the vine decline symptoms often associated with SqVYV infection of watermelon were not observed. All three viruses are vectored by whiteflies and previously described in Florida (Akad et al., 2008; Polston et al., 2008; Adkins et al., 2009). To confirm the presence of these viruses, RNA was isolated from 20 symptomatic samples using the RNeasy Plant Mini Kit (Qiagen, USA) as per protocol. This was followed by RT-PCR (NEB, USA) using gene-specific primers described for CuLCrV, CYSDV and SqVYV (Adkins et al., 2009). Amplicons of expected sizes were obtained for all the viruses with the infection of CuLCrV in 17/20, CYSDV in 16/20, and SqVYV in 8/20 samples. In addition, the presence of cucurbit chlorotic yellows virus (CCYV; genus: Crinivirus, family: Closteroviridae) in mixed infection was confirmed in 4/20 samples (3 leaves and 1 fruit) by RT-PCR with primers specific to the CCYV coat protein (CP), heat shock protein 70 homolog (HSP70h) and RNA dependent RNA polymerase (RdRp) designed based on the available CCYV sequences (Sup Table. 1). The RT-PCR amplification was performed using a symptomatic watermelon sample and the amplicons of RdRp, HSP70h and CP were directly sequenced by Sanger method, and the sequences of the amplicons were deposited in GenBank under the accession number: MW527462 (RdRp, 952 bp), MW527461 (HSP70h, 583 bp) and MW527460 (CP, 852 bp). BLASTn analysis demonstrated that the sequences exhibited an identity of 99% to 100% (RdRp and HSP70h, 100%; and CP, 99%) with the corresponding regions of the CCYV isolate Shanghai from China (accession number: KY400636 and KY400633). The presence of CCYV was further confirmed in the watermelon samples by ELISA (Loewe, Germany) using crude sap extracted from the RT-PCR-positive, symptomatic watermelon samples. CCYV was first identified in Kumamoto, Japan in 2004 on melon plants (Gyoutoku et al. 2009). The CCYV was previously reported on melon from Imperial Valley, California (Wintermantel et al., 2019), and more recently on squash in Tifton, Georgia (Kavalappara et al., 2021) and cantaloupe in Cameron, Texas (Hernandez et al., 2021). To our knowledge, this is the first report of CCYV on field watermelon production in the U.S. Continued monitoring of the CCYV in spring and fall watermelon crop, and cucurbit volunteers and weeds will be critical toward understanding the spread of this virus and its potential risk to watermelon in Florida and other regions of the U.S.

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First Report of Tomato spotted wilt virus in Blackberry Lily in North America

Plant Disease ◽

10.1094/pdis.2003.87.1.102c ◽

2003 ◽

Vol 87 (1) ◽

pp. 102-102 ◽

Cited By ~ 3

Author(s):

S. Adkins ◽

L. Breman ◽

C. A. Baker ◽

S. Wilson

Keyword(s):

North America ◽

Tomato Spotted Wilt Virus ◽

Amino Acid Sequences ◽

South Florida ◽

N Gene ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

First Report ◽

Tomato Spotted Wilt ◽

Wilt Virus

Blackberry lily (Belamcanda chinensis (L.) DC.) is an herbaceous perennial in the Iridaceae characterized by purple-spotted orange flowers followed by persistent clusters of black fruit. In July 2002, virus-like symptoms including chlorotic ringspots and ring patterns were observed on blackberry lily leaves on 2 of 10 plants in a south Florida ornamental demonstration garden. Inclusion body morphology suggested the presence of a Tospovirus. Tomato spotted wilt virus (TSWV) was specifically identified by serological testing using enzyme-linked immunosorbent assay (Agdia, Elkhart, IN). Sequence analysis of a nucleocapsid (N) protein gene fragment amplified by reverse transcription-polymerase chain reaction (RT-PCR) with primers TSWV723 and TSWV722 (1) from total RNA confirmed the diagnosis. Nucleotide and deduced amino acid sequences of a 579 base pair region of the RT-PCR product were 95 to 99% and 95 to 100% identical, respectively, to TSWV N-gene sequences in GenBank. Since these 2-year-old plants were grown on-site from seed, they were likely inoculated by thrips from a nearby source. Together with a previous observation of TSWV in north Florida nursery stock (L. Breman, unpublished), this represents, to our knowledge, the first report of TSWV infection of blackberry lily in North America although TSWV was observed in plants of this species in Japan 25 years ago (2). References: (1) S. Adkins, and E. N. Rosskopf. Plant Dis. 86:1310, 2002. (2) T. Yamamoto and K.-I. Ohata. Bull. Shikoku Agric. Exp. Stn. 30:39, 1977.

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First Report of Cucurbit aphid-borne yellows virus in Iran Causing Yellows on Four Cucurbit Crops

Plant Disease ◽

10.1094/pd-90-0526a ◽

2006 ◽

Vol 90 (4) ◽

pp. 526-526 ◽

Cited By ~ 7

Author(s):

K. Bananej ◽

C. Desbiez ◽

C. Wipf-Scheibel ◽

I. Vahdat ◽

A. Kheyr-Pour ◽

...

Keyword(s):

Citrullus Lanatus ◽

Healthy Plant ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

First Report ◽

Chain Reactions ◽

Reference Isolate ◽

Sigma Chemical ◽

International Mycological Institute ◽

Das Elisa

A survey was conducted from 2001 to 2004 in the major cucurbit-growing areas in Iran to reassess the relative incidence of cucurbit viruses. Severe yellowing symptoms were observed frequently on older leaves of cucurbit plants in various regions in outdoor crops, suggesting the presence of Cucurbit aphid-borne yellows virus (CABYV, genus Polerovirus, family Luteoviridae) (1,2). Leaf samples (n = 1019) were collected from plants of melon (Cucumis melo L.), cucumber (C. sativus L.), squash (Cucurbita sp.), and watermelon (Citrullus lanatus L.) showing various virus-like symptoms (mosaic, leaf deformation, yellowing). All samples, collected from 15 provinces, were screened for the presence of CABYV by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with IgGs and alkaline phosphatase-conjugated IgGs against a CABYV reference isolate (1). Of the 1,019 samples tested, 471 were positive for CABYV using DAS-ELISA. Some of the positive samples had typical severe yellowing symptoms while symptoms in other samples were masked by mosaic or leaf deformations caused by other viruses frequently found in mixed infections (data not shown). During the entire survey, CABYV was detected by DAS-ELISA in 201 of 503 melon samples, 72 of 129 cucumber samples, 158 of 249 squash samples, and 40 of 138 watermelon samples. These results indicate that CABYV is widely distributed on four cucurbit species in the major growing areas of Iran. In order to confirm CABYV identification, total RNA extracts (TRI-Reagent, Sigma Chemical, St Louis, MO) were obtained from 25 samples that were positive using DAS-ELISA originating from Khorasan (n = 4), Esfahan (n = 6), Teheran (n = 3), Hormozgan (n = 4), Azerbaiejan-E-Sharqi (n = 4), and Kerman (n = 4). Reverse transcription-polymerase chain reactions (RT-PCR) were carried out using forward (5′-CGCGTGGTTGTGG-TCAACCC-3′) and reverse (5′-CCYGCAACCGAGGAAGATCC-3′) primers designed in conserved regions of the coat protein gene according to the sequence of a CABYV reference isolate (3) and three other unpublished CABYV sequences. RT-PCR experiments yielded an expected 479-bp product similar to the fragment amplified with extracts from the reference isolate. No amplification of the product occurred from healthy plant extracts. To our knowledge, this is the first report of the occurrence of CABYV in Iran on various cucurbit species. The high frequency (46.2%) with which CABYV was detected in the samples assayed indicates that this virus is one of the most common virus infecting cucurbits in Iran. References: (1) H. Lecoq et al. Plant Pathol. 41:749, 1992 (2) M. A. Mayo and C. J. D'Arcy. Page 15 in: The Luteoviridae. H. G. Smith and H. Barker, eds. CAB International Mycological Institute, Wallingford, UK, 1999. (3) H. Guilley et al. Virology 202:1012, 1994.

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Detection and Characterization of Human Enteroviruses, Human Cosaviruses, and a New Human Parechovirus Type in Healthy Individuals in Osun State, Nigeria, 2016/2017

Viruses ◽

10.3390/v11111037 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1037 ◽

Cited By ~ 3

Author(s):

Folakemi Abiodun Osundare ◽

Oladele Oluyinka Opaleye ◽

Akeem Abiodun Akindele ◽

Samuel Adeyinka Adedokun ◽

Olusola Anuoluwapo Akanbi ◽

...

Keyword(s):

Healthy Individuals ◽

Coding Region ◽

Rt Pcr ◽

Human Parechovirus ◽

Full Genome Sequencing ◽

Three Samples ◽

Two Samples ◽

Stool Samples ◽

Pcr Assays ◽

Species Specific

Human enteroviruses and human parechoviruses are associated with a broad range of diseases and even severe and fatal conditions. For human cosaviruses, the etiological role is yet unknown. Little is known about the circulation of non-polio enteroviruses, human parechoviruses, and human cosaviruses in Nigeria. A total of 113 stool samples were collected from healthy individuals in Osun State between February 2016 and May 2017. RT-PCR assays targeting the 5′ non-coding region (5′ -NCR) were used to screen for human enteroviruses, human parechoviruses, and human cosaviruses. For human enteroviruses, species-specific RT-PCR assays targeting the VP1 regions were used for molecular typing. Inoculation was carried out on RD-A, CaCo-2, HEp-2C, and L20B cell lines to compare molecular and virological assays. Ten samples tested positive for enterovirus RNA with 11 strains detected, including CV-A13 (n = 3), E-18 (n = 2), CV-A20 (n = 1), CV-A24 (n = 1), EV-C99 (n = 1), and EV-C116 (n = 2). Three samples tested positive for human parechovirus RNA, and full genome sequencing on two samples allowed assignment to a new Parechovirus A type (HPeV-19). Thirty-three samples tested positive for cosavirus with assignment to species Cosavirus D and Cosavirus A based on the 5′-NCR region. Screening of stool samples collected from healthy individuals in Nigeria in 2016 and 2017 revealed a high diversity of circulating human enteroviruses, human parechoviruses, and human cosaviruses. Molecular assays for genotyping showed substantial benefits compared with those of cell-culture assays.

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Squash vein yellowing virus Identified in Watermelon (Citrullus lanatus) in Indiana

Plant Disease ◽

10.1094/pdis-91-8-1056b ◽

2007 ◽

Vol 91 (8) ◽

pp. 1056-1056 ◽

Cited By ~ 17

Author(s):

D. S. Egel ◽

S. Adkins

Keyword(s):

Citrullus Lanatus ◽

Amino Acid Sequences ◽

Rt Pcr ◽

Strain Vector ◽

First Report ◽

Pcr Product ◽

Field Samples ◽

Health Progress ◽

Vine Decline ◽

Yellowing Virus

During September 2006, moderate vine decline symptoms including vine collapse and wilt and root rot were observed on numerous watermelon plants growing in a commercial field in Sullivan County, Indiana. No symptoms were observed on the fruit. Six plants displaying typical vine decline symptoms were collected and assayed for potyvirus infection and subsequently for Squash vein yellowing virus (SqVYV) and Papaya ringspot virus type W (PRSV-W). SqVYV is a whitefly-transmitted member of the Potyviridae, recently shown to cause watermelon vine decline in Florida (1,4). Plants infected with SqVYV in Florida are also frequently infected with PRSV-W, although SqVYV is sufficient for watermelon vine decline. The six field samples harbored one or more potyviruses as determined by ELISA (Agdia, Elkhart, IN). Mechanical inoculation of squash (Cucurbita pepo) and watermelon with sap from three of the field samples induced mosaic symptoms in both that are typical of potyviruses. Vein yellowing in squash and plant death in watermelon typical of SqVYV (1) later developed in plants inoculated with one field sample. A coat protein gene fragment was amplified by reverse transcription (RT)-PCR with SqVYV primers (1) from total RNA of five of the six field samples and also from the symptomatic, inoculated plants. Nucleotide and deduced amino acid sequences of a 957-bp region of the RT-PCR product (primer sequences deleted prior to analysis) were 100% identical to SqVYV (GenBank accession No. DQ812125). PRSV-W also was identified in two of the five SqVYV-infected field samples by ELISA (Agdia) and by sequence analysis of a 3′ genome fragment amplified by RT-PCR with previously described degenerate potyvirus primers (3). No evidence for infection by other potyviruses was obtained. To our knowledge, this is the first report of SqVYV in Indiana and the first report of the virus anywhere outside of Florida. The whitefly (Bemisia tabaci, B strain) vector of SqVYV is relatively uncommon in Indiana and the cold winter temperatures make it unlikely that any SqVYV-infected watermelon vines or whiteflies will overseason, necessitating reintroductions of virus and vector each season. We feel that the moderate and restricted occurrence of SqVYV in Indiana observed in September 2006 should pose little or no threat to commercial watermelon production in Indiana and should not cause growers to alter their growing practices. The occurrence of SqVYV in Indiana does not appear to explain the similar symptoms of mature watermelon vine decline (MWVD) that has been observed in Indiana since the 1980s. In contrast with the insect vectored SqVYV, MWVD seems to be caused by a soilborne biological agent (2). References: (1) S. Adkins et al. Phytopathology 97:145, 2007. (2) D. S. Egel et al. Online publication. doi:10.1094/PHP-2000-1227-01-HN. Plant Health Progress, 2000. (3) A. Gibbs and A. Mackenzie. J. Virol. Methods 63:9, 1997. (4) P. Roberts et al. Citrus Veg. Mag. December 12, 2004.

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

Plant Disease ◽

10.1094/pdis-10-20-2254-pdn ◽

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.

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First Report of Cucurbit aphid-borne yellows virus in Tunisia Causing Yellows on Five Cucurbitacious Species

Plant Disease ◽

10.1094/pd-89-0776b ◽

2005 ◽

Vol 89 (7) ◽

pp. 776-776 ◽

Cited By ~ 18

Author(s):

M. Mnari Hattab ◽

J. Kummert ◽

S. Roussel ◽

K. Ezzaier ◽

A. Zouba ◽

...

Keyword(s):

Citrullus Lanatus ◽

Aphis Gossypii ◽

Pcr Amplification ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

Fresh Leaf ◽

First Report ◽

Reference Isolate ◽

Leaf Tissues ◽

Beet Pseudo Yellows Virus

Viruses, distributed worldwide on cucurbits, cause severe damage to crops. Virus surveys in 2003 and 2004 were made in all the major cucurbit-growing areas in Tunisia. Large populations of aphids (Aphis gossypii Glover) and severe yellowing symptoms of older leaves of cucurbits were observed in outdoor and under plastic-tunnel cultivation, suggesting the presence of Cucurbit aphid-borne yellows virus (CABYV, genus Polerovirus, family Luteoviridae). Leaf samples collected from symptomatic and asymptomatic plants of melon (Cucumis melo L.), cucumber (C. sativus L.), squash (Cucurbita pepo L.), watermelon (Citrullus lanatus L.), and ware cucurbit (Ecballium elaterium L. T. Richard) were screened for the presence of CABYV using enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). Reference isolate, CABYV-N (GenBank Accession No. X76931) was provided by H. Lecoq (INRA-Monfavet Cedex, France). Sample extracts from fresh leaf tissues were tested using ELISA with an antiserum prepared against this isolate. In addition, total RNA was extracted from fresh leaf tissues according to the technique of Celix et al. (2) using the Titan RT-PCR kit from Roche Diagnostics (Penzberg, Germany). Forward primer (5′-GAGGCGAAGGCGAAGAAATC-3′) and reverse primer (5′-TCTGGACCTGGCACTTGATG-3′) were designed with the available sequence of the reference isolate. ELISA tests demonstrated that 91 plants were positive among 160 plants tested with severe yellowing symptoms. All asymptomatic plants were negative. RT-PCR results yielded an expected 550-bp product that was amplified from the reference isolate. Of the 160 plants tested using ELISA, 106 plants were screened with RT-PCR including the 91 plants that were positive in ELISA. These 91 plants also were positive after RT-PCR amplification as were 12 more plants. This demonstrated that the RT-PCR test is more sensitive. No amplicons were produced from extracts of asymptomatic plants, RNA preparations of Cucurbit yellow stunting disorder virus (CYSDV), or Beet pseudo yellows virus (BPYV) positive controls provided by B. Falk (University of California, Davis). CYSDV and BPYV can induce similar yellowing symptoms in cucurbits. The results of the ELISA and RT-PCR tests showed that CABYV is widely distributed on five cucurbit species in the major growing areas of Tunisia including the northern, Sahel, central, and southern regions where it was detected, respectively, in 10 of 25, 11 of 21, 24 of 37, and 58 of 77 samples tested. CABYV was detected at the rates of 63 of 72 on melon, 10 of 21 on cucumber, 17 of 24 on squash, 10 of 25 on watermelon, and 3 of 18 on ware cucurbit. CABYV also seems to be widespread throughout the Mediterranean Basin (1,3,4), but to our knowledge, this is the first report of the occurrence of CABYV in Tunisia on different species of cucurbit and ware cucurbit. References: (1) Y. Abou-Jawdah et al. Crop Prot. 19:217, 2000. (2) A. Celix et al. Phytopathology 86:1370, 1996. (3) M. Juarez et al. Plant Dis. 88:907, 2004. (4) H. Lecoq et al. Plant Pathol. 41:749, 1992.

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First Report of Natural Infection of Zucchini by Tomato Chlorosis Virus and Cucurbit Chlorotic Yellows Virus in China

Plant Disease ◽

10.1094/pdis-05-20-0932-pdn ◽

2021 ◽

Author(s):

Xiaohui Sun ◽

Ning Qiao ◽

Xianping Zhang ◽

Lianyi Zang ◽

Dan Zhao ◽

...

Keyword(s):

Natural Infection ◽

Pcr Analysis ◽

Control Group ◽

Rt Pcr ◽

Degenerate Primers ◽

Specific Primers ◽

First Report ◽

Sequence Identity ◽

Tomato Chlorosis Virus ◽

Cucurbit Chlorotic Yellows Virus

Zucchini (Cucurbita pepo) is an extensively cultivated and important economic cucurbit crop in China. In September 2018 and 2019, interveinal chlorosis and yellowing symptoms, suspected to be caused by either tomato chlorosis virus (ToCV; genus Crinivirus) or cucurbit chlorotic yellows virus (CCYV; genus Crinivirus) or by their co-infection, were observed on zucchini plants in a greenhouse in Shandong Province, China. The incidence of the disease in the greenhouse was 20–30%. To identify the causal agent(s) of the disease, leaf samples from 66 zucchini plants were collected in 14 greenhouses in the cities of Shouguang (n = 12), Dezhou (n = 36), Qingzhou (n = 12), and Zibo (n = 6) in Shandong. Four whitefly (Bemisia tabaci) samples and four symptomatic tomato samples were also collected from these sampling sites (one each for each site) because numerous whiteflies were observed in the sampling greenhouses and ToCV was previously reported in greenhouse tomato plants from these regions (Zhao et al. 2014). To determine whether the symptoms were associated with Crinivirus infection, reverse transcription polymerase chain reaction (RT-PCR) using Crinivirus-specific degenerate primers (CriniRdRp251F/CriniRdRp995R) (Wintermantel and Hladky 2010) was performed first on total RNA extracted using the TRIzol protocol (Jordon-Thaden et al. 2015). Thereafter, the RNA samples were subjected to RT-PCR with ToCV- or CCYV-specific primers (Sun et al. 2016; Gan et al. 2019). Of the 66 zucchini samples, 54 tested positive by the degenerate crinivirus primer pair; and among them, 10 tested positive for ToCV only, 40 positive for CCYV only, and 4 positive for both viruses. Interestingly, while both viruses were detected in all B. tabaci samples, only ToCV was detected in the tomato samples (n = 4). To confirm the identity of the viruses, the amplicons of ToCV (four samples each of tomato, B. tabaci and zucchini) and CCYV (four samples each of B. tabaci and zucchini) were Sanger sequenced (Tsingke Biotechnology Co., Ltd., Beijing, China) after cloning into pMD18-T vectors (Takara, Shiga, Japan). BLASTn analysis demonstrated that all sequences were identical to their respective amplicons. The ToCV sequences (GenBank accession numbers: tomato, MN944406; B. tabaci, MN944404; zucchini, MN944405) shared 100% sequence identity with isolates from Beijing (KT751008, KC887999, KR184675, and KP335046), Hebei (KP217196), and Shandong (KX900412). The CCYV sequence (GenBank accession number MT396249) shared 99.9% sequence identity with isolates China (JN126046, JQ904629, KP896506, KX118632, KY400633, and MK568545), Greece (LT716000, LT716001, LT716002, LT716005, and LT716006), and Cyprus (LT992909, LT992910, and LT992911). To assess the transmissibility of ToCV and CCYV, virus-free B. tabaci (n = 30) were placed in ToCV or CCYV-infected zucchini plants for one day for virus acquisition. Thereafter, the whiteflies were transferred into virus-free zucchini seedlings (cv. ‘Zaoqingyidai’, 4-leaf-stage, n = 6 for each of the control, ToCV and CCYV treatment) for one day. Three weeks after inoculation, all plants that were inoculated with either ToCV or CCYV displayed same symptoms as those observed in the greenhouses, whereas plants in the control group remained symptom free. RT-PCR analysis using ToCV- and CCYV-specific primers confirmed the infection of the plants with the respective virus, whereas control plants were free from the viruses. CCYV has been previously reported on zucchini in Algeria (Kheireddine et al. 2020), Iran (LR585225), and Cyprus (LT992910). To our knowledge, this is the first report of CCYV infection in zucchini in China, and moreover the first report of ToCV infection in zucchini in the world. Clearly, stringent management is needed to minimize the losses caused by these viruses in greenhouse operations in the region.

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