Detection of Cucurbit leaf crumple virus in Florida Cucurbits

In October of 2006, yellow straightneck and zucchini squash plants (Cucurbita pepo L.) with crumpled, curled, thickened leaves were found in St. Johns and Marion counties in central Florida, respectively. Both locations had high populations of the whitefly, Bemisia tabaci. Incidences of symptomatic plants were greater than 95% in three squash fields (33 ha total) in St. Johns County and 35% in an experimental plot in Marion County. Twenty-three samples were collected from symptomatic plants (two from St. Johns County and 21 from Marion County). DNA was extracted for PCR and tested for the presence of begomoviruses using the following pairs of degenerate primers: AC1048/AV494, which amplifies a conserved region of the coat protein gene (2), PAR1c496/PAL1v1978, which amplifies a region of the begomovirus A component, and PBL1v2040/PCRc154, which amplifies a hypervariable region of the begomovirus B component (1). All squash samples yielded amplicons of sizes expected for a bipartite begomovirus: 1,159 nt with PAR1c496/PAL1v1978, 550 nt with AC1048/AV494, and 493 nt with PBL1v2040/PCRc154. The 1,159- and 493-nt amplicons obtained from two squash plants were cloned and sequenced. The 1,159 nt sequences from both plants shared 98% sequence identity with each other and 97% identity with equivalent regions of the A component of Cucurbit leaf crumple virus (CuLCrV) from Arizona and California (GenBank Accession Nos. AF256200 and AF224760, respectively). The 493-nt sequences amplified with PBL1v2040/PCRc154 were identical and shared a 96% identity with CuLCrV sequence (GenBank Accession No. AF327559) from Arizona and 97% identity with CuLCrV B component sequence (GenBank Accession No. AF224761) from California. Leaves were collected from eight symptomatic squash plants from Citra, FL and used for whitefly transmission assays. Approximately 100 adults of Bemisia tabaci biotype B were released onto each caged leaf and given a 24-h acquisition access period, after which a healthy squash seedling was introduced. Symptoms developed within 10 days on all test plants, and the presence of CuLCrV was confirmed by PCR assays, (primer pairs PAR1c496/PAL1v1978 and PBL1v2040/PCRc154) followed by sequencing. In 2007, similar symptoms were seen in several locations around the state. The same assays confirmed the presence of CuLCrV in watermelon (Citrullus lanatus L.) and squash in the following counties: Collier and Hendry in southwest Florida and Hillsborough, Manatee, and Sarasota in west-central Florida. To our knowledge, this is the first report of CuLCrV, and the first report of any begomovirus in cucurbits in Florida. References: (1) M. R. Rojas et al. Plant Dis. 77:340, 1993. (2) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.

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Differential transcriptional activity of plant-pathogenic begomoviruses in their whitefly vector (Bemisia tabaci, Gennadius: Hemiptera Aleyrodidae)

Journal of General Virology ◽

10.1099/vir.0.80665-0 ◽

2005 ◽

Vol 86 (5) ◽

pp. 1525-1532 ◽

Cited By ~ 81

Author(s):

Xiomara H. Sinisterra ◽

C. L. McKenzie ◽

Wayne B. Hunter ◽

Charles A. Powell ◽

Robert G. Shatters

Keyword(s):

Bemisia Tabaci ◽

Biological Effects ◽

Transcript Abundance ◽

Genome Replication ◽

Bipartite Begomovirus ◽

New Synthesis ◽

Gene Transcripts ◽

The Difference ◽

Biotype B ◽

Whitefly Vector

Plant-pathogenic begomoviruses have a complex association with their whitefly vector and aspects concerning virus genetic activity (genome replication and gene transcription) within the insect remain highly controversial. Virus transcript abundance was assessed by quantifying selected gene transcripts of Tomato mottle virus (ToMoV, a New World bipartite begomovirus) and Tomato yellow leaf curl virus (TYLCV, an Old World monopartite begomovirus) in whiteflies (Bemisia tabaci biotype B) after feeding on virus-infected tomato plants and after subsequent transfer to cotton, a plant that is immune to the selected begomoviruses. Real-time RT-PCR was performed using specific primers for three ToMoV genes (AV1, BC1 and BV1) and three TYLCV genes (V1, V2 and C3). The ToMoV gene transcripts rapidly became undetectable in whiteflies following transfer from tomato to cotton, probably because degradation was not accompanied by new synthesis. On the other hand, TYLCV transcripts increased after transfer of whiteflies to cotton, indicating active TYLCV transcription. Interestingly, the difference observed in ToMoV and TYLCV transcripts in the vector parallel observations on the different biological effects of these viruses on whiteflies, i.e. TYLCV, but not ToMoV, reduces whitefly fitness.

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Occurrence of Eretmocerus mundus Mercet (Hymenoptera: Aphelinidae) parasitizing Bemisia tabaci (Genn.) biotype B (Hemiptera: Aleyrodidae) in Brazil

Bragantia ◽

10.1590/brag.2014.025 ◽

2014 ◽

Vol 73 (2) ◽

pp. 160-162 ◽

Cited By ~ 2

Author(s):

André Luiz Lourenção ◽

Valmir Antonio Costa ◽

Lillian Silveira Pereira ◽

Juliana Cardoso Prado

Keyword(s):

Bemisia Tabaci ◽

Sao Paulo ◽

First Report ◽

Paulo State ◽

Experimental Station ◽

Eretmocerus Mundus ◽

Cotton Plants ◽

Cotton Genotypes ◽

São Paulo State ◽

Biotype B

The parasitism of Bemisia tabaci (Genn.) biotype B nymphs on cotton plants was observed during a research on resistance of cotton genotypes to this whitefly. The experiment was set in a greenhouse at the Experimental Station of the Instituto Agronômico (IAC), in Campinas, São Paulo State, Brazil. Samples of the parasitized nymphs were collected and maintained in laboratory to monitor the parasitism and obtain the adult parasitoids. A total of 129 adult parasitoids were obtained, including one Encarsia inaron (Walker), 13 En. lutea (Masi), and 115 Eretmocerus mundus Mercet (Hymenoptera: Aphelinidae). This is the first report of Er. mundus in Brazil.

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Recovery from Cucurbit leaf crumple virus (Family Geminiviridae, Genus Begomovirus) Infection Is an Adaptive Antiviral Response Associated with Changes in Viral Small RNAs

Phytopathology ◽

10.1094/phyto-98-9-1029 ◽

2008 ◽

Vol 98 (9) ◽

pp. 1029-1037 ◽

Cited By ~ 37

Author(s):

C. Hagen ◽

M. R. Rojas ◽

T. Kon ◽

R. L. Gilbertson

Keyword(s):

Small Rnas ◽

Defense Mechanism ◽

Enhanced Recovery ◽

Rna Virus ◽

Citrullus Lanatus ◽

Severe Disease ◽

Bipartite Begomovirus ◽

Viral Dna ◽

Virus Family ◽

Cucurbit Leaf Crumple Virus

A strong recovery response occurs in cantaloupe (Cucumis melo) and watermelon (Citrullus lanatus) infected with the bipartite begomovirus Cucurbit leaf crumple virus (CuLCrV). This response is characterized by initially severe symptoms, which gradually become attenuated (almost symptomless). An inverse relationship was detected between viral DNA levels and recovery, indicating that recovered tissues had reduced viral titers. Recovered tissues also were resistant to reinfection with CuLCrV; i.e., recovered leaves reinoculated with the virus did not develop symptoms or have an increased level of viral DNA. In contrast, infection of CuLCrV-recovered leaves with the RNA virus, Cucumber mosaic virus (CMV), disrupted recovery, resulting in the development of severe disease symptoms (more severe than those induced by CMV or CuLCrV alone) and increased CuLCrV DNA levels. Small RNAs with homology to CuLCrV DNA were detected in recovered and nonrecovered tissues; as well as in phloem exudates from infected, but not uninfected plants. Levels of these small RNAs were positively correlated with viral titer; thus, recovered tissues had lower levels than symptomatic tissues. In addition, viral DNA from a host that undergoes strong recovery (watermelon) was more highly methylated compared with that from a host that undergoes limited recovery (zucchini). Furthermore, inoculation of CuLCrV-infected zucchini with a construct expressing an inverted repeat of the CuLCrV common region enhanced recovery and reduced viral symptoms and viral DNA levels in newly emerged leaves. Taken together, these results suggest that recovery from CuLCrV infection is an adaptive antiviral defense mechanism, most likely mediated by gene silencing.

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Cucurbit leaf crumple virus Identified in Common Bean in Florida

Plant Disease ◽

10.1094/pdis-93-3-0320b ◽

2009 ◽

Vol 93 (3) ◽

pp. 320-320 ◽

Cited By ~ 10

Author(s):

S. Adkins ◽

J. E. Polston ◽

W. W. Turechek

Keyword(s):

Common Bean ◽

Blot Hybridization ◽

Cdna Probe ◽

Hypervariable Region ◽

Rt Pcr ◽

Dot Blot ◽

Fresh Market ◽

Degenerate Primers ◽

Pcr Products ◽

Cucurbit Leaf Crumple Virus

Virus-like symptoms of leaf deformation and rugosity, especially of younger leaves, and a mild mosaic were observed on fresh market common (green) bean (Phaseolus vulgaris L.) plants in Hendry County in southwest Florida in December of 2007 and again in February of 2008. All bean fields were adjacent to watermelon fields in which Cucurbit leaf crumple virus (CuLCrV), Squash vein yellowing virus (SqVYV), and Papaya ringspot virus type W (PRSV-W) infections had previously been confirmed (fall of 2007) by PCR, reverse transcription (RT)-PCR, and/or ELISA. Whiteflies, Bemisia tabaci, were observed on both bean and watermelon plants in December and February. Fifteen samples (eleven with symptoms) were collected in December and two (both with symptoms) in February. Initial ELISA assays using commercially available antisera for potyviruses or Cucumber mosaic virus (Agdia, Elkhart, IN) were negative. Total nucleic acids were extracted and used for PCR testing. All samples tested negative by RT-PCR using specific primers for SqVYV, PRSV-W, and Cucurbit yellow stunting disorder virus, and degenerate primers for potyviruses. Ten of fifteen December samples (ten of eleven symptomatic samples) and both February samples yielded PCR products of the expected size with the degenerate begomovirus primers, PAR1c496/PAL1v1978, which amplify a portion of the begomovirus A component (3). PCR products from three December and both February samples were cloned and sequenced. The 1,159-nt PCR products shared 99% identity with each other and 96% identity with the corresponding region of A component sequences of Arizona and California CuLCrV isolates (GenBank Accession Nos. AF256200 and AF224760, respectively). Additional degenerate begomovirus primers PBL1v2040/PCRc154, which amplify a 381-nt portion of the hypervariable region of the begomovirus B component (3), and AC1048/AV494, which amplify a 533-nt portion of a conserved region of the coat protein gene (4), were used to confirm the identity of CuLCrV in the three December samples. The PBL1v2040/PCRc154 PCR products shared 98 to 99% identity with each other and 94 to 95% identity with the corresponding region of B component sequences of Arizona and California CuLCrV isolates (GenBank Accession Nos. AF327559 and AF224761, respectively), whereas the AC1048/AV494 PCR products shared 99% identity with each other and 97% identity with the corresponding region of A component sequences of Arizona and California CuLCrV isolates. Nucleic acid dot-blot hybridization assays of sap from homogenized leaves of the three December samples (from which the PCR product clones were obtained) with a digoxigenin-labeled CuLCrV cDNA probe also confirmed the presence of CuLCrV. Although CuLCrV has been reported to experimentally infect common bean and tobacco (2), to our knowledge, this is the first report of CuLCrV infecting any noncucurbit host in Florida. This finding suggests that CuLCrV may be more widely distributed than previously known in Florida (1) and that common bean (and potentially other legumes) are potential reservoirs for CuLCrV. References: (1) F. Akad et al. Plant Dis. 92:648, 2008. (2) J. K. Brown et al. Phytopathology 92:734, 2002. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993. (4) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.

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Low Frequency of Horizontal and Vertical Transmission of Cucurbit Leaf Crumple Virus in Whitefly Bemisia tabaci Gennadius

Phytopathology ◽

10.1094/phyto-09-19-0337-r ◽

2020 ◽

Vol 110 (6) ◽

pp. 1235-1241 ◽

Cited By ~ 2

Author(s):

Kiran R. Gadhave ◽

Saurabh Gautam ◽

Bhabesh Dutta ◽

Tim Coolong ◽

Scott Adkins ◽

...

Keyword(s):

Bemisia Tabaci ◽

Low Frequency ◽

Transovarial Transmission ◽

Bipartite Begomovirus ◽

Time Points ◽

Virus Accumulation ◽

Modes Of Transmission ◽

Copy Numbers ◽

Viral Copy ◽

Cucurbit Leaf Crumple Virus

Cucurbit leaf crumple virus (CuLCrV), a bipartite begomovirus, is transmitted by whiteflies in a persistent and circulative manner. Like other begomoviruses, CuLCrV transmission via feeding is well understood; however, whether and how CuLCrV is transmitted by horizontal and vertical modes in its vector, Bemisia tabaci, remains unexplored. We studied transovarial and mating transmission of CuLCrV, and comparatively analyzed virus accumulation in whiteflies through feeding and nonfeeding modes. Furthermore, we quantified CuLCrV DNA A accumulation at different time points to determine whether this virus propagates in whiteflies. CuLCrV DNA A was transmitted vertically and horizontally by B. tabaci, with low frequency in each case. Transovarial transmission of CuLCrV DNA A was only 3.93% in nymphs and 3.09% in adults. Similarly, only a single viruliferous male was able to transmit CuLCrV DNA A to its nonviruliferous female counterparts via mating. In contrast, viruliferous females were unable to transmit CuLCrV DNA A to nonviruliferous males. Additionally, the recipient adults that presumably acquired CuLCrV transovarially and via mating were not able to transmit the virus to squash plants. We further report that the CuLCrV DNA A viral copy numbers were significantly lower in nonfeeding modes of transmission than in feeding ones. The viral copy numbers significantly decreased at succeeding time points throughout adulthood, suggesting no CuLCrV propagation in B. tabaci. Altogether, the low frequency of nonfeeding transmission, reduced virus accumulation in whiteflies, and absence of plant infectivity through nonfeeding transmission suggest that transovarial and mating CuLCrV transmission might not substantially contribute to CuLCrV epidemics.

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First Report of Tomato yellow leaf curl virus in Louisiana

Plant Disease ◽

10.1094/pdis.2001.85.2.230d ◽

2001 ◽

Vol 85 (2) ◽

pp. 230-230 ◽

Cited By ~ 11

Author(s):

R. A. Valverde ◽

P. Lotrakul ◽

A. D. Landry ◽

J. E. Boudreaux

Keyword(s):

The United States ◽

Tomato Yellow Leaf ◽

Yield Reduction ◽

Yellow Leaf ◽

Degenerate Primers ◽

Tomato Plants ◽

First Report ◽

Biotype B ◽

Leaf Curl Virus ◽

Leaf Curl

Tomato yellow leaf curl virus (TYLCV) is a begomovirus (Geminiviridae) that causes a serious disease of tomato throughout the world. In 1997, the strain from Israel of TYLCV (TYLCV-IS) was found infecting tomatoes in Florida for the first time in the United States (1). During late spring of 2000, approximately 90% of the tomato plants (Lycopersicon esculentum) in a farm near New Orleans exhibited severe stunting, leaf cupping, and chlorosis. Symptoms were similar to those caused by TYLCV. Whiteflies (Bemisia tabaci biotype B) were present in the field but in relatively low numbers. The effect on yield reduction varied from negligible (late infections) to 100% (early infections). Six selected plants showing symptoms were assayed by polymerase chain reaction (PCR) using begomovirus-specific primers. Capsicum frutescens infected with an isolate of Texas pepper virus from Costa Rica was used as positive control. DNA was extracted using Plant DNAzol Reagent (GIBCO BRL). PCR was conducted using degenerate primers AV494/AC1048 that amplify the core coat protein region of most begomoviruses (2). PCR yielded a DNA fragment of approximately 550 bp, suggesting that a begomovirus was associated with the disease. The amplified DNA of one field isolate was cloned and the nucleotide (nt) sequence determined. Sequence comparisons with other begomoviruses in the GenBank Database indicated that the Louisiana isolate shared 100% nt identity with TYLCV-IS (GenBank Accession X76319). Successful transmission (100%) to Bonny Best tomato were obtained with four groups of 10 whiteflies each (B. tabaci biotype B) that fed on TYLCV-IS infected tomato plants. Acquisition and transmission feedings were for 2 days. In all cases, the virus was diagnosed by the ability to reproduce typical TYLCV-like symptoms in tomato and PCR. The virus was also successfully graft-transmitted to tomato cv. Bonny Best, Nicotiana benthamiana, and tomatillo (Physalis ixocarpa) using scions from tomato plants infected with a whitefly transmitted virus isolate. This is the first report of TYLCV-IS in Louisiana. References: (1) J. E. Polston et al. Plant Dis. 83:984–988, 1999. (2) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288–1293, 1996.

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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

Plant Disease ◽

10.1094/pdis-06-21-1141-pdn ◽

2021 ◽

Author(s):

Katherine Hendricks ◽

Regina Nicole Hernandez ◽

Pamela Roberts ◽

Thomas Isakeit ◽

Olufemi Joseph Alabi

Keyword(s):

Citrullus Lanatus ◽

South Florida ◽

Rt Pcr ◽

First Report ◽

Three Samples ◽

One Step ◽

Complete Coat Protein ◽

Species Specific ◽

Cucurbit Leaf Crumple Virus ◽

Cucurbit Chlorotic Yellows Virus

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 Watermelon chlorotic stunt virus Infecting Watermelon in Saudi Arabia

Plant Disease ◽

10.1094/pdis-06-14-0583-pdn ◽

2014 ◽

Vol 98 (10) ◽

pp. 1451-1451 ◽

Cited By ~ 5

Author(s):

M. A. Al-Saleh ◽

M. H. Ahmad ◽

I. M. Al-Shahwan ◽

J. K. Brown ◽

A. M. Idris

Keyword(s):

Saudi Arabia ◽

Nucleic Acids ◽

Citrullus Lanatus ◽

Rolling Circle Amplification ◽

Bipartite Begomovirus ◽

High Sequence Identity ◽

Stem Loop ◽

First Report ◽

Sequence Identity ◽

Watermelon Chlorotic Stunt Virus

In the Saudi Arabian deserts, watermelon [Citrullus lanatus (Thunb.)] is cultivated in the lowlands and wadis (washes) where water accumulates following rainfall, and where heat, salt, and drought stress are common constraints on production. During the spring of 2014, watermelon leaves exhibited yellowing and severe chlorotic mottling symptoms. The foliar symptoms were reminiscent of Watermelon chlorotic stunt virus (WmCSV), a bipartite begomovirus previously reported in several neighboring countries (1,3). Ten samples were collected from three farms in the Leith region, where 100% of the watermelon plants were symptomatic. Total nucleic acids were extracted from the symptomatic watermelon plants and were subjected to PCR using WmCSV DNA-A specific primers designed based on a complete genome sequence (GenBank Accession No. AJ012081), WmCSVF-3′-CGTGCTGTTGCCCCCACTGT-5′ and WmCSVR-3′-CCTGCATATCTCGTGCCAGAATC-5′ to obtain an expected size fragment of 1,111 bp located between the nucleotide (nt) coordinates 400-1510. The amplicons (one per sample) were cloned, and the DNA sequence was determined for each and used to search the NCBI database. The top hits for sequences obtained from all 10 samples were to WmSCV sequences, with shared nt identity values of 97 to 98%. To clone the full-length begomoviral DNA-A and DNA-B components, nucleic acids were subjected to rolling circle amplification (RCA) (2). The RCA products were cloned into the pGEM7 plasmid vector using the unique restriction sites, identified as HindIII for DNA-A, and as EcoRI for DNA-B, respectively. Ten DNA-A clones and two DNA-B component clones were sequenced. The DNA-A components ranged in size from 2,751 (KM066100) to 2,752 bp (KJ939448), whereas the DNA-B components were 2,757 bp in size (KJ939447). Analysis of the viral sequences from the DNA-A clones indicated they had the characteristics of a typical genome of a begomovirus DNA-A component that consist of a hairpin stem-loop structure in the intergenic region, two tandem copies of the iteron (TGGAGAC) located between the nt coordinates 2675 and 2680, 2682 and 2688 predicted to be involved in Rep binding, and six predicted genes encoding proteins with high sequence identity to those encoded by other WmCSV isolates. The 10 DNA-A component sequences were aligned with sequences for previously described WmCSV isolates available in GenBank using Muscle, followed by pairwise comparisons using SDT software (4). The analysis revealed that the cloned DNA-A components shared 99 to 100% nt sequence identity with each other, and 97 to 98% nt identity with WmCSV isolates reported from Yemen (AJ012081), Jordan (EU561237), Iran (AJ245652), and Sudan (AJ245650). Further, the WmCSV DNA-B from Saudi Arabia shared the highest nt identity with sequences from Yemen (AJ012082) at 96%, Iran (AJ245653) at 95%, and only 94% with DNA-B from both Sudan (AJ245651) and Jordan (EU561236). To our knowledge, this is the first report of WmCSV in Saudi Arabia. WmCSV poses a serious threat to the production of this highly valued crop in Saudi Arabia and considerably reduces crop yield (1). References: (1) I. D. Bedford et al. Eur. J. Plant Pathol. 100:243, 1994. (2) A. Idris et al. Plant Dis. 97:910, 2007. (3) A. Kheyr-Pour et al. Phytopathology 90:629, 2000. (4) B. Muhire et al. Arch. Virol. 158:1411, 2013.

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

Plant Disease ◽

10.1094/pdis-07-10-0550 ◽

2011 ◽

Vol 95 (1) ◽

pp. 73-73 ◽

Cited By ~ 18

Author(s):

Q. S. Gu ◽

Y. H. Liu ◽

Y. H. Wang ◽

W. G. Huangfu ◽

H. F. Gu ◽

...

Keyword(s):

Bemisia Tabaci ◽

Disease Incidence ◽

Pcr Primers ◽

Degenerate Primers ◽

First Report ◽

Representative Sequence ◽

Pcr Product ◽

Pcr Products ◽

Symptomatic Leaf ◽

Cucurbit Chlorotic Yellows Virus

Systemic foliar chlorosis of melon, watermelon, and cucumber plants grown in plastichouses was first observed in Shanghai, China in 2008. By the end of October 2009, this symptom had become prevalent across 13,000 ha of plastichouses in Shanghai, Ningbo in Zhejiang Province, and Shouguang in Shandong Province. By mid-October, disease incidence ranged from 50 to 100% and losses were estimated between 10 and 20% across 100 plastichouses. Initial disease symptoms were chlorosis beginning at the base and middle portion of the older leaves followed by development of chlorotic spots on the lamina. Within 4 to 5 days, the entire leaf lamina was bright yellow and the veins remained green. The whitefly, Bemisia tabaci, was frequently observed colonizing plants in all plastichouses included in this study. Leaf samples were collected from six symptomatic cucumber, melon, and watermelon plants from individual plastichouses in Shanghai, Ningbo, and Shouguang. A pair of degenerate primers, F-5′-GGN TTA GAN TTC GGN ACN AC-3′ and R-5′-TCA AAN GTN CCN CCN CCN AA-3′, that were specific for the genera Crinivirus and Closterovirus, family Closteroviridae (2) were used to amplify a 636-bp fragment of the viral heat shock protein 70 gene (Hsp70). A PCR product of the expected size was amplified from RNA extracted with TaKaRa RNAiso Reagent (TaKaRa, Dalian, China) from symptomatic leaf samples: 3 of 3 melon, 2 of 2 watermelon, and 1 of 1 cucumber, and from 5 of 5 Bemisia tabaci adults collected from plants in five plastichouses in Shanghai, Ningbo, and Shouguang. No PCR product was obtained from RNA extracted from cucumber leaves grown in a virus-free facility at the Fruit Research Institute, Zhengzhou. PCR products were sequenced from representative plants samples and the sequences were submitted to GenBank (Nos. GU721105 to GU721107, GU72118 to GU721110, and GU721111. The six Hsp70 sequences shared 99.8 to 100% identity with Cucurbit chlorotic yellows virus (CCYV) (GenBank No. AB523789) from Japan. Using the complete CCYV sequence (1), PCR primers were designed to amplify the complete CCYV coat protein (Cp): Cp F-5′-CGCAATCAATAAGGCGGCGACC-3′ and Cp R-5′-ACTACAACCTCCCGGTGCCAACT-3′ (804 bp), minor Cp (Cpm): Cpm-F-5′-TGATGAANTGCCANGCTNTGAAA-3′ and Cpm-R5′-ACAANTGATTCACATTNACAAT-3′ (1,632 bp); and Hsp70: Hsp F-5′-TGCAACCGATGTCAGGTTCAGCG-3′ with Hsp R-5′-TGGATAATTGGTCACGACCTCCAGT-3′ (1,947 bp). One PCR amplicon was obtained for each target gene using RNA extracted from a cucumber collected in Ningbo. Three of the PCR amplicons were cloned and the DNA sequence was determined. A representative sequence for each gene was deposited in GenBank as: cp (HM581658), cpm (HM581657), and hsp70 (HM581659). The cp, cpm, and hsp70 sequences shared 99.7, 99.9, and 99.9% nt identity with the respective genes of CCYV (AB523789), whereas they shared only 62.5, 49.9, and 69.6% identity with the respective gene sequences for Cucurbit yellow stunting disorder virus (CYSDV; NC004810), suggesting the two viruses are divergent crinivirus species. Although this virus was first reported to infect cucurbits in Japan in 2009 (1), to our knowledge, this is the first report of CCYV in China. Eradication and management efforts are therefore paramount to reducing the spread of the disease. References: (1) M. Okuda et al. Phytopathology 100:560, 2010. (2) T. Tian et al. Phytopathology 86:1167, 1996.

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