Squash Leaf Curl China Virus Research Articles

Development and application of triple antibody sandwich enzyme-linked immunosorbent assays for begomovirus detection using monoclonal antibodies against Tomato yellow leaf curl Thailand virus

BackgroundTomato yellow leaf curl Thailand virus, TYLCTHV, is a begomovirus that causes severe losses of tomato crops in Thailand as well as several countries in Southeast and East Asia. The development of monoclonal antibodies (MAbs) and serological methods for detecting TYLCTHV is essential for epidemiological studies and screening for virus-resistant cultivars.MethodsThe recombinant coat protein (CP) of TYLCTHV was expressed in Escherichia coli and used to generate MAbs against TYLCTHV through hybridoma technology. The MAbs were characterized and optimized to develop triple antibody sandwich enzyme-linked immunosorbent assays (TAS-ELISAs) for begomovirus detection. The efficiency of TAS-ELISAs for begomovirus detection was evaluated with tomato, pepper, eggplant, okra and cucurbit plants collected from several provinces in Thailand. Molecular identification of begomoviruses in these samples was also performed through PCR and DNA sequence analysis of the CP gene.ResultsTwo MAbs (M1 and D2) were generated and used to develop TAS-ELISAs for begomovirus detection. The results of begomovirus detection in 147 field samples indicated that MAb M1 reacted with 2 begomovirus species, TYLCTHV and Tobacco leaf curl Yunnan virus (TbLCYnV), whereas MAb D2 reacted with 4 begomovirus species, TYLCTHV, TbLCYnV, Tomato leaf curl New Delhi virus (ToLCNDV) and Squash leaf curl China virus (SLCCNV). Phylogenetic analyses of CP amino acid sequences from these begomoviruses revealed that the CP sequences of begomoviruses recognized by the narrow-spectrum MAb M1 were highly conserved, sharing 93% identity with each other but only 72–81% identity with MAb M1-negative begomoviruses. The CP sequences of begomoviruses recognized by the broad-spectrum MAb D2 demonstrated a wider range of amino acid sequence identity, sharing 78–96% identity with each other and 72–91% identity with those that were not detected by MAb D2.ConclusionsTAS-ELISAs using the narrow-specificity MAb M1 proved highly efficient for the detection of TYLCTHV and TbLCYnV, whereas TAS-ELISAs using the broad-specificity MAb D2 were highly efficient for the detection of TYLCTHV, TbLCYnV, ToLCNDV and SLCCNV. Both newly developed assays allow for sensitive, inexpensive, high-throughput detection of begomoviruses in field plant samples, as well as screening for virus-resistant cultivars.

The occurrence and distribution of major viruses infecting cucurbits in Tamil Nadu state, India

Field surveys were conducted during 2012–14 to document the occurrence and distribution of viruses infecting cucurbits in all the seven agroclimatic zones of Tamil Nadu state, south India. Samples collected from various types of cucurbits showing virus-like symptoms were tested for Cucumber mosaic virus (CMV), Papaya ringspot virus (PRSV), Zucchini yellow mosaic virus (ZYMV), Cucumber green mottle mosaic virus (CGMMV) and begomoviruses using serological (Dot Immuno Binding Assay, DIBA) and molecular (RT-PCR for RNA viruses and PCR for DNA viruses) methods. Results indicated higher incidence (98.6%) of begomoviruses, followed by PRSV (32.1%) and CGMMV (22.2%). ZYMV and CMV were detected at a lower frequency of 7.1% and 5%, respectively. Mixed infections with two or three of these viruses were observed in more than 50% of the samples analyzed. Sequencing of begomovirus-specific amplicons indicated the presence of sequences highly similar to Tomato leaf curl New Delhi virus (ToLCNDV) and Squash leaf curl China virus (SLCCNV).

Laporan Pertama Infeksi Begomovirus pada Tanaman Mentimun di Bali

Leaf yellowing symptoms was commonly found in cucumber plants in Bali provinces, i.e. in Apuan and Bangli villages recently. Begomovirus infection is suspected as the causal agent, due to similar symptoms previously reported from cucumber plants in Java. In addition, Bemisia tabaci was observed in the field. The objective of this research was to identify the causal agent of leaf yellowing disease of cucumber in Bali. Virus detection and identification was conducted by polymerase chain reaction method using universal primers for Begomovirus , i.e. SPG1/SPG2. DNA fragment of 912 bp in size was successfully amplified from leaf samples. Analysis of nucleotide sequencing indicated that Begomovirus infecting cucumber plants in Bali has the highest homology (91%) with Squash leaf curl China virus (SLCCNV) isolate from Malaysia. This is the first report of SLCCNV infection in Bali.

New record of bipartite Squash leaf curl China virus (SLCCNV) and Croton yellow vein mosaic beta satellite associated with yellow vein disease of ash gourd in India

Benincasa hispida commonly known as winter melon or ash gourd is the only member of its genus belonging to the family Cucurbitaceae. It is one of the most commercially important vegetable crops cultivated in the Indian subcontinent,…

Squash leaf curl China virus. [Distribution map

Abstract A new distribution map is provided for Squash leaf curl China virus. Geminiviridae: Begomovirus. Hosts: Cucurbita spp., Cucumis spp. Information is given on the geographical distribution in Asia (China, Guangxi, Zhejiang, India, Tamil Nadu, Uttar Pradesh, Malaysia, Peninsular Malaysia, Pakistan, Philippines, Thailand, Vietnam).

First report on a variant of Squash leaf curl China virus (SLCCNV) infecting Benincasa hispida in India

Benincasa hispida (family Cucurbitaceae), known as winter melon, white gourd, winter gourd or ash gourd, is one of the commercially most important vegetable crops cultivated in the Indian subcontinent. Leaf curl disease of winter melon has become a serious problem in the state of Tamil Nadu, which is one of the major growing areas for this crop in India. Severe disease incidence is found in the Perambalur district state of Tamil Nadu, with up to 100% crop loss recorded over the period December 2012 to May 2013. Infected plant leaves exhibited typical symptoms of upward leaf curling, crinkling, puckering, yellowing and reduced leaf blade area (Fig. 1). The bipartite Squash leaf curl China virus-India (SLCCNV-IN) (Muniyappa et al., 2; Singh et al., 5), genus Begomovirus in the family Geminiviridae, is one of the major disease-causing factors severely affecting winter melon in India. Total DNA used as template for PCR was isolated from symptom-bearing and symptomless leaf samples using the Dellaporta method. A 771 bp fragment of a begomoviral DNA-A component was amplified using coat protein gene-specific degenerate primers designed for this study (FP: 5'-ATGTCSAAGCGWCCRGCAGA-3, RP: 5'-ATTCTTMACAGTWGCAGTGC-3'). Rolling circle amplification (RCA) was performed using an Illustra Templiphi 100 Amplification Kit (GE Healthcare Limited, UK) according to the manufacturer's protocol and digested with BamHI. A resulting ~2.7 kb fragment was purified via gel electrophoresis and cloned into BamHI -linearised pBluescript II KS (+) (Fig. 2). No such DNA band was observed for symptomless plant samples treated in parallel. The cloned fragment represented full-length viral DNA-A as identified by sequencing to reveal 2,739 bases with 8 ORFs (AV1, AV2, AV3, AC1, AC2, AC3, AC4, AC5 according to the designation of Padidam et al., 3) (GenBank Accession No. KF188433). BLASTn was performed for the viral genomic sequence (KF188433) against the non-redundant/nucleotide (nr/nt) databases, showing 98% and 91-96% nucleotide sequence identity with DNA-A components of Squash leaf curl China virus-India [India:Coimbatore:Pumpkin] (AY184487) (Brown et al., 1) and other Cucurbitaceae-infecting Squash leaf curl China virus-India isolates (EU573715, DQ026296, GU967381, AM286794, JN587811, KC857509, AF509743, AB330078, AM260205, KC171648, HM566112, AM260206, AF509741, AB027465, EF197940, EU543562, EU487031), respectively. According to Brown et al. (1), we report that the Begomovirus causing high losses of Benincasa hispida in India is a variant of SLCCNV-IN [IN:Coi:Pum] and has been named Squash leaf curl China virus - India isolate KP1 according to its segment DNA-A. A phylogenetic tree (Fig. 3) was calculated by the neighbour-joining method with MEGA version 5.2. (Tamura et al., 6). The variant SLCCNV-IN (KF188433) is supported by a bootstrap value of 100% and is grouped within the SLCCNV-IN [IN:Coi:Pum] isolates from Coimbatore in cluster B whereas SLCCNV-CN [TH:NP:WG:07] isolates (EU543562) from Benincasa hispida in Thailand (Sawangjit, 4) were found to be grouped in a separate cluster A. We thus found that the Begomovirus SLCCNV-IN KP1 infection in Benincasa hispida (KF188433) in India is divergent from that in Thailand. This report describes the existence of SLCCNV-IN infection in Benincasa hispida in India for the first time. The authors thank University Grant Commission (UGC), Government of India for financial assistance. S.U.M. thanks UGC-NRCBS, Madurai Kamaraj University, Madurai, Tamil Nadu, India for providing training. Special thanks to Dr. Amit Gal-on, ARO, Bet Dagan, Israel for valuable suggestions and ideas.

Mixed infections of begomoviruses in pumpkins with yellow vein mosaic disease in north India

Yellow vein mosaic disease of pumpkin (Cucurbita moschata) is a serious problem for the cultivation of pumpkin throughout India. Symptomatic samples collected from Varanasi region of North India showed mixed infection of the three begomoviruses which could represent up to three species namely Tomato leaf curl New Delhi virus, Squash leaf curl China virus and Tomato leaf curl Palampur virus.

Molecular identification and genetic diversity among six Begomovirus isolates affecting cultivation of cucurbitaceous crops in Uttar Pradesh, India

Begomoviruses cause a number of serious diseases of cultivated crops and are considered as the major constraint for the cultivation of several crops all over the world. During a survey in the years 2007 and 2008, the typical symptoms of Begomovirus (yellow mosaic and yellow vein) were observed on six cucurbitaceous crops, viz. bitter gourd (Momordica charantia L.), pointed gourd (Trichosanthes dioica), pumpkin (Cucurbita pepo), pumpkin (Cucurbita maxima), sponge gourd (Luffa cylindrica) and ridged gourd (Luffa acutangula) being cultivated in northern India. Begomovirus infection was suspected due to significant infestation of whiteflies (Bemisia tabaci, the known vector of Begomoviruses) on these species. The presence of Begomovirus was detected from the total DNA extracted from six infected leaf samples of these species by polymerase chain reaction using the specific primers of a well-characterised Begomovirus. The ∼800 bp amplicons of these isolates were cloned, sequenced and the data obtained were compared with each other and with sequence database available in GenBank for best sequence identities and phylogenetic relationships. Based on highest 97–99% sequence identities and closest phylogenetic relationships, four representative Begomovirus species were identified as Ageratum enation virus (from T. dioica), Squash leaf curl China virus (from C. maxima), Tomato leaf curl New Delhi virus (from M. charantia, L. cylindrica and L. acutangula) and Tomato leaf curl Palampur virus (from C. pepo). These results suggested the existence of a high genetic diversity among Begomoviruses infecting cucurbitaceous crops.

First report of Squash leaf curl china virus causing mosaic symptoms on summer squash (Cucurbita pepo) grown in Varanasi district of India

Severe incidence of a mosaic disease was observed on summer squash (Cucurbita pepo), commonly called pepo, grown in Varanasi during June–September of Khariff season 2007. Symptoms observed were mosaic, puckering on the leaves, wartiness on fruits, general stunting of plants and low yield. PCR amplification with degenerate primers designed to target the conserved sequences of coat protein gene of whitefly transmitted geminiviruses showed ∼800 bp fragment in all symptomatic samples tested, indicating the association of a geminivirus with the disease. Nucleotide sequence analysis of the amplified fragment showed 99% identity with pumpkin isolate of squash leaf curl china virus (SLCCNV) from Lucknow. It showed 85–96.7% homology with other isolates of SLCCNV from India and abroad. Phylogenetic analysis revealed the isolate on pepo from Varanasi clustered with SLCCNV isolates on pumpkin from Lucknow and Coimbatore.

Complete nucleotide sequences of a distinct bipartite begomovirus, bitter gourd yellow vein virus, infecting Momordica charantia

Momordica charantia (Cucurbitaceae), a vegetable crop commonly cultivated throughout Pakistan, and begomoviruses, a serious threat to crop plants, are natives of tropical and subtropical regions of the world. Leaf samples of M. charantia with yellow vein symptoms typical of begomovirus infections and samples from apparently healthy plants were collected from areas around Lahore in 2004. Full-length clones of a bipartite begomovirus were isolated from symptomatic samples. The complete nucleotide sequences of the components of one isolate were determined, and these showed the arrangement of genes typical of Old World begomoviruses. The complete nucleotides sequence of DNA A showed the highest nucleotide sequence identity (86.9%) to an isolate of Tomato leaf curl New Delhi virus (ToLCNDV), confirming it to belong to a distinct species of begomovirus, for which the name Bitter gourd yellow vein virus (BGYVV) is proposed. Sequence comparisons showed that BGYVV likely emerged as a result of inter-specific recombination between ToLCNDV and tomato leaf curl Bangladesh virus (ToLCBDV). The complete nucleotide sequence of DNA B showed 97.2% nucleotide sequence identity to that of an Indian strain of Squash leaf curl China virus.

Molecular Characterization of Tomato leaf curl Palampur virus and Pepper leaf curl betasatellite Naturally Infecting Pumpkin (Cucurbita moschata) in India

Pumpkin cultivation in India is affected by severe incidence of a yellow vein mosaic disease. Tomato leaf curl New Delhi virus and Squash leaf curl China virus are known to be associated with this disease in India. We were able to identify a third begomovirus-Tomato leaf curl Palampur virus (ToLCPMV), from pumpkin showing typical symptoms of the disease at Varanasi based on the sequence of complete DNA-A genome of the virus. The complete DNA-A sequence of the virus shared more than 99% sequence identity with other ToLCPMV isolates available in the GenBank and clustered with them in the phylogenetic analysis. This betasatellite amplified from the same infected sample has been identified as Pepper leaf curl betasatellite (PepLCB) which also infects chilli in India. There was 92% sequence identity between the two isolates. This is the first report of natural infection of ToLCPMV on pumpkin and association of PepLCB with yellow vein mosaic disease of pumpkin in India.

Chlorotic curly stunt: a severe begomovirus disease of bottle gourd in northern India.

Bottle gourd (Lagenaria siceraria) an important vegetable crop in India was observed to be affected by a chlorotic curly stunt disease (CCSD) during 2003-2006 in the vegetable growing areas of Delhi and adjoining state of Haryana. The affected plants are severely stunted and bear very small chlorotic and mildly curled leaves. Incidence of the disease varied from 4.7 to 36%. The disease could be easily transmitted by whitefly, Bemisia tabaci but not by sap. The causal virus was found to be a Begomovirus on the basis of whitefly transmission and sequence identity of putative coat protein (CP) and replication initiator protein (Rep) genes. The virus was transmitted to Cucumis sativus, Luffa acutangula, L. cylndrica, Lycopersicon esculentum, Nicotiana tabacum and Praecitrullus fistulosus but not to Citrullus lunatus, Cucumis melo, Cucurbita moschata and Vigna unguiculata. The N-terminal 60 amino acids of CP of the virus had 100% sequence identity with all the isolates of Tomato leaf curl New Delhi virus (ToLCNDV) and two isolates of Squash leaf curl China virus (SLCCV). The full length amino acid sequence of the CP and Rep genes had 100% similarity with ToLCNDV-Svr and -Luffa isolates. The phylogenetic analysis showed that the virus associated with CCSD of bottle gourd belongs to ToLCNDV cluster of the begomoviruses. This is the first record of emergence of a Begomovirus associated severe disease in bottle gourd in India.

First report ofSquash leaf curl China virusin Pakistan

Leaf samples of Cucurbita pepo plants showing yellow mosaic symptoms were collected from Lahore, Pakistan. Full-length clones of a bipartite begomovirus were isolated and sequenced in their entirety. The sequences showed the highest levels of nucleotide sequence identity (98.4% for the DNA-A and 89.6% for the DNA-B) to the ‘Indian’ strain of Squash leaf curl China virus (SLCCNV).

Biological and molecular characterization of a begomovirus associated with yellow mosaic vein mosaic disease of pumpkin from Northern India

The biological and molecular properties of Squash leaf curl China virus from Varanasi, India (SLCCNV-IN[IN:Var:Pum]) were characterized. SLCCNV-IN[IN:Var:Pum] could be transmitted by grafting and through whitefly transmission. The complete DNA-A and DNA-B components were amplified through PCR using specific DNA-A and DNA-B primers. The DNA-A of the isolate was comprised of 2,738 nucleotides, encoding typical six open reading frames, and DNA-B of 2,704 nucleotides, encoding two ORFs. Genome organization of the isolate was typical of an old world bipartite begomovirus. Comparisons showed that DNA-A and its intergenic region have the highest sequence identity (97.6 and 97.4%, respectively) with the SLCCNV-IN[IN:Luc:Pum]; (DQ026296). This data suggested that the isolate is a same begomovirus species for which the name Squash leaf curl China virus-India[India:Varanasi:Pumpkin] is proposed. DNA-B showed maximum sequence identity (89.2%) with SLCCNV-IN[IN:Coi:Pum] (AY184488). Phylogenetic analysis of the present isolate showed close relationship to other cucurbit-infecting geminiviruses. This is the first evidence of occurrence of the bipartite Squash leaf curl China virus associated with severe yellow mosaic disease of pumpkin in northern India.

Yellow leaf curl disease of pumpkin in Thailand is associated with Squash leaf curl China virus

Yellow leaf curl disease of pumpkin in Thailand is associated with <i>Squash leaf curl China virus</i>

Molecular Characterization of a Strain of Squash leaf curl China Virus from North India

AbstractA Begomovirus causing yellow vein mosaic disease of pumpkin (Cucurbita maxima L.) was characterized at molecular level by cloning and sequence analysis of its complete DNA‐A genome. The DNA‐A of the isolate contains 2758 nucleotides which encode six open reading frames (ORFs): AV1 and AV2 in the virion‐sense and AC1, AC2, AC3 and AC4 in the complementary‐sense. Based on the highest (96%) sequence identities and close phylogenetic relationships with Squash leaf curl China virus species, the Begomovirus was identified as strain of Squash leaf curl China virus. The presence of DNA‐B genome of the virus strain was also detected by dot blot hybridization test using DNA‐B specific probe.

Complete nucleotide sequence of a new isolate of tomato leaf curl New Delhi virus infecting cucumber, bottle gourd and muskmelon in Thailand

Geminiviruses have monopartite or bipartite circular sin-gle-stranded DNA (ssDNA) genomes, encapsidated ingeminate particles. The family Geminiviridae is composedof four genera, distinguished by their host range, insectvector, and genome organization [2]. Most of the charac-terized geminiviruses are transmitted by whiteflies(Bemisia tabaci Genn.) and belong to the genus Begomo-virus. Begomoviruses generally have bipartite genomes(designated as DNA-A and DNA-B) and infect dicotyle-donous plants. Some begomoviruses, however, have asingle genomic DNA molecule. Yellow leaf curl diseasecauses heavy losses to cucurbit plants in Thailand.Recently, we reported that yellow leaf curl disease ofpumpkin (Cucurbita pepa L.) is associated with squash leafcurl China virus [4]. Cucumber plants showing severeyellow leaf disease symptoms were observed during 1996.The disease was transmitted by grafting and whiteflyinoculations (unpublished results). To date, only partialDNA fragments of begomoviruses from diseased cucum-ber, muskmelon (Cucumis melo L.), cantaloupe (Cucumismelo var. reliculatus) and wax gourd (Benincase hispidaCogn.) have been sequenced [6–8]. The complete nucleo-tide sequences of DNA-A and DNA-B were obtained fromcucumber plants. DNA-A shares 97% with isolates oftomato leaf curl New Delhi virus (ToLCNDV) and thusnamed tomato leaf curl New Delhi virus-[Cucum-ber:Thailand] (ToLCNDV-[Cuc:Tha]). This virus has alsobeen detected on bottle gourd (Lagenaria leucantha Rosby)and muskmelon exhibiting yellow leaf symptoms collectedfrom fields in the same region. The sequences obtainedfrom bottle gourd and muskmelon isolates share more than99% sequence identity with ToLCNDV-[Cuc:Tha]. Thispaper describes for the first time an isolate of ToLCNDVinfecting cucumber, bottle gourd and muskmelon samplesand reports its complete nucleotide sequence. However, noDNA-B component has been described for ToLCNDVoriginating from Thailand. Here, we describe briefly thatyellow leaf curling of cucumber is associated with andcaused by a bipartite begomovirus.In 1996, Cucumis sativus L. plants exhibiting yellow leafsymptoms were collected from fields at Kamphaengsaen,Nakorn Pathom, Thailand. In addition, samples exhibitingyellow symptoms on bottle guard and muskmelon were alsocollected from nearby fields and were maintained on sus-ceptible cucumber plants by grafting/whitefly inoculation inan insect-proof glasshouse. Leaf tissue was desiccated withsilica gel for 2 weeks and stored at 4 C until use. Totalnucleic acids were extracted from the leaf tissues of infectedcucumber plants, as described by Kon et al. [5]. The poly-merase chain reaction (PCR) was carried out as describedby Sharma et al. [9]. Initially, 2.6 kbp viral DNA fragmentswere obtained from diseased cucumber plants using be-gomovirus DNA-A-specific primers UPV1 and UPC2 [1]and cloned into pGEM-T Easy Vector (Promega, Madison,WI) and sequenced. Recombinant plasmids containing anapproximately full-length insert of the PCR product were

Pumpkin yellow vein mosaic disease is caused by two distinct begomoviruses: complete viral sequences and comparative transmission by an indigenous Bemisia tabaci and the introduced B‐biotype

AbstractPumpkin yellow vein mosaic disease (PYVMD) causes significant damage to pumpkin production throughout India. A begomovirus causing PYVMD in South India was characterized recently but the nature of virus causing the disease in North India was not known. Samples of PYVMD were obtained from North India and two putative begomoviruses were PCR‐amplified and sequenced. Comparison of complete DNA‐A sequences indicated that PYVMD in North and South India were caused by two distinct begomoviruses and shared only approximately 88% DNA‐A nucleotide identity. The South Indian isolate was most closely related to Squash leaf curl China virus between 91 and 96% identities, and the two North Indian isolates to Tomato leaf curl New Delhi virus between 94 and 96% identities. The South Indian isolate was previously shown to be transmitted by the indigenous biotype of Bemisia tabaci, however, the situation has since changed with the introduction of the B‐biotype to South India in 1999. Comparative transmission experiments between the indigenous biotype v/s the introduced B‐biotype for the time required for virus acquisition (30 min v/s 15 min), inoculation (15 min v/s 10 min) and incubation (30 min v/s 4 h) have indicated that the B‐biotype transmits the virus quickly and more efficiently than the indigenous biotype. An epidemic of PYVMD was recorded for the first time in South India in 2004 with disease incidences of up to 100% and significant yield losses. This may be due to a combination of several factors including the large numbers of B‐biotype populations, the ability of the B‐biotype to transmit the virus efficiently and the cultivation of susceptible varieties. These possibilities and the threat to pumpkin cultivation associated with the spread of the B‐biotype in India are discussed.

Molecular Characterization of a Strain of Squash Leaf Curl China Virus from the Philippines

AbstractThe complete nucleotide sequence of infectious cloned DNA components (A and B) of the causal agent of squash leaf curl disease in the Philippines was determined. DNA‐A and DNA‐B comprise 2739 and 2705 nucleotides, respectively; the common region is 174 bases in length. Five ORFs were found in DNA‐A and two in DNA‐B. Partial dimeric clones containing DNA‐A and DNA‐B, constructed in a binary vector and transformed into Agrobacterium tumefaciens, induced systemic infection in agro‐inoculated pumpkin plants (Cucurbita moschata). The total DNA‐A sequence was most closely related to that of Squash leaf curl China virus (SLCCNV) (88% identity), although the existence of B component of SLCCNV has not been reported. The deduced coat protein was like that of SLCCNV (98% amino acid sequence identity) and the Philippines virus has low sequence identity to Squash leaf curl virus (SLCV) and Squash mild leaf curl virus (SMLCV) (63 and 64% total nucleotide sequence identities, respectively). From these results, we propose that the Philippines virus be designated Squash leaf curl China virus‐[Philippines] (SLCCNV‐[PH]).