Detection and Molecular Characterization of Squash leaf curl virus (SLCV) in Jordan

Squash leaf curl virus (SLCV) was detected for the first time in Jordan using degenerated oligonucleotide primers. Two isolates of the virus, SLCV‐E and SLCV‐R, were detected using specific oligonucleotide primers in symptomatic Cucurbita pepo. SLCV was also found to occur naturally in Malva parviflora, which showed severe leaf curling, yellowing and stunting of the whole plants. The full‐length genomes of Squash leaf curl virus‐Malva (SLCV‐Malva) isolate were amplified using the bacteriophage Φ DNA polymerase enzyme. Nucleotide sequence analysis showed that SLCV‐Malva shared high nucleotide identity (98% and 97%) with SLCV‐EG and SLCV‐E from Egypt and USA, respectively. A survey using dot‐blot hybridization indicated that squash leaf curl disease occurred in all surveyed areas. The highest disease incidence (95%) was recorded in Dir Alla area, whereas disease incidence did not exceed 69% in squash samples collected from North Ghor.     

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

A 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.     

Molecular Characterization of a Distinct Begomovirus Infecting Euphorbia pulcherrima in China

Virus isolate G35 was obtained from Euphorbia pulcherrima showing leaf curl and vein thickening symptoms in Tianyang, Guangxi Province, China. The virus was transmitted by whiteflies to Nicotiana tabacum, Lycopersicon esculentum, Datura stramonium and E. pulcherrima. DNA‐A contains 2746 nucleotides, with two open reading frames (ORFs) in the virion‐sense DNA and four ORFs in the complementary‐sense DNA. When compared with the DNA‐A sequence of other begomoviruses, the total DNA‐A of isolate G35 was most closely related to that of Ageratum enation virus (79.9% sequence identity). However, the deduced coat protein of G35 is most like that of Pepper leaf curl virus from Bangladesh (94.9% amino acid sequence identity), and the AC1 of G35 is most like that of Cotton leaf curl Multan virus‐Okra (87.2% amino acid sequence identity). The molecular data showed that G35 is a distinct Begomovirus species, for which the name Euphorbia leaf curl virus (ELCV) is proposed.     

Host range, vector relationships and sequence comparison of a begomovirus infecting hibiscus in India

Hibiscus leaf curl disease (HLCuD) occurs widely in India. Infected hibiscus plants show vein thickening, upward curling of leaves and enations on the abaxial leaf surface, reduction in leaf size and stunting. The commonly‐occurring weeds (Ageratum conyzoides, Croton bonplandianum and Euphorbia geniculata), Nicotiana benthamiana, Nicotiana glutinosa and Nicotiana tabacum (var. Samsun, Xanthi), cotton and tomato were shown to be susceptible to HLCuD. One of the four species of hibiscus (Hibiscus rosa‐sinensis) and 75 of the 101 commercial hybrids/varieties grown in the Bangalore area of southern India were also susceptible. Two virus isolates associated with HLCuD from Bangalore, South India (Ban), and Bhubaneswar, North India (Bhu), were detected serologically and by PCR‐mediated amplification of virus genomes. The isolates were characterised by sequencing a fragment of DNA‐A component (1288 nucleotides) and an associated satellite DNA molecule of 682 nucleotides. Phylogenetic analyses of these DNA‐A sequences clustered them with Old World cotton‐infecting begomoviruses and closest to Cotton leaf curl Multan virus (CLCuMV) at 95–97% DNA‐A nucleotide identities. The 682‐nucleotide satellite DNA molecules associated with the HLCuD samples Ban and Bhu shared 96.9% sequence identity with each other and maximum identity (93.1–93.9% over positions 158–682) with ～1350‐nucleotide DNA‐β satellite molecules associated with cotton leaf curl disease in Pakistan and India (accession nos AJ298903, AJ316038). HLCuD in India, therefore, appears to be associated with strains of CLCuMV, a cotton‐infecting begomovirus from Pakistan, which is transmitted in a persistent manner by Bemisia tabaci.     

Tomato yellow leaf curl viruses: ménage à trois between the virus complex,the plant and the whitefly vector

Tomato yellow leaf curl disease (TYLCD) is one of the most devastating viral diseases affecting tomato crops in tropical, subtropical and temperate regions of the world. Here, we focus on the interactions through recombination between the different begomovirus species causing TYLCD, provide an overview of the interactions with the cellular genes involved in viral replication, and highlight recent progress on the relationships between these viruses and their vector, the whitefly Bemisia tabaci. Taxonomy: The tomato yellow leaf curl virus‐like viruses (TYLCVs) are a complex of begomoviruses (family Geminiviridae, genus Begomovirus) including 10 accepted species: Tomato yellow leaf curl Axarquia virus (TYLCAxV), Tomato yellow leaf curl China virus (TYLCCNV), Tomato yellow leaf curl Guangdong virus (TYLCGuV), Tomato yellow leaf curl Indonesia virus (TYLCIDV), Tomato yellow leaf curl Kanchanaburi virus (TYLVKaV), Tomato yellow leaf curl Malaga virus (TYLCMalV), Tomato yellow leaf curl Mali virus (TYLCMLV), Tomato yellow leaf curl Sardinia virus (TYLCSV), Tomato yellow leaf curl Thailand virus (TYLCTHV), Tomato yellow leaf curl Vietnam virus (TYLCVNV) and Tomato yellow leaf curl virus(TYLCV). We follow the species demarcation criteria of the International Committee on Taxonomy of Viruses (ICTV), the most important of which is an 89% nucleotide identity threshold between full‐length DNA‐A component nucleotide sequences for begomovirus species. Strains of a species are defined by a 93% nucleotide identity threshold. Host range: The primary host of TYLCVs is tomato (Solanum lycopersicum), but they can also naturally infect other crops [common bean (Phaseolus vulgaris), sweet pepper (Capsicum annuum), chilli pepper (C. chinense) and tobacco (Nicotiana tabacum)], a number of ornamentals [petunia (Petunia×hybrida) and lisianthus (Eustoma grandiflora)], as well as common weeds (Solanum nigrum and Datura stramonium). TYLCVs also infect the experimental host Nicotiana benthamiana. Disease symptoms: Infected tomato plants are stunted or dwarfed, with leaflets rolled upwards and inwards; young leaves are slightly chlorotic; in recently infected plants, fruits might not be produced or, if produced, are small and unmarketable. In common bean, some TYLCVs produce the bean leaf crumple disease, with thickening, epinasty, crumpling, blade reduction and upward curling of leaves, as well as abnormal shoot proliferation and internode reduction; the very small leaves result in a bushy appearance.     

Identification and Molecular Characterization of a New Geminivirus Betasatellite Infecting Malvastrum coromandelianum in China

The complete nucleotide sequence of a satellite molecule associated with Malvastrum leaf curl Guangdong virus (MLCuGdV) infecting M. coromandelianum plants exhibiting leaf curl symptoms in a suburb of Guangzhou, Guangdong Province of China, is described and analysed. The molecule has typical features of betasatellites, containing a single ORF in the complementary‐sense strand, an A‐rich region, the satellite‐conserved region and a stem–loop structure. Compared with the geminivirus betasatellites in GenBank database, this molecule shows the highest nucleotide sequence identity of 71.9% with Tomato leaf curl Philippine betasatellite isolate Laguna1 (ToLCPB, AB307732). Phylogenetic analysis indicates that it is more related to isolate Laguna 1 and Laguna 2 of ToLCPB. According to the proposed species demarcation threshold of betasatellites (78% nucleotide identity), it is a novel betasatellite species, for which we propose the name Malvastrum leaf curl Guangdong betasatellite (MLCuGdB).     

Evidence of Cotton leaf curl Burewala virus Variant and its Associate Betasatellite Causing Yellow Mosaic of Eggplant (Solanum melongena) in Pakistan

Eggplant (Solanum melongena L.) plants with severe leaf mosaic and mottling were found in a kitchen garden near cotton fields in Pakistan. Rolling Circle Amplification products from six of the naturally infected eggplant plants, subjected to PCR, successfully amplified expected products of 2.8 and 1.4 kb using begomovirus and betasatellite‐specific primers, respectively. Based on 99% nucleotide sequence identity, the virus was identified as a variant of Cotton leaf curl Burewala virus (CLCuBuV) (GenBank Accession No. HG428709). Likewise, the sequenced betasatellite with a maximum of 97% nucleotide sequence identity was recognized as a new variant of Cotton leaf curl Multan betasatellite (CLCuMuB^Mul) (GenBank Accession No. HG428708). The symptomatic induction of Cotton leaf curl disease in CLCuBuV susceptible cotton genotype CIM‐496 by back‐indexing further confirmed the presence of CLCuBuV in eggplant. This is the first report of CLCuBuV and its associate betasatellite in naturally infected plants of eggplant.     

Begomoviruses Associated with Leaf Curl Disease of Tomato in Java,Indonesia*

We report that several begomoviruses are associated with tomato leaf curl disease in Java, Indonesia. Tomato plants with leaf curl symptoms were collected from Bandung (west Java), Purwokerto (central Java), Magelang (central Java) and Malang (east Java) of Indonesia, the major tomato‐growing areas of the country. Viruses were detected using the polymerase chain reaction (PCR), with universal primers for the genus Begomovirus. PCR‐amplified fragments were cloned and sequenced. Based on sequence comparisons and phylogenetic analyses, the viruses were divided into three groups. With respect to amino acid (aa) identities of the N‐terminal halves of the coat proteins compared in this study, group I was most closely related to Ageratum yellow vein virus (AYVV) (97%), Ageratum yellow vein China virus‐[Hn2] (AYVCNV‐[Hn2]) (96%) and Ageratum yellow vein virus‐[Taiwan] (AYVV‐[Tai]) (95%), and ageratum‐infecting begomovirus from Java (99%). Group II had high sequence identity with a tentative species of tomato leaf curl Java virus (ToLCJAV) (96% aa) for the CP. Group III was most closely related to a proposed species of Pepper yellow leaf curl Indonesia virus (PepYLCIDV) (90% aa identity) by its partial CP sequence.     

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.     

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.     

A New Alphasatellite Molecule Associated with Ageratum yellow vein China virus in the Philippines

From Synedrella nodiflora plants with leaf curling, vein swelling and enation symptoms on Samal Island, the Philippines, a begomoviral DNA‐A and its associated alphasatellite molecule were cloned and sequenced. The begomovirus was identified as an isolate of Ageratum yellow vein China virus (AYVCNV) with 91% nucleotide sequence identity to AYVCNV‐[P157] (EU487045), while the alphasatellite molecule was most closely related to tobacco curly shoot alphasatellite‐Y99 (TbCSA‐Y99, AJ579347) with 74.5% nucleotide sequence identity. The satellite molecule has the typical features of alphasatellites, with a single gene in the virion sense, an A‐rich region and a 33‐bp predicted stem‐loop structure. According to the proposed species demarcation threshold of alphasatellites (83% nucleotide sequence identity), the alphasatellite molecule represents a new species, herein named ‘Ageratum yellow vein China alphasatellite’ ( KF785752 ).     

Complete Nucleotide Sequence and Construction of an Infectious Clone of a Tunisian Isolate of Tomato yellow leaf curl Sardinia virus

The full‐length genome of a Tunisian isolate of Tomato yellow leaf curl Sardinia virus (TYLCSV) was engineered and submitted to sequence analysis. The Tunisian isolate has 99% sequence identity with TYLCSV‐Sicilian (Sic), results thus providing further evidence for the inclusion of this isolate in the TYLCSV‐Sic group. A 1.7‐mer construct of the virus was obtained and efficiently agroinfiltrated into tomato and tobacco plants to induce symptoms indistinguishable from those of natural infection.     

The circulative pathway of begomoviruses in the whitefly vector Bemisia tabaci— insights from studies with Tomato yellow leaf curl virus

Our current knowledge concerning the transmission of begomoviruses by the whitefly vector Bemisia tabaci is based mainly on research performed on the Tomato yellow leaf curl virus (TYLCV) complex and on a number of viruses originating from the Old World, such as Tomato leaf curl virus, and from the New World, including Abutilon mosaic virus, Tomato mottle virus, and Squash leaf curl virus. In this review we discuss the characteristics of acquisition, transmission and retention of begomoviruses by the whitefly vector, concentrating on the TYLCV complex, based on both published and recent unpublished data. We describe the cells and organs encountered by begomoviruses in B. tabaci. We show immunolocalisation of TYLCV to the B. tabaci stylet food canal and to the proximal part of the descending midgut, and TYLCV‐specific labelling was also associated with food in the lumen. The microvilli and electron‐dense material in the epithelial cells of the gut wall were also labelled by the anti TYLCV serum, pointing to a possible virus translocation route through the gut wall and to a putative site of long‐term virus storage. We describe the path of begomoviruses in their vector B. tabaci and in the non‐vector whitefly Trialeurodes vaporariorum, and we follow the rate of virus translocation in these insects. We discuss TYLCV transmission between B. tabaci during mating, probably by exchange of haemolymph. We show that following a short acquisition access to infected tomato plants, TYLCV remains associated with the B. tabaci vector for weeks, while the virus is undetectable after a few hours in the non‐vector T. vaporariorum. The implications of the long‐term association of TYLCV with B. tabaci in the light of interactions of the begomovirus with insect receptors are discussed.     

Virus–vector Relationships,Host Range,Detection and Sequence Comparison of Chilli leaf curl virus Associated with an Epidemic of Leaf Curl Disease of Chilli in Jodhpur,India

An epidemic of chilli leaf curl disease was recorded in 2004 in Jodhpur, a major chilli‐growing area in Rajasthan, India. Several isolates were efficiently transmitted by the whitefly (Bemisia tabaci), all of which induced severe leaf curl symptoms in chilli. A single whitefly was capable of transmitting the virus, and eight or more whiteflies per plant resulted in 100% transmission. The minimum acquisition access period (AAP) and inoculation access period (IAP) were 180 and 60 min, respectively. The virus persisted in whiteflies for up to 5 days postacquisition. Of 25 species tested, the virus infected only five (Capsicum annuum, Carica papaya, Solanum lycopersicum, Nicotiana tabacum and N. benthamiana). The virus was identified as Chilli leaf curl virus (ChiLCV), which shared the closest sequence identity (96.1%) with an isolate of ChiLCV from potato in Pakistan and showed sequence diversity up to 12.3% among the ChiLCV isolates reported from India and Pakistan. A betasatellite was identified, which resembled most closely (97.3%) that of Tomato leaf curl Bangladesh betasatellite previously reported from chilli and tomato leaf curl in India. The betasatellite was very different from that reported from chilli leaf curl in Pakistan, indicating that different betasatellites are associated with chilli leaf curl in India and Pakistan. We describe here for the first time the virus–vector relationships and host range of ChiLCV.     

A mixture of begomoviruses in leaf curl-affected tobacco in Karnataka, South India

Tobacco leaf curl is widespread in several states in India including Andhra Pradesh, Gujarat, Karnataka, Bihar and West Bengal. Tobacco leaf curl virus (TbLCV) isolates collected from five different parts of India induced four distinct symptom phenotypes (group I, II, III & IV) on tobacco cultivars Samsun and Anand 119 (Valand & Muniyappa, 1992). PCR was performed on DNA extracted from group I and IV leaf curl‐affected tobacco from Karnataka, India using degenerate begomovirus‐specific primers. Subsequent cloning and sequencing of PCR products revealed preliminary evidence for the presence of at least three begomoviruses in the affected material following alignment of a 333 bp region of the coat protein gene (CP). The complete CP and common region (CR) of two putative begomoviruses, Tobacco leaf curl virus‐Karnataka1 (TbLCV‐Kar1) and Tobacco leaf curl virus‐Karnataka2 (TbLCV‐Kar2), were sequenced using PCR clones obtained with designed sequence‐specific primers. Phylogenetic analysis of the CP and CR of TbLCV‐Kar1 and TbLCV‐Kar2 placed them in the Asian Old World begomovirus cluster. The two viruses differed from each other significantly in both the CP gene and the CR (< 90% nucleotide sequence identity). This difference, in conjunction with distinct iterative sequences strongly suggests that these begomoviruses are distinct from one another. Group I and IV tobacco were also found to harbour a possible third begomovirus following the 333 bp CP alignment. Comparison of TbLCV‐Kar1 and TbLCV‐Kar2 with other geminiviruses, showed that both sequences shared high nucleotide sequence identity (> 90%) with other begomoviruses in either the CP or CR, thereby suggesting these viruses to be possible strains of other reported begomoviruses. Combined comparison of the CP and CR sequences however, suggests that the two viruses are not strains of other reported begomoviruses, but may be distinct begomoviruses that could have arisen through recombination events during mixed infections. Phylogenetic comparison demonstrated no significant homology between the Indian tobacco begomoviruses and a tobacco‐infecting begomovirus from Zimbabwe, again showing that as with other geminiviruses, there is a geographic basis for phylogenetic relationships rather than an affiliation with tobacco as a host.     

Complete Nucleotide Sequence of Ageratum enation virus and an Alphasatellite Infecting a New Host Glycine max in India

During 2011, leaf crumpling, yellowing and stunting were observed on soya bean (Glycine max) in Himachal Pradesh, India. PCR‐based detection confirmed the presence of a begomovirus. The viral genome was amplified by rolling circle amplification, cloned and sequenced. The complete nucleotide sequence of DNA‐A showed highest nucleotide identity to an isolate of Ageratum enation virus infecting a weed Ageratum conyzoides. In addition, a DNA molecule was found which shared 95% nucleotide identity with an alphasatellite infecting ageratum. Neither beta satellite nor DNA‐B was detected in the infected samples.     

Molecular Evidence for the Association of Tomato leaf curl Gujarat virus with a Leaf Curl Disease of Phaseolus vulgaris L.

A leaf curl disease with symptoms typical of begomoviruses was observed in bean (Phaseolus vulgaris) at the Main Research Farm of the Indian Institute of Pulses Research, Kanpur, India. Infected plants had severe distortion of leaves and the plants were unproductive. PCR indicated the involvement of French bean leaf curl virus (JQ866297), a recently described Begomovirus, and Tomato leaf curl Gujarat virus (ToLCGV). The full‐length genome of ToLCGV associated with leaf curl disease of bean was 2757 nucleotides long and had maximum identity (97–98%) with seven isolates of ToLCGV (AY234383, AF449999, EU573714, GQ994098, AY190290, FR819708, AF413671) and is designated as Tomato leaf curl Gujarat virus‐(IN:Knp:Bean:2013) (KF440686). To the best of our knowledge, this is the first record of ToLCGV infecting a leguminous host, P. vulgaris.     

Molecular Characterization of a Distinct Begomovirus and its Associated Satellite DNA Molecule Infecting Sida acuta in China*

Three viral isolates Hn8, Hn40 and Hn41 were obtained from Sida acuta showing yellow mosaic symptom in the Hainan province, China. Comparison of partial DNA‐A sequences amplified with degenerate primers confirmed the existence of single type of Begomovirus. The complete nucleotide sequence of the DNA‐A‐like molecule of Hn8 was determined to be 2749 nucleotides, having a typical genetic organization of a Begomovirus. Hn8 DNA‐A had the highest sequence identity (78%) with that of Ageratum yellow vein China virus‐[G13] ( AJ558120 ), and had less sequence identity with other begomoviruses. Based on the above molecular data, Hn8 was thus considered as a new Begomovirus species, for which the name Sida yellow mosaic China virus (SiYMCNV) is proposed. Satellite DNA‐β molecules (Hn8‐β, Hn40‐β and Hn41‐β) were found to be associated with Hn8, Hn40 and Hn41 and their complete nucleotide sequences were determined. Sequence analysis showed that Hn8‐β, Hn40‐β and Hn41‐β shared more than 84% nucleotide sequence identity, and they were different from other characterized DNA‐β, sharing the highest nucleotide sequence identity (47.8%) with DNA‐β of Ageratum yellow vein virus.     

Biology and interactions of two distinct monopartite begomoviruses and betasatellites associated with radish leaf curl disease in India

Background

Emerging whitefly transmitted begomoviruses are major pathogens of vegetable and fibre crops throughout the world, particularly in tropical and sub-tropical regions. Mutation, pseudorecombination and recombination are driving forces for the emergence and evolution of new crop-infecting begomoviruses. Leaf curl disease of field grown radish plants was noticed in Varanasi and Pataudi region of northern India. We have identified and characterized two distinct monopartite begomoviruses and associated beta satellite DNA causing leaf curl disease of radish (Raphanus sativus) in India.

Results

We demonstrate that RaLCD is caused by a complex of two Old World begomoviruses and their associated betasatellites. Radish leaf curl virus-Varanasi is identified as a new recombinant species, Radish leaf curl virus (RaLCV) sharing maximum nucleotide identity of 87.7% with Tomato leaf curl Bangladesh virus-[Bangladesh:2] (Accession number AF188481) while the virus causing radish leaf curl disease-Pataudi is an isolate of Croton yellow vein mosaic virus-[India] (CYVMV-IN) (Accession number AJ507777) sharing 95.8% nucleotide identity. Further, RDP analysis revealed that the RaLCV has a hybrid genome, a putative recombinant between Euphorbia leaf curl virus and Papaya leaf curl virus. Cloned DNA of either RaLCV or CYVMV induced mild leaf curl symptoms in radish plants. However, when these clones (RaLCV or CYVMV) were individually co-inoculated with their associated cloned DNA betasatellite, symptom severity and viral DNA levels were increased in radish plants and induced typical RaLCD symptoms. To further extend these studies, we carried out an investigation of the interaction of these radish-infecting begomoviruses and their associated satellite, with two tomato infecting begomoviruses (Tomato leaf curl Gujarat virus and Tomato leaf curl New Delhi virus). Both of the tomato-infecting begomoviruses showed a contrasting and differential interaction with DNA satellites, not only in the capacity to interact with these molecules but also in the modulation of symptom phenotypes by the satellites.

Conclusion

This is the first report and experimental demonstration of Koch's postulate for begomoviruses associated with radish leaf curl disease. Further observations also provide direct evidence of lateral movement of weed infecting begomovirus in the cultivated crops and the present study also suggests that the exchange of betasatellites with other begomoviruses would create a new disease complex posing a serious threat to crop production.