Complete genome sequence of hollyhock vein yellowing virus, a novel monopartite begomovirus infecting hollyhock in Pakistan.

Hollyhock (Alcea rosea, family Malvaceae) is an ornamental plant grown widely in gardens across South Asia. In a bed of ornamental plants near the village of Chakri (Punjab Province, Pakistan) in 2014, hollyhock plants showing two distinct symptom types were identified: yellow vein mosaic and leaf crumple. PCR amplification with universal primers amplified a begomovirus from separate nucleic acid extracts of single plants of each type but amplified a betasatellite only from the plant with the yellow vein mosaic symptoms. No potential begomovirus DNA B component or alphasatellite could be identified in either sample. After cloning, the genome sequences of two viruses, one from a plant of each symptom type, were determined and shown to share 99.9% nucleotide sequence identity with each other but less than 91% nucleotide sequence identity with all previously characterized begomoviruses, with the highest identity (90%) to an isolate of pedilanthus leaf curl virus (PeLCV). This indicates that the two hollyhock plants were infected with a newly identified begomovirus for which the name "hollyhock vein yellowing virus" (HoVYV) is proposed. HoVYV likely has a recombinant origin. The betasatellite showed the highest nucleotide sequence identity to an isolate of cotton leaf curl Multan betasatellite (CLCuMuB), a betasatellite associated with cotton leaf curl disease across Pakistan and northwestern India. These findings add to the diversity of known begomoviruses in South Asia and again highlight the role of hollyhock as a reservoir of the cotton leaf curl begomovirus betasatellite complex. The results also suggest that the yellow vein mosaic symptoms in hollyhock are due to the betasatellite rather than the virus.

Codiaeum variegatum in Pakistan harbours pedilanthus leaf curl virus and papaya leaf curl virus as well as a newly identified betasatellite.

Codiaeum variegatum (common name, garden croton) is an ornamental plant grown for its bright yellow variegated leaf morphology. Two C. variegatum plants with upward leaf curling and vein swelling symptoms were collected in Faisalabad, Pakistan. Sequencing of clones obtained by PCR amplification with specific primers showed one plant infected with the monopartite begomoviruses pedilanthus leaf curl virus (PeLCV) and papaya leaf curl virus (PaLCuV) and the other to be infected with only PeLCV. Both plants also harboured a betasatellite that was distinct from all previously identified betasatellites, for which the name "codiaeum leaf curl betasatellite" (CoLCuB) is proposed. This is the first identification of a begomovirus and an associated betasatellite infecting C. variegatum in Pakistan. Both PeLCV and PaLCuV cause problems in a number of crop plants, and C. variegatum may act as a reservoir for these agriculturally important viruses. The precise impact and geographical distribution of the newly identified CoLCuB will be investigated.

Molecular and biological characterization of Chilli leaf curl virus and associated Tomato leaf curl betasatellite infecting tobacco in Oman.

BACKGROUND: In Oman tobacco (Nicotiana tabacum; family Solanaceae) is a minor crop, which is produced only for local consumption. In 2015, tobacco plants exhibiting severe downward leaf curling, leaf thickening, vein swelling, yellowing and stunting were identified in fields of tobacco in Suhar Al-Batina region, Oman. These symptoms are suggestive of begomovirus (genus Begomovirus, family Geminiviridae) infection. METHODS: Circular DNA molecules were amplified from total DNA extracted from tobacco plants by rolling circle amplification (RCA). Viral genomes were cloned from RCA products by restriction digestion and betasatellites were cloned by PCR amplification from RCA product, using universal primers. The sequences of full-length clones were obtained by Sanger sequencing and primer walking. Constructs for the infectivity of virus and betasatellite were produced and introduced into plants by Agrobacterium-mediated inoculation. RESULTS: The full-length sequences of 3 begomovirus and 3 betasatellite clones, isolated from 3 plants, were obtained. Analysis of the full-length sequences determined showed the virus to be a variant of Chilli leaf curl virus (ChiLCV) and the betasatellite to be a variant of Tomato leaf curl betasatellite (ToLCB). Both the virus and the betasatellite isolated from tobacco show the greatest levels of sequence identity to isolates of ChiLCV and ToLCB identified in other hosts in Oman. Additionally clones of ChiLCV and ToLCB were shown, by Agrobacterium-mediated inoculation, to be infectious to 3 Nicotiana species, including N. tabacum. In N. benthamiana the betasatellite was shown to change the upward leaf rolling symptoms to a severe downward leaf curl, as is typical for many monopartite begomoviruses with betasatellites. CONCLUSIONS: The leaf curl disease of tobacco in Oman was shown to be caused by ChiLCV and ToLCB. This is the first identification of ChiLCV with ToLCB infecting tobacco. The study shows that, despite the low diversity of begomoviruses and betasatellites in Oman, the extant viruses/betasatellites are able to fill the niches that present themselves.

Molecular characterization of Chickpea chlorotic dwarf virus strain D in Chickpea (Cicer arietinum) from District Dera Ismail Khan Khyber Pakhtunkhwa, Pakistan.

Chickpea chlorotic dwarf virus (CpCDV), a member of genus Mastrevirus (family Geminiviridae) is an important viral pathogen of chickpea and other legume crops in Middle East, North Africa, India and Pakistan. Among sixteen known strains of CpCDV three are known to infect legume crops in Punjab province of Pakistan. In this study diversity of CpCDV was explored in Khyber Pakhtunkhwa (KP) province of Pakistan. In year 2016, during a survey in the chickpea growing areas of district Dera Ismail Khan, CpCDV infected plants were identified. Leaf samples were collected, and a diagnostic PCR confirmed mastreivrus infection in 4 out of 100 samples. From these samples full-length genome of CpCDV was amplified using specific back-to-back primers. Virus molecules were sequenced to their entirety and sequence analysis of a molecule KRF4 (GenBank accession # KY952837) showed the highest pair wise sequence identity of 97% with a CpCDV molecule (KM229787) isolated from chickpea plant. An SDT analysis revealed it to be the D strain of CpCDV and a recombination detection program (RDP) showed it to be a recombinant between C (KM229780) and L (KT634301) strains of CpCDV. Thus, further supporting the intra-species recombination for CpCDV and presence of the same strain in chickpea growing areas of Pakistan other than Punjab province. This is the first identification of CpCDV (genus Mastrevirus family Geminiviridae) from chickpea (Cicer arietinum) plants in District Dera Ismail Khan, KP province, Pakistan.

Alphasatellitidae: a new family with two subfamilies for the classification of geminivirus- and nanovirus-associated alphasatellites.

Nanoviruses and geminiviruses are circular, single stranded DNA viruses that infect many plant species around the world. Nanoviruses and certain geminiviruses that belong to the Begomovirus and Mastrevirus genera are associated with additional circular, single stranded DNA molecules (~ 1-1.4 kb) that encode a replication-associated protein (Rep). These Rep-encoding satellite molecules are commonly referred to as alphasatellites and here we communicate the establishment of the family Alphasatellitidae to which these have been assigned. Within the Alphasatellitidae family two subfamilies, Geminialphasatellitinae and Nanoalphasatellitinae, have been established to respectively accommodate the geminivirus- and nanovirus-associated alphasatellites. Whereas the pairwise nucleotide sequence identity distribution of all the known geminialphasatellites (n = 628) displayed a troughs at ~ 70% and 88% pairwise identity, that of the known nanoalphasatellites (n = 54) had a troughs at ~ 67% and ~ 80% pairwise identity. We use these pairwise identity values as thresholds together with phylogenetic analyses to establish four genera and 43 species of geminialphasatellites and seven genera and 19 species of nanoalphasatellites. Furthermore, a divergent alphasatellite associated with coconut foliar decay disease is assigned to a species but not a subfamily as it likely represents a new alphasatellite subfamily that could be established once other closely related molecules are discovered.

Transmission of cotton leaf curl disease: answer to a long-standing question.

Cotton leaf curl disease (CLCuD) has been a major constraint to cotton production across Pakistan and northwestern India since the early 1990s. The disease is caused by a number of begomoviruses, including Cotton leaf curl Multan virus (CLCuMuV), which associate with a specific host range and symptom determining betasatellite known as Cotton leaf curl Multan betasatellite (CLCuMuB). Bemisia tabaci is a complex of cryptic species that consists of numerous (> 44) morphologically indistinguishable and, at least partially, reproductively isolated species. CLCuD has recently been introduced into parts of China but has not, at least so far, become a problem in the major cotton regions. The disease in China has been shown to be caused by CLCuMuV with CLCuMuB, which is believed to have been introduced from South Asia in ornamental plants. To understand the basis for this lack of spread of CLCuD into the cotton-growing areas of China, Pan et al. (Phytopathology 108:1172-1183, 2018) investigated the transmission of CLCuMuV/CLCuMuB by B. tabaci. The study showed that, of the four cryptic B. tabaci species investigated, only the cryptic species Asia II 1 was able to efficiently transmit CLCuMuV/CLCuMuB. Significantly, Asia II 1 is not present in the major cotton-growing regions of China. The results of Pan et al. (Phytopathology 108:1172-1183, 2018) are discussed with particular emphasis on the situation of CLCuD in Pakistan and northwestern India, which differs significantly from the situation in China.

ICTV Virus Taxonomy Profile: Geminiviridae.

The geminiviruses are a family of small, non-enveloped viruses with single-stranded, circular DNA genomes of 2500-5200 bases. Geminiviruses are transmitted by various types of insect (whiteflies, leafhoppers, treehoppers and aphids). Members of the genus Begomovirus are transmitted by whiteflies, those in the genera Becurtovirus, Curtovirus, Grablovirus, Mastrevirus and Turncurtovirus are transmitted by specific leafhoppers, the single member of the genus Topocuvirus is transmitted by a treehopper and one member of the genus Capulavirus is transmitted by an aphid. Geminiviruses are plant pathogens causing economically important diseases in most tropical and subtropical regions of the world. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Geminiviridae which is available at www.ictv.global/report/geminiviridae.

Capulavirus and Grablovirus: two new genera in the family Geminiviridae.

Geminiviruses are plant-infecting single-stranded DNA viruses that occur in most parts of the world. Currently, there are seven genera within the family Geminiviridae (Becurtovirus, Begomovirus, Curtovirus, Eragrovirus, Mastrevirus, Topocuvirus and Turncurtovirus). The rate of discovery of new geminiviruses has increased significantly over the last decade as a result of new molecular tools and approaches (rolling-circle amplification and deep sequencing) that allow for high-throughput workflows. Here, we report the establishment of two new genera: Capulavirus, with four new species (Alfalfa leaf curl virus, Euphorbia caput-medusae latent virus, French bean severe leaf curl virus and Plantago lanceolata latent virus), and Grablovirus, with one new species (Grapevine red blotch virus). The aphid species Aphis craccivora has been shown to be a vector for Alfalfa leaf curl virus, and the treehopper species Spissistilus festinus is the likely vector of Grapevine red blotch virus. In addition, two highly divergent groups of viruses found infecting citrus and mulberry plants have been assigned to the new species Citrus chlorotic dwarf associated virus and Mulberry mosaic dwarf associated virus, respectively. These species have been left unassigned to a genus by the ICTV because their particle morphology and insect vectors are unknown.

Further changes in the cotton leaf curl disease complex: an indication of things to come?

Cotton leaf curl disease (CLCuD) has been a problem for cotton production in Pakistan and India since the early 1990s. The disease is caused by begomoviruses associated with a specific satellite, the cotton leaf curl Multan betasatellite (CLCuMB). In 2001, resistance introduced into cotton was broken by a recombinant begomovirus, Cotton leaf curl Kokhran virus strain Burewala (CLCuKoV-Bur). Unusually, in resistant cotton, this virus lacked an intact transcriptional activator protein (TrAP) gene, with the capacity to encode only 35 of the usual ~134 amino acids. Recently, isolates of CLCuKoV-Bur with a longer, but still truncated, TrAP gene have been identified in cotton breeding lines lacking the earlier resistance. This suggests that more pathogenic viruses with a full TrAP could return to cotton if the earlier resistance is not maintained in ongoing breeding efforts to produce CLCuD-resistant cotton varieties. This conclusion is supported by recent studies showing the reappearance of pre-resistance-breaking begomoviruses, with full-length TrAP genes, in cotton.

The 35-amino acid C2 protein of Cotton leaf curl Kokhran virus, Burewala, implicated in resistance breaking in cotton, retains some activities of the full-length protein.

With one exception, all the begomoviruses characterized so far encode an ~134-amino acid (aa) (A)C2 protein. The exception is the "Burewala" strain of Cotton leaf curl Kokhran virus (CLCuKoV-Bu), associated with resistance breaking in cotton across Pakistan and northwestern India, that encodes a truncated 35-aa C2. The C2 protein encoded by begomoviruses performs multiple functions including suppression of post-transcriptional gene silencing (PTGS), modulating microRNA (miRNA) expression and may be a pathogenicity determinant. The study described here was designed to investigate whether the CLCuKoV-Bu 35-aa C2 retains the activities of the full-length C2 protein. The results showed the 35-aa C2 of CLCuKoV-Bu acts as a pathogenicity determinant, suppresses PTGS and upregulates miRNA expression when expressed from a Potato virus X vector in Nicotiana benthamiana. The symptoms induced by expression of full-length C2 were more severe than those induced by the 35-aa C2. The accumulation of most developmental miRNAs decreases with the full-length C2 protein and increases with the 35-aa peptide of CLCuKoV-Bu. The study also revealed that 35-aa peptide of CLCuKoV-Bu maintains suppressor of silencing activity at a level equal to that of full-length C2. The significance of the results with respect to virus fitness and resistance breaking is discussed.

RNA interference-based resistance in transgenic tomato plants against Tomato yellow leaf curl virus-Oman (TYLCV-OM) and its associated betasatellite.

BACKGROUND: Tomato yellow leaf curl virus (TYLCV), a monopartite begomovirus (family Geminiviridae) is responsible for heavy yield losses for tomato production around the globe. In Oman at least five distinct begomoviruses cause disease in tomato, including TYLCV. Unusually, TYLCV infections in Oman are sometimes associated with a betasatellite (Tomato leaf curl betasatellite [ToLCB]; a symptom modulating satellite). RNA interference (RNAi) can be used to develop resistance against begomoviruses at either the transcriptional or post-transcriptional levels. RESULTS: A hairpin RNAi (hpRNAi) construct to express double-stranded RNA homologous to sequences of the intergenic region, coat protein gene, V2 gene and replication-associated gene of Tomato yellow leaf curl virus-Oman (TYLCV-OM) was produced. Initially, transient expression of the hpRNAi construct at the site of virus inoculation was shown to reduce the number of plants developing symptoms when inoculated with either TYLCV-OM or TYLCV-OM with ToLCB-OM to Nicotiana benthamiana or tomato. Solanum lycopersicum L. cv. Pusa Ruby was transformed with the hpRNAi construct and nine confirmed transgenic lines were obtained and challenged with TYLCV-OM and ToLCB-OM by Agrobacterium-mediated inoculation. For all but one line, for which all plants remained symptomless, inoculation with TYLCV-OM led to a proportion (≤25%) of tomato plants developing symptoms of infection. For inoculation with TYLCV-OM and ToLCB-OM all lines showed a proportion of plants (≤45%) symptomatic. However, for all infected transgenic plants the symptoms were milder and virus titre in plants was lower than in infected non-transgenic tomato plants. CONCLUSIONS: These results show that RNAi can be used to develop resistance against geminiviruses in tomato. The resistance in this case is not immunity but does reduce the severity of infections and virus titer. Also, the betasatellite may compromise resistance, increasing the proportion of plants which ultimately show symptoms.

Identification of an Australian-like dicot-infecting mastrevirus in Pakistan.

Although members of five distinct viral species in the genus Mastrevirus (family Geminiviridae) infect dicotyledonous plants in Australia, in the remainder of the world, only a single dicot-infecting mastrevirus, chickpea chlorotic dwarf virus (CpCDV) has ever been identified. This virus has been found infecting leguminous hosts in Africa, the Middle East and the Indian subcontinent. To further explore the diversity of CpCDV in Pakistan, ten full mastrevirus genome sequences from chickpea and lentil plants were determined. Eight of these genomes were from previously described CpCDV strains and included the first reported strain D and H isolates in Pakistan. Two other genomes derived from infected chickpea plants are more closely related to dicot-infecting mastreviruses found in Australia than they are to CpCDV. These two divergent genomes shared less than 75 % genome-wide nucleotide sequence identity with other characterised mastreviruses and therefore are likely to belong to a second species of dicot-infecting mastreviruses outside of Australia. We propose naming this species Chickpea yellow dwarf virus. We discuss how the presence of chickpea yellow dwarf virus (CpYDV) in Pakistan weakens the hypothesis that Australia is the geographical origin of the dicot-infecting mastreviruses.

Revision of Begomovirus taxonomy based on pairwise sequence comparisons.

Viruses of the genus Begomovirus (family Geminiviridae) are emergent pathogens of crops throughout the tropical and subtropical regions of the world. By virtue of having a small DNA genome that is easily cloned, and due to the recent innovations in cloning and low-cost sequencing, there has been a dramatic increase in the number of available begomovirus genome sequences. Even so, most of the available sequences have been obtained from cultivated plants and are likely a small and phylogenetically unrepresentative sample of begomovirus diversity, a factor constraining taxonomic decisions such as the establishment of operationally useful species demarcation criteria. In addition, problems in assigning new viruses to established species have highlighted shortcomings in the previously recommended mechanism of species demarcation. Based on the analysis of 3,123 full-length begomovirus genome (or DNA-A component) sequences available in public databases as of December 2012, a set of revised guidelines for the classification and nomenclature of begomoviruses are proposed. The guidelines primarily consider a) genus-level biological characteristics and b) results obtained using a standardized classification tool, Sequence Demarcation Tool, which performs pairwise sequence alignments and identity calculations. These guidelines are consistent with the recently published recommendations for the genera Mastrevirus and Curtovirus of the family Geminiviridae. Genome-wide pairwise identities of 91 % and 94 % are proposed as the demarcation threshold for begomoviruses belonging to different species and strains, respectively. Procedures and guidelines are outlined for resolving conflicts that may arise when assigning species and strains to categories wherever the pairwise identity falls on or very near the demarcation threshold value.

Identification of a distinct strain of cotton leaf curl Burewala virus.

Cotton leaf curl disease (CLCuD) is the major biotic limitation to cotton production in Pakistan and northwestern India. The disease is caused by various distinct viruses of the genus Begomovirus (family Geminiviridae) in association with a disease-specific betasatellite - cotton leaf curl Multan betasatellite (CLCuMB). Since 2001, when resistance to CLCuD in cotton was broken, only one virus was consistently identified in cotton exhibiting CLCuD symptoms in Punjab province (Pakistan) - cotton leaf curl Burewala virus (CLCuBuV). An analysis of all CLCuBuV isolates available in the databases showed these to represent only a single strain, based on currently applicable criteria. Virus and betasatellite clones were obtained from a leaf sample (isolate C49) collected from a CLCuD symptomatic cotton plant in Layyah district, Punjab province, in 2012. Analysis of the sequence of the betasatellite showed this to be an isolate of CLCuMB containing the recombinant fragment typical of this satellite post-resistance-breaking. The virus was shown it to be an isolate of CLCuBuV but to be distinct from all previously characterised isolates and to represent a distinct strain. In common with previous CLCuBuV isolates, the virus from C49 is a recombinant containing sequences derived from viruses of two species that were prevalent in cotton pre-resistance-breaking but with distinct recombination break sites. As was the case with the earlier CLCuBuV, the newly identified strain of CLCuBuV lacks an intact transcriptional activator protein.

Identification of a disease complex involving a novel monopartite begomovirus with beta- and alphasatellites associated with okra leaf curl disease in Oman.

Okra leaf curl disease (OLCD) is an important viral disease of okra in tropical and subtropical areas. The disease is caused by begomovirus-satellite complexes. A begomovirus and associated betasatellite and alphasatellite were identified in symptomatic okra plants from Barka, in the Al-Batinah region of Oman. Analysis of the begomovirus sequences showed them to represent a new begomovirus most closely related to cotton leaf curl Gezira virus (CLCuGeV), a begomovirus of African origin. The sequences showed less than 85 % nucleotide sequence identity to CLCuGeV isolates. The name okra leaf curl Oman virus (OLCOMV) is proposed for the new virus. Further analysis revealed that the OLCOMV is a recombinant begomovirus that evolved by the recombination of CLCuGeV isolates with tomato yellow leaf curl virus-Oman (TYLCV-OM). An alpha- and a betasatellite were also identified from the same plant sample, which were also unique when compared to sequences available in the databases. However, although the betasatellite appeared to be of African origin, the alphasatellite was most closely related to alphasatellites originating from South Asia. This is the first report of a begomovirus-satellite complex infecting okra in Oman.

A distinct strain of chickpea chlorotic dwarf virus (genus Mastrevirus, family Geminiviridae) identified in cotton plants affected by leaf curl disease.

As part of a study to determine the diversity of whitefly-transmitted viruses (genus Begomovirus, family Geminiviridae) associated with cotton leaf curl disease in Pakistan, leaf samples from cotton plants showing typical leaf curl disease symptoms were collected in various locations of Punjab province. Sequence analysis of full-length virus clones (~2.7 kb) showed plants to be infected with the begomovirus cotton leaf curl Burewala virus, the only virus identified in cotton in the Punjab since 2001. Surprisingly, a second virus, the leafhopper-transmitted chickpea chlorotic dwarf virus (CpCDV) of the genus Mastrevirus (family Geminiviridae), was identified in a small number of plants. The sequences of four CpCDV isolates from cotton originating from geographically distinct areas in Punjab were obtained. Analysis of the sequences showed them to represent a distinct, newly identified strain of CpCDV with the highest levels of nucleotide sequence identity to isolates of CpCDV strains C and D that have been identified previously in Pakistan. CpCDV has not been identified previously in cotton. The significance of this finding is discussed.

Recent evolution of a novel begomovirus causing tomato leaf curl disease in the Al-Batinah region of Oman.

For last two decades, begomoviruses (family Geminiviridae) have been a major constraint for tomato production in Oman, particularly in the Al-Batinah region, the major agricultural area of Oman. Farms in the Al-Batinah region were surveyed during January-March and November-December in 2012 and January-February in 2013. Leaf samples of tomato plants showing typical leaf curl disease symptoms were collected and analyzed for begomoviruses. Out of fifteen begomovirus clones sequenced, seven were shown to be tomato yellow leaf curl virus strain Oman (TYLCV-OM); three, chili leaf curl virus strain Oman (ChLCV-OM); and one, tomato leaf curl Oman virus (ToLCOMV) - viruses that have previously been shown to occur in Oman. Four sequences were shown to have relatively low percent identity values to known begomoviruses, with the highest (86 %) to isolates of pepper leaf curl Lahore virus, indicating that these should be included in a new species, for which the name "Tomato leaf curl Al Batinah virus" (ToLCABV) is proposed. Although the betasatellite tomato leaf curl betasatellite (ToLCB; 7 full-length sequences isolated) was identified with some isolates of ChLCV-OM, TYLCV-OM and ToLCOMV, it was not identified in association with any of the ToLCABV isolates. Analysis of the sequences of the TYLCV-OM and ToLCOMV isolates characterized here did not show them to differ significantly from previously characterized isolates of these viruses. The three isolates of ChLCV-OM characterized were shown to have a recombination pattern distinct from earlier characterized isolates. ToLCABV was shown to have resulted from recombination between ChLCV-OM and ToLCOMV. A clone of ToLCABV was infectious by Agrobacterium-mediated inoculation to Nicotiana benthamiana and tomato, inducing symptoms typical of those seen in tomato in the field. Additionally, ToLCABV was shown to be able to interact in planta with ToLCB, resulting in a change in symptom phenotype, although the betasatellite did not appear to affect viral DNA levels.

Temporal changes in the levels of virus and betasatellite DNA in B. tabaci feeding on CLCuD affected cotton during the growing season.

Cotton, a key source of income for Pakistan, has suffered significantly by cotton leaf curl disease (CLCuD) since 1990. This disease is caused by a complex of phylogenetically-related begomovirus (genus Begomovirus, family Geminiviridae) species and a specific betasatellite (genus Betasatellite, family Tolecusatellitidae), cotton leaf curl Multan betasatellite. Additionally, another DNA satellite called alphasatellite (family Alphasatellitidae), is also frequently associated. All these virus components are vectored by a single species of whitefly (Bemisia tabaci). While many factors affect cotton productivity, including cotton variety, sowing time, and environmental cues such as temperature, humidity, and rainfall, CLCuD is a major biotic constraint. Although the understanding of begomoviruses transmission by whiteflies has advanced significantly over the past three decades, however, the in-field seasonal dynamics of the viruses in the insect vector remained an enigma. This study aimed to assess the levels of virus and betasatellite in whiteflies collected from cotton plants throughout the cotton growing season from 2014 to 2016. Notably, begomovirus levels showed no consistent pattern, with minimal variations, ranging from 0.0017 to 0.0074 ng.µg-1 of the genomic DNA in 2014, 0.0356 to 0.113 ng.µg-1 of the genomic DNA in 2015, and 0.0517 to 0.0791 ng.µg-1 of the genomic DNA in 2016. However, betasatellite levels exhibited a distinct pattern. During 2014 and 2015, it steadily increased throughout the sampling period (May to September). While 2016 showed a similar trend from the start of sampling (July) to September but a decline in October (end of sampling). Such a study has not been conducted previously, and could potentially provide valuable insights about the epidemiology of the virus complex causing CLCuD and possible means of controlling losses due to it.

Cotton leaf curl disease - an emerging threat to cotton production worldwide.

Cotton leaf curl disease (CLCuD) is a serious disease of cotton which has characteristic symptoms, the most unusual of which is the formation of leaf-like enations on the undersides of leaves. The disease is caused by whitefly-transmitted geminiviruses (family Geminiviridae, genus Begomovirus) in association with specific, symptom-modulating satellites (betasatellites) and an evolutionarily distinct group of satellite-like molecules known as alphasatellites. CLCuD occurs across Africa as well as in Pakistan and north-western India. Over the past 25 years, Pakistan and India have experienced two epidemics of the disease, the most recent of which involved a virus and satellite that are resistance breaking. Loss of this conventional host-plant resistance, which saved the cotton growers from ruin in the late 1990s, leaves farmers with only relatively poor host plant tolerance to counter the extensive losses the disease causes. There has always been the fear that CLCuD could spread from the relatively limited geographical range it encompasses at present to other cotton-growing areas of the world where, although the disease is not present, the environmental conditions are suitable for its establishment and the whitefly vector occurs. Unfortunately recent events have shown this fear to be well founded, with CLCuD making its first appearance in China. Here, we outline recent advances made in understanding the molecular biology of the components of the disease complex, their interactions with host plants, as well as efforts being made to control CLCuD.

A recombinant begomovirus resulting from exchange of the C4 gene.

A begomovirus isolated from Malvastrum coromandelianum and tomato originating from Yunnan province (China) was shown to be representative of a new begomovirus species, for which the name tomato leaf curl Yunnan virus (TLCYnV) is proposed. TLCYnV has high levels of sequence identity to tomato yellow leaf curl China virus (TYLCCNV) across the whole genome, except for sequences encompassing the C4 gene. Agrobacterium-mediated inoculation showed TLCYnV to be highly infectious to a range of plant species but poorly infectious to M. coromandelianum. In contrast to TYLCCNV, TLCYnV was shown to infect tomato in the absence of a betasatellite. In field-collected samples, TLCYnV was identified most frequently in tomato in which it was not associated with a betasatellite. Transgenic expression in Nicotiana benthamiana showed that the C4 protein of TYLCCNV did not induce developmental abnormalities, whereas the C4 of TLCYnV induced severe developmental abnormalities, reminiscent of virus symptoms. The genome of TLCYnV was shown to be significantly less methylated in plants than that of TYLCCNV and the C4 protein of TLCYnV was shown to suppress post-transcriptional gene silencing and transcriptional gene silencing more effectively than the C4 of TYLCCNV. The results indicate that TLCYnV evolved from TYLCCNV by recombination, acquiring a more virulent C4, allowing it to dispense with the requirement for a betasatellite. The implications of these findings in relation to the evolution of monopartite begomoviruses are discussed.