Begomovirus-betasatellite Complexes Research Articles

The Role of Satellites in the Evolution of Begomoviruses.

Begomoviruses have emerged as destructive pathogens of crops, particularly in the tropics and subtropics, causing enormous economic losses and threatening food security. Epidemics caused by begomoviruses have even spread in regions and crops that were previously free from these viruses. The most seriously affected crops include cassava; cotton; grain legumes; and cucurbitaceous, malvaceous, and solanaceous vegetables. Alphasatellites, betasatellites, and deltasatellites are associated with the diseases caused by begomoviruses, but begomovirus-betasatellite complexes have played significant roles in the evolution of begomoviruses, causing widespread epidemics in many economically important crops throughout the world. This article provides an overview of the evolution, distribution, and approaches used by betasatellites in the suppression of host plant defense responses and increasing disease severity.

Sentinel plot surveillance of cotton leaf curl disease in Pakistan- a case study at the cultivated cotton-wild host plant interface

A sentinel plot case study was carried out to identify and map the distribution of begomovirus-betasatellite complexes in sentinel plots and commercial cotton fields over a four-year period using molecular and high-throughput DNA ‘discovery’ sequencing approaches. Samples were collected from 15 study sites in the two major cotton-producing areas of Pakistan. Whitefly- and leafhopper-transmitted geminiviruses were detected in previously unreported host plant species and locations. The most prevalent begomovirus was cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu). Unexpectedly, a recently recognized recombinant, cotton leaf curl Multan virus-Rajasthan (CLCuMuV-Ra) was prevalent in five of 15 sites. cotton leaf curl Alabad virus (CLCuAlV) and cotton leaf curl Kokhran virus-Kokhran, ‘core’ members of CLCuD-begomoviruses that co-occurred with CLCuMuV in the ‘Multan’ epidemic were detected in one of 15 sentinel plots. Also identified were chickpea chlorotic dwarf virus and ‘non-core’ CLCuD-begomoviruses, okra enation leaf curl virus, squash leaf curl virus, and tomato leaf curl New Delhi virus. Cotton leaf curl Multan betasatellite (CLCuMuB) was the most prevalent CLCuD-betasatellite, and less commonly, two ‘non-core’ betasatellites. Recombination analysis revealed previously uncharacterized recombinants among helper virus-betasatellite complexes consisting of CLCuKoV, CLCuMuV, CLCuAlV and CLCuMuB. Population analyses provided early evidence for CLCuMuV-Ra expansion and displacement of CLCuKoV-Bu in India and Pakistan from 2012-2017. Identification of ‘core’ and non-core CLCuD-species/strains in cotton and other potential reservoirs, and presence of the now predominant CLCuMuV-Ra strain are indicative of ongoing diversification. Investigating the phylodynamics of geminivirus emergence in cotton-vegetable cropping systems offers an opportunity to understand the driving forces underlying disease outbreaks and reconcile viral evolution with epidemiological relationships that also capture pathogen population shifts.

Temporal Dynamic of the Ratio between Monopartite Begomoviruses and Their Associated Betasatellites in Plants, and Its Modulation by the Viral Gene βC1.

The begomovirus-betasatellite complex constantly threatens crops in Asia. However, the quantitative relationship between begomoviruses and betasatellites remains largely unknown. The quantities of tobacco curly shoot virus (TbCSV) and its betasatellite (TbCSB) and their ratio varied significantly in initial infection, and thereafter, the ratio tended to become constant. The TbCSB/TbCSV ratio in agrobacteria inoculum significantly affected that in plants in the initial infection but not thereafter. Null-mutation of βC1 that encodes a multifunctional protein important for pathogenesis in TbCSB significantly reduced the TbCSB/TbCSV ratio in plants. Viral inoculum plants with higher TbCSB/TbCSV ratios promoted whitefly transmission of the virus. The expression of AV1 encoded by TbCSV, βC1 encoded by TbCSB and the βC1/AV1 ratio varied significantly in the initial infection and thereafter the ratio tended to become constant. Additionally, the temporal dynamics of the ratio between another begomovirus and its betasatellite was similar to that of TbCSV and was positively regulated by βC1. These results indicate that the ratio between monopartite begomoviruses and betasatellites tend to become constant as infection progresses, and is modulated by βC1, but a higher betasatellite/begomovirus ratio in virally inoculated plants promotes virus transmission by whiteflies. Our findings provide novel insights into the association between begomoviruses and betasatellites.

Diverse begomovirus-betasatellite complexes cause tomato leaf curl disease in the western India

In the Indian sub-continent, tomato leaf curl disease (ToLCD) of tomato caused by begomoviruses has emerged as a major limiting factor for tomato cultivation. Despite the spread of this disease in the western India, a systematic study on the characterization of virus complexes with ToLCD is lacking. Here, we report the identification of a complex of begomoviruses including 19 DNA-A and 4 DNA-B as well as 15 betasatellites with ToLCD in the western part of the country. Additionally, a novel betasatellite and an alphasatellite were also identified. The recombination breakpoints were detected in the cloned begomoviruses and betasatellites. The cloned infectious DNA constructs cause disease on the tomato (a moderately virus-resistant cultivar) plants, thus fulfilling Koch's postulates for these virus complexes. Further, the role of non-cognate DNA B/betasatellite with ToLCD-associated begomoviruses on disease development was demonstrated. It also emphasizes the evolutionary potential of these virus complexes in breaking disease resistance and plausible expansion of its host range. This necessitates to investigate the mechanism of the interaction between resistance breaking virus complexes and the infected host.

Brugmansia arborea reported as a new host of a begomovirus and betasatellite from Pakistan

Symptoms of severe leaf curling, typical of begomovirus infection, were observed on a plant of Brugmansia arborea grown in lawns of Quaid-i-Azam university Islamabad, Pakistan. A begomovirus and its associated betasatellite were amplified and sequenced from infected plants. Nucleotide sequence showed that a monopartite begomovirus named Chilli leaf curl virus (ChLCV) and Chilli leaf curl betasatellite (ChLCB) are associated with the disease and it shared highest sequence identity 99% and 91% respectively with previously reported clones. Our data adds to recent reports that show an expansion of the host range of begomovirus-betasatellite complexes in the Old World.

Molecular characterization and identification of bhendi yellow vein mosaic virus with satellite DNAs infecting okra plants of district Mardan, Pakistan

Abstract Background Bhendi or okra (Abelmoschus esculentus) is an important crop widely cultivated in the Indian subcontinent. The production of okra in tropical regions is constrained by several abiotic and biotic factors. Among biotic stresses, yellow vein mosaic disease (YVMD), transmitted by the whitefly (Bemisia tabaci Genn.), causes significant production losses. Results Leaves showing symptoms of YVMD were collected from okra plants in the district of Mardan, Pakistan, from which the total nucleic acid was extracted. It was found that the viral genome was 2739 bp in the length and had seven conserved open reading frames, comparable to those of monopartite begomovirus species from the Old World. Nucleotide sequence comparison revealed that the genome has 97.7% identity with bhendi yellow vein mosaic virus (BYVMV). Additionally, alpha- and betasatellite DNA components were amplified and sequenced. The alpha- and betasatellite DNA sequences were 1367 and 1346 nt in length, respectively. Sequence analysis revealed that the alpha- and betasatellite sequences shared 97.9 and 98.7% similarity with cotton leaf curl Multan alphasatellite DNA (CLCuMuA) and croton yellow vein mosaic betasatellite DNA (CroYVMB), respectively. Conclusions Based on the prevailing classification system, the isolate was identified as a variant of BYVMV, CLCuMuA, and CroYVB. Alphasatellite presence in the begomovirus betasatellite complex detected in the present study indicated a recent mobilization into the viral complex infecting okra in this region. The study findings may facilitate the design of new management strategies to protect this valuable crop against begomovirus infection.

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.

Two distinct monopartite begomovirus-betasatellite complexes in western India cause tomato leaf curl disease

In India, begomovirus infection causing tomato leaf curl disease (ToLCD) is a major constraint for tomato productivity. Here, we have identified two distinct monopartite begomovirus and betasatellite complexes causing ToLCD in the western part of India. A new monopartite begomovirus (Tomato leaf curl Mumbai virus, ToLCMumV) and betasatellite (Tomato leaf curl Mumbai betasatellite, ToLCMumB) were isolated from the Mumbai sample. A distinct Tomato leaf curl Gandhinagar virus (ToLCGanV) and Tomato leaf curl Gandhinagar betasatellite (ToLCGanB) were identified from the Gandhinagar sample. Both of the cloned begomoviruses were recombinants. The demonstration of systemic infection caused by begomovirus (ToLCGanV or ToLCMumV) alone in N. benthamiana and tomato (a virus resistant variety) emphasizes that they were monopartite begomoviruses. Co-inoculation of cognate begomovirus and betasatellite reduces the incubation period and increases symptom severity. Thus, Koch’s postulates were satisfied for these virus complexes. Further, an enhanced accumulation of ToLCGanV was detected in the presence of cognate ToLCGanB, however ToLCMumB did not influence the level of ToLCMumV in the agro-inoculated tomato plants. Our results indicate that the cloned viruses form potential virus resistance breaking disease complexes in India. This necessitates to investigate the spread of these disease complexes to major tomato growing regions in the country.

Characterization of a Begomovirus-Betasatellite Complex, Producing Defective Molecules in Spinach (Spinacia oleracea L.), a New Host for Begomovirus and Betasatellite Complex in Pakistan.

Spinach is a vegetable crop which is widely grown over a large area especially in Punjab province of Pakistan. Leaf curling and enations on spinach plant collected shown to be associated with the begomovirus Pedilanthus leaf curl virus (PeLCV) and Shahdadpur strain of Cotton leaf curl Multan betasatellite (CLCuMBSha). Defective molecules of half and quarter size derived from monopartite begomoviruses are usually generated by the deletion of virion-sense sequences. Characterization of defective molecules of PeLCV from spinach revealed that the molecules of half the size are derived from the deletion of complementary-sense genes while quarter size molecule appears to have evolved by further deletion. This is the first report of the begomovirus-betasatellite complex on spinach and unusual defective molecules derived from deletion of complementary-sense genes in Pakistan.

Association of chili leaf curl betasatellite with tomato leaf curl disease

Leaf samples of Solanum lycopersicum exhibiting the tomato leaf curl disease (ToLCD) symptoms were collected from different genotypes. PCR amplifications confirmed the presence of Tomato leaf curl New Delhi virus (ToLCNDV) and betasatellite. Molecular analysis of betasatellite nucleotide sequences showed high level of sequence identity (99%) with chili leaf curl betasatellite (ChLCB) previously reported from chili in Pakistan. To the best of our knowledge, this is the first natural occurrence of ChLCB with ToLCD in Pakistan. Therefore, the association of ToLCNDV with ChLCB is also a component of begomovirus-betasatellite complex that may also affect other cropping systems.

Occurrence of a recombinant molecule carrying sequences derived from an alphasatellite and the helper virus in cotton affected with cotton leaf curl disease

Cotton leaf curl disease (CLCuD), a serious constraint to cotton production across Pakistan and northwestern India, is caused by begomoviruses in association with a specific betasatellite, Cotton leaf curl Multan betasatellite (CLCuMB). Begomovirus-betasatellite complexes are also frequently associated with satellite-like components known as alphasatellites, the most commonly identified of which, in cotton, is Cotton leaf curl Multan alphasatellite (CLCuMA). In Pakistan, the Burewala strain of Cotton leaf curl Kokhran virus (CLCuKoV-Bu) is the only begomovirus consistently identified in CLCuD affected cotton. Analysis of a CLCuD-affected cotton plant originating from Bhakkar (Punjab province, Pakistan) showed the presence of CLCuKoV-Bu, CLCuMB and CLCuMA, which were all cloned. However, a further clone obtained was shown to be a recombinant molecule consisting of sequences derived from CLCuMA, a small amount of sequence derived from a second alphasatellite and sequence derived from CLCuKoV-Layyah, another strain of the virus that is less frequently encountered. This study highlights the ease with which sequences can be exchanged between components of begomovirus-betasatellite-alphasatellite complexes.

Molecular diagnostic development for begomovirus-betasatellite complexes undergoing diversification: A case study

At least five begomoviral species that cause leaf curl disease of cotton have emerged recently in Asia and Africa, reducing fiber quality and yield. The potential for the spread of these viruses to other cotton-vegetable growing regions throughout the world is extensive, owing to routine, global transport of alternative hosts of the leaf curl viruses, especially ornamentals. The research reported here describes the design and validation of polymerase chain reaction (PCR) primers undertaken to facilitate molecular detection of the two most-prevalent leaf curl-associated begomovirus-betasatellite complexes in the Indian Subcontinent and Africa, the Cotton leaf curl Kokhran virus-Burewala strain and Cotton leaf curl Gezira virus, endemic to Asia and Africa, respectively. Ongoing genomic diversification of these begomoviral-satellite complexes was evident based on nucleotide sequence alignments, and analysis of single nucleotide polymorphisms, both factors that created new challenges for primer design. The additional requirement for species and strain-specific, and betasatellite-specific primer design, imposes further constraints on primer design and validation due to the large number of related species and strains extant in ‘core leaf curl virus complex’, now with expanded distribution in south Asia, the Pacific region, and Africa-Arabian Peninsula that have relatively highly conserved coding and non-coding regions, which precludes much of the genome-betasatellite sequence when selecting primer ‘targets’. Here, PCR primers were successfully designed and validated for detection of cloned viral genomes and betasatellites for representative ‘core leaf curl’ strains and species, distant relatives, and total DNA isolated from selected plant species. The application of molecular diagnostics to screen plant imports prior to export or release from ports of entry is expected to greatly reduce the likelihood of exotic leaf curl virus introductions that could dramatically affect the production of cotton as well as vegetable and ornamental crop hosts.

Molecular diversity, recombination and population structure of alphasatellites associated with begomovirus disease complexes

The genus, begomovirus (family Geminiviridae) includes a large number of viruses infecting a wide range of plant species worldwide. The majority of monopartite begomoviruses are associated with satellites (betasatellites) and/or satellite-like molecules (alphasatellites). In spite of the Indo-China region being regarded as the centre of origin of begomoviruses and satellites, a detailed study on the emergence and evolution of alphasatellites in India has not yet conducted. Our present analysis indicated the association of 22 alphasatellites with monopartite and bipartite begomovirus-betasatellite complexes in India. Based on sequence pairwise identity, these alphasatellites were categorized into five distinct groups: Cotton leaf curl alphasatellite, Gossypium darwinii symptomless alphasatellite, Gossypium mustelinum symptomless alphasatellite, Okra leaf curl alphasatellite and an unreported Chilli leaf curl alphasatellite (ChiLCA). Furthermore, infectivity analysis of the cloned ChiLCA along with the viral components of either cognate or non-cognate chilli-infecting begomoviruses on Nicotiana benthamiana suggested that ChiLCA is dispensable for leaf curl disease development. It is noteworthy that in the presence of ChiLCA, a marginal decrease in betasatellite DNA level was noticed. Additionally, high genetic variability and diverse recombination patterns were detected among these alphasatellites, and the nucleotide substitution rate for the Rep gene of ChiLCA was determined to be 2.25×10−3nucleotides/site/year. This study highlights the genetic distribution, and likely contribution of recombination and nucleotide diversity in facilitating the emergence of alphasatellites.

A novel East African monopartite begomovirus-betasatellite complex that infects Vernonia amygdalina

The complete genomes of a monopartite begomovirus (genus Begomovirus, family Geminiviridae) and an associated betasatellite found infecting Vernonia amygdalina Delile (family Compositae) in Uganda were cloned and sequenced. Begomoviruses isolated from two samples showed the highest nucleotide sequence identity (73.1% and 73.2%) to an isolate of the monopartite begomovirus tomato leaf curl Vietnam virus, and betasatellites from the same samples exhibited the highest nucleotide sequence identity (67.1% and 68.2%) to vernonia yellow vein Fujian betasatellite. Following the current taxonomic criteria for begomovirus species demarcation, the isolates sequenced here represent a novel begomovirus species. Based on symptoms observed in the field, we propose the name vernonia crinkle virus (VeCrV) for this novel begomovirus and vernonia crinkle betasatellite (VeCrB) for the associated betasatellite. This is the first report of a monopartite begomovirus-betasatellite complex from Uganda.

Characterization of a new begomovirus and betasatellite associated with chilli leaf curl disease in India.

Chilli leaf curl disease (ChiLCD) is a serious problem and a major limitation to chilli (Capsicum spp.) cultivation in India. Leaves of a chilli plant showing leaf curl symptoms were collected from the Gonda district in Uttar Pradesh, India, in April, 2013. Full-length genomes of a begomovirus and an associated betasatellite were amplified, cloned and sequenced. The size of the begomovirus genome and the betasatellite were 2760 bp and 1374 bp, respectively. The nucleotide sequence of the begomovirus genome shared maximum identity (89%) with pepper leaf curl Bangladesh virus-India isolate Chhapra (PepLCBV, JN663853), below the threshold for species demarcation. Sequence analysis showed that the begomovirus is a potential recombinant between viruses related to PepLCBV and chilli leaf curl virus (ChiLCV). The name chilli leaf curl Gonda virus (ChiLCGV) is being proposed. The betasatellite associated with ChiLCGV was identified as tomato leaf curl Bangladesh betasatellite (ToLCBDB). Agroinoculation of the viral genome along with betasatellite induced severe leaf curl symptoms in Nicotiana benthamiana. ChiLCGV and ToLCBDB characterized in this study represent a new begomovirus-betasatellite complex infecting Capsicum.

Role of Alphasatellite in Begomoviral Disease Complex

A circular single stranded satellite, called as alphasatellite (initially identified as DNA-1), wascharacterised and confirmed to be associated with the Geminivirus begomovirus-betasatellite complexes.Alphasatellites are single stranded DNA (ssDNA) components, frequently accompanying with monopartitebegomovirus or some time with bipartite begomovirus and/or betasatellite complex. The genome ofalphasatellite DNAs are nearly half size of its helper virus genome and have no similarity with it. Furthermore,their function in begomovirus-betasatellite complex is still unclear. Recent advances in application ofmolecular tools helped in finding new viruses and allied satellite components that further help in advancingour understanding o f this satellite DNA and this evolution

Sesbania bispinosa, a new host of a begomovirus-betasatellite complex in Pakistan

Symptoms of severe leaf curling, yellowing and vein thickening, typical of begomovirus infection, were observed on Sesbania bispinosa grown in cotton leaf curl disease affected cotton fields in Pakistan. A begomovirus and its associated betasatellite were amplified and sequenced from these affected plants. Complete nucleotide sequence showed that a monopartite begomovirus named Pedilanthus leaf curl virus (PeLCV) is associated with the disease and it shared highest sequence identity (98.73%) with a clone previously reported from soybean. Characterization of the betasatellite showed that it had highest sequence identity (96–97%) with Tobacco leaf curl betasatellite (TbLCB), previously isolated from soybean. Our data shows the expansion of the host range of begomovirus-betasatellite complexes. To the best of our knowledge, this is the first occurrence of PeLCV and TbLCB on S. bispinosa.

Three gene products of a begomovirus-betasatellite complex restore expression of a transcriptionally silenced green fluorescent protein transgene in Nicotiana benthamiana.

Single-stranded DNA geminiviruses replicate via double-stranded DNA intermediates forming mini-chromosomes that are targets for transcriptional gene silencing (TGS) in plants. The ability of the cotton leaf curl Kokhran virus (CLCuKoV)-cotton leaf curl Multan betasatellite (CLCuMuB) proteins, replication-associated protein (Rep), transcriptional activator protein (TrAP), C4, V2 and βC1, to suppress TGS was investigated by using the Nicotiana benthamiana line 16-TGS (16-TGS) harbouring a transcriptionally silenced green fluorescent protein (GFP) transgene. Inoculation of 16-TGS plants with a recombinant potato virus X vector carrying Rep, TrAP or βC1 resulted in re-expression of GFP. Northern blot analysis confirmed that the observed GFP fluorescence was associated with GFP mRNA accumulation. These results indicated that Rep, TrAP and βC1 proteins of CLCuKoV-CLCuMuB can re-activate the expression of a transcriptionally silenced GFP transgene in N. benthamiana. Although Rep, TrAP, or βC1 proteins have, for other begomoviruses or begomoviruses-betasatellites, been previously shown to have TGS suppressor activity, this is the first report demonstrating that a single begomovirus-betasatellite complex encodes three suppressors of TGS.

Advances in Understanding Begomovirus Satellites

Begomoviruses are numerous and geographically widespread viruses that cause devastating diseases in many crops. Monopartite begomoviruses are frequently associated with betasatellites or alphasatellites. Both betasatellite and alphasatellite DNA genomes are approximately half the size of begomovirus DNA genomes. Betasatellites are essential for induction of typical disease symptoms. The βC1 genes encoded by the betasatellites have important roles in symptom induction, in suppression of transcriptional and posttranscriptional gene silencing, and they can affect jasmonic acid responsive genes. Host plants of begomoviruses have evolved diverse innate defense mechanisms against the βC1 protein to counter these challenges. Alphasatellites have been identified mainly in monopartite begomoviruses that associate with betasatellites and have no known contributions to pathogenesis of begomovirus-betasatellite disease complexes. Applications of current molecular tools are facilitating viral diagnosis and the discovery of novel species of geminiviruses and satellite DNAs and are also advancing our understanding of the global diversity and evolution of satellite DNAs.

A Leaf Curl Disease in Germplasm of Rapeseed‐Mustard in India: Molecular Evidence of a Weed‐Infecting Begomovirus–Betasatellite Complex Emerging in a New Crop

AbstractEvaluation of 130 accessions of rapeseed‐mustard germplasm grown at the National Bureau of Plant Genetic Resources, New Delhi, India during the winter season (2011–2012) revealed the occurrence of a leaf curl disease in seven accessions. The occurrence of the disease was observed in another 62 of 525 accessions evaluated during 2012–2013. The association of a monopartite begomovirus and betasatellite was established with the symptomatic plants by whitefly transmission and PCR amplification. The complete nucleotide sequences of the begomovirus (JX270684, 2745 nucleotides), obtained by rolling circle amplification, showed the highest sequence identity (98.1%) with the weed‐infecting begomovirus, Croton yellow vein mosaic virus. Analysis of recombination indicated the probable occurrence of many overlapping inter‐ and intraspecific recombination events. The sequence of betasatellite (JX270685, 1355 nucleotides) showed the highest sequence identity (95.7%) with Croton yellow vein mosaic betasatellite. Begomoviruses were not previously known to naturally infect rapeseed‐mustard. This is the first report of the emergence of a weed‐infecting begomovirus–betasatellite complex in rapeseed‐mustard germplasm in India and raises the concern on utilization of such susceptible germplasm in crop improvement programmes.