Nanopore sequencing of tomato mottle leaf distortion virus, a new bipartite begomovirus infecting tomato in Brazil.

During a survey in a tomato field in Luziânia (Goiás State, Brazil), a single plant with mottling, chlorotic spots, and leaf distortion was found. A new bipartite begomovirus sequence was identified using nanopore sequence technology and confirmed by Sanger sequencing. The highest nucleotide sequence identity match of the DNA-A component (2596 bases) was 81.64% with tomato golden leaf deformation virus (HM357456). Due to the current species demarcation criterion of 91% nucleotide sequence identity for DNA-A, we propose this virus to be a new member of the genus Begomovirus, named "tomato mottle leaf distortion virus".

A New Type of Satellite Associated with Cassava Mosaic Begomoviruses.

Cassava mosaic disease (CMD), which is caused by single-stranded DNA begomoviruses, severely limits cassava production across Africa. A previous study showed that CMD symptom severity and viral DNA accumulation increase in cassava in the presence of a DNA sequence designated SEGS-2 (sequence enhancing geminivirus symptoms). We report here that when SEGS-2 is coinoculated with African cassava mosaic virus (ACMV) onto Arabidopsis thaliana, viral symptoms increase. Transgenic Arabidopsis with an integrated copy of SEGS-2 inoculated with ACMV also display increased symptom severity and viral DNA levels. Moreover, SEGS-2 enables Cabbage leaf curl virus (CaLCuV) to infect a geminivirus-resistant Arabidopsis thaliana accession. Although SEGS-2 is related to cassava genomic sequences, an earlier study showed that it occurs as episomes and is packaged into virions in CMD-infected cassava and viruliferous whiteflies. We identified SEGS-2 episomes in SEGS-2 transgenic Arabidopsis. The episomes occur as both double-stranded and single-stranded DNA, with the single-stranded form packaged into virions. In addition, SEGS-2 episomes replicate in tobacco protoplasts in the presence, but not the absence, of ACMV DNA-A. SEGS-2 episomes contain a SEGS-2 derived promoter and an open reading frame with the potential to encode a 75-amino acid protein. An ATG mutation at the beginning of the SEGS-2 coding region does not enhance ACMV infection in A. thaliana. Together, the results established that SEGS-2 is a new type of begomovirus satellite that enhances viral disease through the action of an SEGS-2-encoded protein that may also be encoded by the cassava genome. IMPORTANCE Cassava is an important root crop in the developing world and a food and income crop for more than 300 million African farmers. Cassava is rising in global importance and trade as the demands for biofuels and commercial starch increase. More than half of the world's cassava is produced in Africa, where it is primarily grown by smallholder farmers, many of whom are from the poorest villages. Although cassava can grow under high temperature, drought, and poor soil conditions, its production is severely limited by viral diseases. Cassava mosaic disease (CMD) is one of the most important viral diseases of cassava and can cause up to 100% yield losses. We provide evidence that SEGS-2, which was originally isolated from cassava crops displaying severe and atypical CMD symptoms in Tanzanian fields, is a novel begomovirus satellite that can compromise the development of durable CMD resistance.

Detection of Begomovirus in chilli and tomato plants using functionalized gold nanoparticles.

Begomoviruses are a major class of Geminiviruses that affects most dicotyledonous plants and causes heavy economic losses to farmers. Early detection of begomovirus is essential to control the spread of the disease and prevent loss. Many available detection methods like ELISA, immunosorbent electron microscopy, PCR or qPCR require expertise in handling sophisticated instruments, complex data interpretation and costlier chemicals, enzymes or antibodies. Hence there is a need for a simpler detection method, here we report the development of a visual detection method based on functionalized gold nanoparticles (AuNP assay). The assay was able to detect up to 500 ag/µl of begomoviral DNA (pTZCCPp3, a clone carrying partial coat protein gene) suspended in MilliQ water. Screening of chilli plants for begomoviral infection by PCR (Deng primers) and AuNP assay showed that AuNP assay (77.7%) was better than PCR (49.4%). The AuNP assay with clccpi1 probe was able to detect begomoviral infection in chilli, tomato, common bean, green gram and black gram plants which proved the utility and versatility of the AuNP assay. The specificity of the assay was demonstrated by testing with total DNA from different plants that are not affected by begomoviruses.

Interspecies Recombination Has Driven the Macroevolution of Cassava Mosaic Begomoviruses.

Begomoviruses (family Geminiviridae, genus Begomovirus) significantly hamper crop production and threaten food security around the world. The frequent emergence of new begomovirus genotypes is facilitated by high mutation frequencies and the propensity to recombine and reassort. Homologous recombination has been especially implicated in the emergence of novel cassava mosaic begomovirus (CMB) genotypes, which cause cassava mosaic disease (CMD). Cassava (Manihot esculenta) is a staple food crop throughout Africa and an important industrial crop in Asia, two continents where production is severely constrained by CMD. The CMD species complex is comprised of 11 bipartite begomovirus species with ample distribution throughout Africa and the Indian subcontinent. While recombination is regarded as a frequent occurrence for CMBs, a revised, systematic assessment of recombination and its impact on CMB phylogeny is currently lacking. We assembled data sets of all publicly available, full-length DNA-A (n = 880) and DNA-B (n = 369) nucleotide sequences from the 11 recognized CMB species. Phylogenetic networks and complementary recombination detection methods revealed extensive recombination among the CMB sequences. Six out of the 11 species descended from unique interspecies recombination events. Estimates of recombination and mutation rates revealed that all species experience mutation more frequently than recombination, but measures of population divergence indicate that recombination is largely responsible for the genetic differences between species. Our results support that recombination has significantly impacted the CMB phylogeny and has driven speciation in the CMD species complex. IMPORTANCE Cassava mosaic disease (CMD) is a significant threat to cassava production throughout Africa and Asia. CMD is caused by a complex comprised of 11 recognized virus species exhibiting accelerated rates of evolution, driven by high frequencies of mutation and genetic exchange. Here, we present a systematic analysis of the contribution of genetic exchange to cassava mosaic virus species-level diversity. Most of these species emerged as a result of genetic exchange. This is the first study to report the significant impact of genetic exchange on speciation in a group of viruses.

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.

Development of a triple antibody sandwich enzyme-linked immunosorbent assay for cassava mosaic disease detection using a monoclonal antibody to Sri Lankan cassava mosaic virus.

BACKGROUND: Cassava mosaic disease (CMD) is one of the most devastating viral diseases for cassava production in Africa and Asia. Accurate yet affordable diagnostics are one of the fundamental tools supporting successful CMD management, especially in developing countries. This study aimed to develop an antibody-based immunoassay for the detection of Sri Lankan cassava mosaic virus (SLCMV), the only cassava mosaic begomovirus currently causing CMD outbreaks in Southeast Asia (SEA). METHODS: Monoclonal antibodies (MAbs) against the recombinant coat protein of SLCMV were generated using hybridoma technology. MAbs were characterized and used to develop a triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) for SLCMV detection in cassava leaves and stems. Assay specificity, sensitivity and efficiency for SLCMV detection was investigated and compared to those of a commercial ELISA test kit and PCR, the gold standard. RESULTS: A TAS-ELISA for SLCMV detection was successfully developed using the newly established MAb 29B3 and an in-house polyclonal antibody (PAb) against begomoviruses, PAb PK. The assay was able to detect SLCMV in leaves, green bark from cassava stem tips, and young leaf sprouts from stem cuttings of SLCMV-infected cassava plants without cross-reactivity to those derived from healthy cassava controls. Sensitivity comparison using serial dilutions of SLCMV-infected cassava sap extracts revealed that the assay was 256-fold more sensitive than a commercial TAS-ELISA kit and 64-fold less sensitive than PCR using previously published SLCMV-specific primers. In terms of DNA content, our assay demonstrated a limit of detection of 2.21 to 4.08 × 106 virus copies as determined by quantitative real-time PCR (qPCR). When applied to field samples (n = 490), the TAS-ELISA showed high accuracy (99.6%), specificity (100%), and sensitivity (98.2%) relative to the results obtained by the reference PCR. SLCMV infecting chaya (Cnidoscolus aconitifolius) and coral plant (Jatropha multifida) was also reported for the first time in SEA. CONCLUSIONS: Our findings suggest that the TAS-ELISA for SLCMV detection developed in this study can serve as an attractive tool for efficient, inexpensive and high-throughput detection of SLCMV and can be applied to CMD screening of cassava stem cuttings, large-scale surveillance, and screening for resistance.

Identification of the Begomoviruses Squash Leaf Curl Virus and Watermelon Chlorotic Stunt Virus in Various Plant Samples in North America.

Geminiviruses are a group of plant-infecting viruses with single-stranded DNA genomes. Within this family, viruses in the genus Begomovirus are known to have a worldwide distribution causing a range of severe diseases in a multitude of dicotyledonous plant species. Begomoviruses are transmitted by the whitefly Bemisia tabaci, and their ssDNA genomes can be either monopartite or bipartite. As part of a viral survey, various plants including those in the families Alliaceae, Amaranthaceae, Apiaceae, Asteraceae, Brassicaceae, Cactaceae, Cucurbitaceae, Lamiaceae, Lauraceae, Malvaceae, Oleaceae and Solanaceae were sampled and screened for begomoviruses using both a high-throughput sequencing and a begomovirus-specific primer pair approach. Based on the sequences derived using these approaches, the full-length genome of various begomoviruses were amplified from plants using abutting primers. Squash leaf curl virus (SLCV) and watermelon chlorotic stunt virus (WCSV) were identified in Cactaceae (n = 25), Solanaceae (n = 7), Cucurbitaceae (n = 2) and Lamiaceae (n = 1) samples. WCSV is an Old World bipartite begomovirus that has only recently been discovered infecting watermelons in the Americas. Our discovery of WCSV in the USA is the first indication that it has reached this country and indicates that this virus might be widespread throughout North America. Phylogenetic analysis suggests WCSV was introduced to the New World twice. The detection of begomoviruses in cactus plants suggests possible spillover events from agricultural areas into native vegetation. Since WCSV and SLCV have previously been found in mixed infections, pseudo-recombination infection experiments were conducted. We demonstrate that WCSV DNA-B is successfully trans-replicated by SLCV DNA-A despite very low degree of similarity between the replication-associated iterative sequences present in their common region, an essential feature for binding of the replication associated protein. This study highlights the importance of viral surveys for the detection of spillover events into native vegetation, but also suggests the need for more surveillance of WCSV in the USA, as this virus is a serious threat to watermelon cultivation in the Middle East.

LAMP-Coupled CRISPR-Cas12a Module for Rapid and Sensitive Detection of Plant DNA Viruses.

One important factor for successful disease management is the ability to rapidly and accurately identify the causal agent. Plant viruses cause severe economic losses and pose a serious threat to sustainable agriculture. Therefore, optimization of the speed, sensitivity, feasibility, portability, and accuracy of virus detection is urgently needed. Here, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid diagnostic method utilizing the CRISPR-Cas12a system for detecting two geminiviruses, tomato yellow leaf curl virus (TYLCV) and tomato leaf curl New Delhi virus (ToLCNDV), which have single-stranded DNA genomes. Our assay detected TYLCV and ToLCNDV in infected plants with high sensitivity and specificity. Our newly developed assay can be performed in ~1 h and provides easy-to-interpret visual readouts using a simple, low-cost fluorescence visualizer, making it suitable for point-of-use applications.

Complete genome sequence of a previously undescribed monopartite begomovirus and betasatellite infecting Malvastrum coromandelianum in Cambodia.

A previously undescribed monopartite begomovirus was identified in Kampot province, Cambodia, in Malvastrum coromandelianum plants exhibiting yellow vein symptoms characteristic of begomovirus infections. The apparently full-length viral component was cloned and sequenced following enrichment of circular DNA by rolling-circle amplification and restriction enzyme digestion. The genome of the virus was 2737 nucleotides in length (KP188831) and exhibited an organization like that of other monopartite begomoviruses, sharing the highest nucleotide sequence similarity (87.7% identity) with ageratum yellow vein virus (AM940137). A satellite molecule was amplified from total DNA by PCR amplification, using the betasatellite-specific primer pair ß01/ß02. The satellite molecule (1346 nt, KP188832) had structural characteristics like those of other betasatellites associated with begomoviruses and shared the highest nucleotide sequence similarity (84.8% identity) with malvastrum yellow vein betasatellite (MN205547). According to the criteria established for species demarcation for classification of begomoviruses (family Geminiviridae) and betasatellites (family Tolecusatellitidae), respectively, the virus isolate from M. coromandelianum in Cambodia is a previously undescribed novel monopartite begomovirus, for which the name "malvastrum yellow vein Cambodia virus" (MaYVCV) is proposed, and the betasatellite is a previously undescribed novel betasatellite, for which the name "malvastrum yellow vein Cambodia betasatellite" (MaYVKHB) is proposed.

Muntingia yellow spot virus: a novel New World begomovirus infecting Muntingia calabura L.

The whole genome sequence of a begomovirus (family Geminiviridae) infecting Muntingia calabura L. (family Muntingiaceae) from the province of Guayas in Ecuador was determined in this work. The major symptom observed on this plant species was yellow spots on leaves. The nucleotide sequences of three DNA-A clones and one DNA-B clone were compared to those of other begomoviruses. The DNA-A clones displayed the highest similarity to isolates of pepper leafroll virus (PepLRV), with 87.4 to 88.1% sequence identity. Likewise, the DNA-B clone showed the highest similarity (79.3-79.6% sequence identity) to PepLRV isolates. According to the demarcation criteria for begomovirus species, the begomovirus described in this work, for which we propose the name "muntingia yellow spot virus", represents a novel species. To our best knowledge, this is the first report of a begomovirus infecting a plant of the family Muntingiaceae.

Occurrence of Cotton leaf curl Multan virus and associated betasatellites with leaf curl disease of Bhut-Jolokia chillies (Capsicum chinense Jacq.) in India.

Geminiviridae comprises the largest family of plant viruses which causes severe crop losses in India. The highest pungency chilli Bhut-Jolokia or ghost pepper (Capsicum chinense Jaqc.) hails from North-East region of India and is used in many dishes to add flavors and also for its medicinal value. However, this chilli variety is also affected by viruses leading to crop and economic losses. The present study reports the identification of begomoviruses in the infected chilli Bhut-Jolokia leaf samples collected from eight different places of North-East region (Manipur) of India. The infected leaf samples were screened for the presence of viral genome by rolling circle amplification (RCA) followed by PCR using degenerate primer pairs. The subsequent analyses using restriction fragment length polymorphism and sequencing revealed the presence of Cotton leaf curl Multan virus (CLCuMuV), and Tomato leaf curl Patna betasatellite (ToLCPaB). The findings focus on the phylogenetic relatedness, probable recombinational hot-spots and evolutionary divergence of the viral DNA sequences with the current reported begomoviral genome. To the best of our knowledge, this is the first report showing the presence of CLCuMuV, and associated non-cognate ToLCPaB with leaf curl disease of Bhut-Jolokia chillies. The study reveals potential recombination sites on both viral genome and betsatellite which, during the course of evolution, may have aided the virus to progress and successfully establish infection in chilli plants. Taken together, our results suggest a possible spread of CLCuMuV to the hitherto non-host crop in the North-East region of India.

Two different begomovirus species are associated with yellow vein mosaic disease of okra in Sri Lanka.

Yellow vein mosaic disease is the major biotic constraint of okra cultivation in Sri Lanka. Identification and detailed molecular characterization of associated pathogen is needed for effective disease management. The genome of the begomovirus and betasatellite were amplified in symptomatic plant samples using specific degenerate primers. DNA-A genome of twelve isolates representing different locations in Sri Lanka were cloned, sequenced and deposited in GenBank database (Accession No- KX698087- KX698092 and MH455207- MH455212). Size of the complete nucleotide sequences ranged from 2735 to 2786 bp. The genome organization showed characteristics of begomoviruses. The pairwise sequence identity revealed the association of two different begomovirus species. Five of the isolates showed > 91% of sequences identity with Bhendi yellow vein mosaic virus, and the rest of the seven isolates were around 92% of identity with Okra enation leaf curl virus. This is further supported by phylogenetic analysis where both of these group of isolates were in different cluster. Recombination analysis showed the presence of recombinant fragments in the virus isolates associated with okra yellow vein mosaic disease (OYVMD) in Sri Lanka. Attempts to amplify DNA- B were failed in any of the samples tested. However, both type of the begomovirus species associated with betasatellite species, Bhendi yellow vein mosaic betasatellite. The present study has revealed the association of two distinct monopartite begomovirus species, Bhendi yellow vein mosaic virus or Okra enation leaf curl virus, with OYVMD in Sri Lanka.

Metagenomic analyses and genetic diversity of Tomato leaf curl Arusha virus affecting tomato plants in Kenya.

BACKGROUND: Tomato production is threatened worldwide by the occurrence of begomoviruses which are associated with tomato leaf curl diseases. There is little information on the molecular properties of tomato begomoviruses in Kenya, hence we investigated the population and genetic diversity of begomoviruses associated with tomato leaf curl in Kenya. METHODS: Tomato leaf samples with virus-like symptoms were obtained from farmers' field across the country in 2018 and Illumina sequencing undertaken to determine the genetic diversity of associated begomoviruses. Additionally, the occurrence of selection pressure and recombinant isolates within the population were also evaluated. RESULTS: Twelve complete begomovirus genomes were obtained from our samples with an average coverage of 99.9%. The sequences showed 95.7-99.7% identity among each other and 95.9-98.9% similarities with a Tomato leaf curl virus Arusha virus (ToLCArV) isolate from Tanzania. Analysis of amino acid sequences showed the highest identities in the regions coding for the coat protein gene (98.5-100%) within the isolates, and 97.1-100% identity with the C4 gene of ToLCArV. Phylogenetic algorithms clustered all Kenyan isolates in the same clades with ToLCArV, thus confirming the isolates to be a variant of the virus. There was no evidence of recombination within our isolates. Estimation of selection pressure within the virus population revealed the occurrence of negative or purifying selection in five out of the six coding regions of the sequences. CONCLUSIONS: The begomovirus associated with tomato leaf curl diseases of tomato in Kenya is a variant of ToLCArV, possibly originating from Tanzania. There is low genetic diversity within the virus population and this information is useful in the development of appropriate management strategies for the disease in the country.

Diversity of tomato-infecting begomoviruses and spatiotemporal dynamics of an endemic viral species of the Brazilian Atlantic rain forest biome.

Yield losses induced by a complex of begomoviruses are observed across all major tomato-producing areas in Brazil. Tomato severe rugose virus (ToSRV) is the most widespread begomovirus in the country. Conversely, tomato common mosaic virus (ToCmMV) displays a more restricted geographical distribution to areas associated with the Atlantic Rain Forest (ARF) biome, encompassing the States of Espírito Santo-ES, Minas Gerais-MG, and Rio de Janeiro-RJ. Here, we characterized 277 tomato-infecting isolates collected in fields located within the ARF biome from 2006 to 2018. ToSRV displayed the highest prevalence (n = 157), followed by ToCmMV (n = 95) and tomato interveinal chlorosis virus (n = 14). Four other begomoviruses were also detected, but with very low incidences. ToCmMV was the predominant begomovirus in the ARF biome up to 2014-2015 with very low ToSRV incidence. Subsequently, ToSRV became the most prevalent species in ES and RJ, but ToCmMV was still predominating in the "Zona da Mata" meso-region in MG. Due to the remarkable endemic distribution of ToCmMV, we carried out phylogeographical studies of this virus using information from all 28 available isolates with complete DNA-A sequences. The closest common ancestor of ToCmMV was more likely originated around Coimbra-MG area ≈ 25 years before the formal report of this viral species. So far, all surveys indicated tomatoes as the only natural hosts of ToCmMV with outbreaks occurring mainly (but not exclusively) in highland areas. ToSRV shows a more widespread incidence across both highland and lowland areas of the ARF biome.

Tomato yellow vein streak virus and Tomato golden vein virus: a reappraisal of the classification status of two South American Begomovirus species based upon genome-wide pairwise identity of multiple isolates.

Tomato yellow vein streak virus (ToYVSV) and tomato golden vein virus (TGVV) are begomoviruses reported infecting tomatoes and other hosts across South America. However, their close phylogenetic relationship has generated uncertainties about their taxonomic status and nomenclature. In fact, genomic DNA-A identity levels of isolates reported with an identical virus name may range from 89-100%. In view of the potential inaccuracy regarding the classification status of these viruses (strains vs. distinct species), we carried out a comprehensive set of analyses employing all 45 available isolates with complete DNA-A sequences with either ToYVSV or TGVV designation. Two clear-cut clusters were identified and they were consistent with the current criteria for Begomovirus species demarcation. Moreover, our reappraisal confirmed a large array of misnamed isolates and recognized a distinctive set of virus species-specific genomic, biological, and ecological features. Hence, the present work gives support to the notion that these viruses are closely-related, but they are distinct and valid Begomovirus species. From the breeding standpoint, this information will be useful in guiding germplasm screening strategies searching for sources of large-spectrum resistance to isolates of both viruses.

Metagenomics of Neotropical Single-Stranded DNA Viruses in Tomato Cultivars with and without the Ty-1 Gene.

A complex of begomoviruses (Geminiviridae) can cause severe tomato yield losses in the neotropics. Here, next-generation sequencing was employed for large-scale assessment of single-stranded (ss)DNA virus diversity in tomatoes either harboring or lacking the large-spectrum begomovirus tolerance Ty-1 gene. Individual leaf samples exhibiting begomovirus-like symptoms (n = 107) were field-collected, circular DNA-enriched, subdivided into pools (with and without Ty-1), and Illumina-sequenced. Virus-specific PCR and Sanger dideoxy sequencing validations confirmed 15 distinct ssDNA virus/subviral agents (occurring mainly in mixed infections), which highlight the potential drawbacks of employing virus-specific resistance in tomato breeding. More viruses (14 versus 6 species) were observed in tomatoes without the Ty-1 gene. A gemycircularvirus (Genomoviridae), a new alpha-satellite, and two novel Begomovirus species were identified exclusively in samples without the Ty-1 gene. A novel begomovirus was found only in the Ty-1 pool, being the only species associated with severe symptoms in Ty-1 plants in our survey. Our work is the first step towards the elucidation of the potential begomovirus adaptation to Ty-1 and its specific filtering effects on a subset of ssDNA viral/subviral agents.

Identification and characterization of Ageratum yellow vein Malaysia virus (AYVMV) and an associated betasatellite among begomoviruses infecting Solanum lycopersicum in Malaysia.

The study describes results of a survey of tomato fields for the presence of begomoviruses from different regions of Peninsular Malaysia. An ORF-based (C2 and C3) study was performed to determine the distribution of begomoviruses associated with a severe leaf curl disease in tomato-growing areas of Peninsular Malaysia. Viral DNA was isolated from symptomatic tomato plants, and begomovirus association was confirmed by PCR using DNA-A degenerate primers. The C2 and C3 sequences of the putative begomoviruses were similar to two corresponded ORFs of different geographically separated strains of begomoviruses: Pepper yellow leaf curl Indonesia virus and Tomato yellow leaf curl Kanchanaburi virus. The present study also identified a unique isolate, Ageratum yellow vein Malaysia virus (AYVMV) among above mentioned survey. It has a single-stranded DNA component and its associated betasatellite. The single-stranded DNA component is consisting of 2750 nt with six open reading frames and an organization resembling that of monopartite geminiviruses. The full length of viral single-stranded DNA component genome obtained using next generation sequencing (NGS) showed the highest sequence identity (99%) with Ageratum yellow vein virus (AYVV-BA). The betasatellite component genome obtained by NGS has 1342 nt and showed the highest sequence identity (91%) with the Pepper yellow leaf curl betasatellite. Following ICTV guidelines, Ageratum yellow vein Malaysia virus was assigned the abbreviation AYVMV with sequence and phylogenetic analysis indicating that it might have evolved by recombination of two or more viral ancestors.

Association of a begomovirus-satellite complex with yellow vein and leaf curl disease of hollyhock (Alcea rosea) in India.

Geminiviruses cause considerable yield loss in several crop plants worldwide. In 2016, several hollyhock plants displaying yellow mosaic and leaf curling symptoms were noticed in a nursery of Jawaharlal Nehru University, New Delhi, India. Analysis of the collected samples indicated an association of monopartite and bipartite begomoviruses with satellites. Three begomoviruses (including a member of a new begomovirus species), two alphasatellites, and a betasatellite were isolated from yellow-mosaic-disease-affected plants. Similarly, a begomovirus, two alphasatellites, and a betasatellite were found to be associated with leaf curl disease of hollyhock. These begomoviruses and satellites were found to be recombinants. By harboring diverse begomoviruses and satellite DNAs, hollyhock may serve as a potential source of virus inoculum.

A new begomovirus isolated from a potyvirus-infected bean plant causes asymptomatic infections in bean and N. benthamiana.

In this work, a begomovirus isolated from a bean plant coinfected with the potyviruses bean common mosaic virus and bean common mosaic necrosis virus was characterized. The three viruses were detected by high-throughput sequencing and assembly of total small RNAs, but the begomovirus-related contigs did not allow precise identification. Molecular analysis based on standard DNA amplification techniques revealed the presence of a single bipartite virus, which is a novel begomovirus according to the current taxonomic criteria. Infectious clones were generated and agroinoculated into Phaseolus vulgaris and Nicotiana benthamiana plants. In all cases, viral DNA-A and DNA-B were detected in new growths, but no symptoms were observed, thus indicating that this virus produces asymptomatic infections in both host species.

Surveillance and diagnostics of the emergent Sri Lankan cassava mosaic virus (Fam. Geminiviridae) in Southeast Asia.

Emergent agricultural pathogens cause severe damage worldwide and their invasive potential is significantly increased by global trade, crop intensification and climate change. Standard surveillance and diagnostic protocols need to be evaluated and implemented, particularly with diseases caused by a wide range of pathogens that induce similar symptoms. Such is the case with Cassava Mosaic Disease (CMD) present in Africa and Asia, and associated with mixed virus infections and recombinant and re-assorted virus strains. CMD has been recently reported in Southeast Asia (SEA) and is already widely spread throughout this region. This communication offers an update on protocols and tools used to track the distribution of CMD and to characterize the pathogen associated with it in SEA.