Begomovirus Infection Research Articles

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.

FIRST REPORT OF Begomovirus INFECTION ON PAPAYA IN BENGKULU, INDONESIA

First report of Begomovirus infection on papaya in Bengkulu, Indonesia. A field survey was conducted during 2019, wefound a severe systemic yellow mosaic, striped green mosaic on leaves petiole, green spots on the fruit of papaya, leafmalformation, and stunting symptoms on three papaya cultivation area in Rejang Lebong, Kepahiang, Bengkulu Tengah, andSeluma, Bengkulu Province, Indonesia. A begomo-like virus was inferred to be the possible cause of the virus-disease-likesymptoms. The study aimed to identify the causal of those typical symptoms on papaya. PCR using universal primer fortranscriptional activator protein (TrAp) and replication-associated protein (Rep) gene of Begomovirus successfully amplifiedthe DNA fragments of 900 bp in all 10 detected samples, except for samples with leaf malformation and stunting symptoms. Itis indicating that those typical symptoms on papaya is associated with Begomovirus infection, while the causal of leafmalformation and stunting is unknown yet. This work is the first report of Begomovirus infected papaya in Indonesia. Severedisease incidence caused by this pathogen was observed on papaya plants in Bengkulu Province that was in the range of 42–100%. This finding is a precious information to be used for identification, and characterization the species of the virus,determining control strategies against the disease.

Characterization of novel French bean leaf curl virus and betasatellite associated with leaf curl disease of French bean (Phaseolus vulgaris L.)

Leaves from French bean plants with severe leaf curl (ten samples) and from asymptomatic plants from 10 fields (FB1–10) in the Western Ghat region of Karnataka, India were collected. Virus isolate FB01 from French bean was shown to be transmitted by whitefly Bemisia tabaci cryptic species Asia I. DNA was isolated from the samples (ten field samples and one sample infected with FB01 via whitefly) and subjected to PCR to confirm begomovirus infection using specific primers. All 11 were positive for the expected 1.2-kb amplicon. The sequence analysis of the 1.2-kb amplicon showed that all isolates shared more than 98% nucleotide identity. Further, the complete genome from isolate FB01 was amplified and sequenced and shown to share a maximum nucleotide identity (89.3%) with Ageratum yellow vein Sri Lanka virus. As per the ICTV species threshold for begomoviruses, the virus is considered as a new species, with the proposed name French bean leaf curl virus (FbLCV). The samples were subjected to PCR using specific primers for DNA B, betasatellite and alphasatellite; only betasatellites were amplified. The detected betasatellite shared maximum nucleotide identity (92.6%) with Synedrella yellow vein betasatellite. Recombination analysis indicated that FbLCV and betasatellite were recombinants. The significance of these findings are discussed.

Molecular characterization of viruses associated to leaf curl disease complex on zucchini squash in Iraq reveals Deng primer set could distinguish between New and Old World Begomoviruses.

The emergence of begomoviruses has led to significant losses in vegetable production in the recent years. Squash leaf curl disease (SqLCD) is caused by begomoviruses, and the infected plants show leaf curl symptoms on zucchini squash. In this study, we characterized the begomoviruses responsible for SqLCD symptoms and economic losses in zucchini major growing area near Baghdad and Babylon provinces. PCR amplification was performed to screen for begomovirus infection using Deng (a begomovirus specific) primer set. Sequence comparison confirmed the detection of two begomoviruses; Tomato leaf curl Palampur virus (ToLCPMV) and Squash leaf curl virus (SLCuV), in symptomatic zucchini samples when shared 99.14 and 99.50% maximum nucleotide (nt) identities with coat protein CP gene, respectively. All samples collected from Baghdad/Al-Jadriya were ToLCPMV infected while those collected from Babylon/Jibela were SLCuV infected. Moreover, mixed infection of the two viruses was detected in all zucchini squash samples collected from Baghdad/Yusufiyah. Evidence is provided here of the relevance of the wild species Malva neglecta and Datura stramonium as reservoir of begomoviruses that cause epedemics of leaf curl disease in zucchini squash in Iraq. Neighbor joining (NJ) Phylogenetic analysis confirmed the relatedness when diverged virus sequences in separated groups based on CP gene. The high nt identity suggests the two begomoviruses may recently be introduced to Iraq and could be a serious threatening to squash cultivation.

Estimation of Genetic Parameters of Cayenne Peppers (Capsicum Annuum L.) from IPB University for Its Resistance Against Begomoviruses in Aceh

The productivity of pepper plants is strongly influenced by climatic and weather conditions as well as pests and diseases. One of the major losses in chili farming is the pest attack, such as whiteflies (Bemisia tabaci Genn.), a virus vector. Evaluation of the resistance of pepper plant germplasm against Begomovirus infection needs to be carried out, evaluate genetic diversity, estimate heritability values, and obtain a selection character for the resistance of chili plants Begomovirus. This research was conducted at Experimental Garden, Faculty of Agriculture, Universitas Syiah Kuala, and the Laboratory of Genetics and Plant Breeding, Faculty of Agriculture, Universitas Syiah Kuala from January to May 2020. Three varieties Gada, Anies and Selekta with seven test genotypes i.e F6 Gada, Seloka 4-10-2-2, F5136074-1-4-3, F5074136-2-3-2, F5074035-2-1-2, F5074035-2-4-2, F5074077-1-1-3 were screened for their resistance against Begomovirus. This study was designed using a non-factorial Randomized Block Design (RBD) and scoring parameter Begomovirus symptom. Tukey’s HSD, the standard deviation of genetic variance, heritability, and correlation were performed to analyze the data. The lowest disease intensity occurred in genotype F6 Gada with a percentage of 12.08% disease intensity (slightly resistant), and the highest level of disease intensity occurred in F5074035-2-4-2 with a percentage of 30.94% (susceptible). Variables symptom scoring, disease intensity, and incubation period were the most effective variables for selecting pepper resistance against Begomovirus infection based on broad-sense heritability values.

New record of a monopartite begomovirus and papaya leaf curl betasatellite infecting Mirabilis jalapa in Himachal Pradesh, India

Mirabilis jalapa is an important plant species for its ornamental and medicinal values. In September 2013, M. jalapa leaf samples showing vein yellowing symptoms were collected from Kangra district of Himachal Pradesh and examined for begomovirus infection. PCR analysis using DNA-A and betasatellite specific primers gave desired amplification in all infected samples. However, no PCR amplification was obtained for DNA-B that suggested monopartite nature of the infecting virus. Sequence analysis showed that the partial begomovirus sequence was most similar (92%) to mirabilis leaf curl virus (MLCV). The full-length betasatellite (1374 bp) showed highest identity (99%) to papaya leaf curl betasatellite (PaLCuB), reported from Valeriana jatamansi. Phylogenetic analysis clustered begomovirus sequence with MLCV whereas the betasatellite sequence clustered with various isolates of PaLCuB reported from India and Pakistan. This is the first report of a monopartite begomovirus and papaya leaf curl betasatellite on a new host, M. jalapa.

Natural Infection of Tomatoes (Solanum lycopersicum) by Euphorbia yellow mosaic virus Isolates across Four Brazilian States.

Severe yield losses induced by a complex of whitefly-transmitted Begomovirus species (family Geminiviridae) have been reported in tomatoes in Brazil (Reis et al. 2020). Nine isolates were obtained from tomato plants exhibiting begomovirus-like symptoms (viz. apical and interveinal chlorosis, yellow spots, and stunting) during independent field surveys: one isolate in Sumaré, São Paulo-SP State (isolate SP-066) in 2001, two in Serra Negra, Minas Gerais-MG (MG-012 and MG-016) in 2002, five in Caxias do Sul, Rio Grande do Sul-RS (RS-039, RS-045, RS-046, RS-047 and RS-058) in 2011 and one in Domingos Martins, Espírito Santo-ES (ES-148) in 2016. Disease incidence across all sampled fields ranged from 30% (in Domingos Martins-ES) to 90% in Sumaré-SP. Total DNA extraction was done by a modified CTAB method (Boiteux et al., 1999). Begomovirus infection was confirmed in all isolates by selective amplification of viral DNA-A segments using the primer pairs 'PAL1v1978 / PAR1c496' (Rojas et al., 1993) and 'BegomoAFor1' / 'BegomoARev1' (Ha et al., 2006), which produce two large and non-overlapping segments (≈1120 bp and ≈1205 bp, respectively). These PCR amplicons were initially characterized via direct Sanger dideoxy sequencing at CNPH. BLASTn analysis of the partial DNA-A genomes of these nine isolates indicated identity levels of 95-97% to three euphorbia yellow mosaic virus (EuYMV) reference isolates (= KY559532, JF756674, and KY559583) found infecting the weed Euphorbia heterophylla L. The entire DNA-A (2,609 nts = MN746971) and DNA-B (2,579 nts = MN746970) components of the MG-016 isolate were obtained via high-performance sequencing using Illumina HiSeq 2500 system (Macrogen Inc., South Korea). Sequences were assembled with the CLC Genomics Workbench program 10. Contigs were validated by BLASTx and BLASTn and compared to the ssDNA virus database at NCBI (www.ncbi.nlm.nih.gov). The fully-characterized MG-016 isolate displayed identity levels ranging from 97 to 99% to the EuYMV reference isolates as well as similar genomic features such as the conserved TATA box, nonanucleotide, and iterons (that were in agreement with a cognate nature of the DNA-A and DNA-B components). A partial sequence of the DNA-B genome was also obtained for the MG-012 isolate (MT7831942). The isolates MG-012 and MG-016 were found in mixed infections with tomato severe rugose virus (ToSRV) and tomato golden vein virus (TGVV), respectively. In addition, the complete DNA-A genomes of ES-148 (MN746972) and SP-066 (MN782438) were also obtained via a combination of primer walking and Sanger dideoxy sequencing, displaying 96-98% identity to EuYMV isolates. To our knowledge, this is the first report of multiple and independent events of natural infection of tomatoes by EuYMV isolates. Our results confirm the natural host status of tomatoes to EuYMV isolates as indicated in previous infectivity assays using biolistic inoculation (Barreto et al., 2013). The weed E. heterophylla is widely disseminated and very often present within tomato fields due to its higher levels of tolerance to the major herbicide (metribuzin) employed in this crop. Therefore, this weed may act as a persistent reservoir of tomato-infecting EuYMV isolates, which may allow the selection of viral populations potentially more adapted to this vegetable crop.

Characterization of Tomato leaf curl New Delhi virus associated with leaf curl and yellowing disease of Watermelon and development of LAMP assay for its detection.

Diseases caused by begomoviruses are becoming the major limiting factors for the production of watermelon in India. Survey for the incidence of plants showing symptoms typical to begomovirus infection was conducted in watermelon fields. The study revealed that 40% of the watermelon plants were showing the yellowing and downward curling symptoms. Twenty infected samples were collected from the different farmer's fields to know the association of begomoviruses. The PCR amplification using begomovirus-specific primers resulted in an expected 1.2kb PCR product indicating the begomovirus association with the watermelon samples. The sequence comparison results of 1.2kb representing partial genome revealed that all sequences obtained from watermelon samples have a nucleotide (nt) identity of more than 98% among them and are maximum homology with Tomato leaf curl New Delhi virus (ToLCNDV). One watermelon sample (WM1) was selected for complete genome amplification using RCA method (rolling-circle amplification). Amplification of DNA B and no amplification of betasatellites and alphasatellite indicated this virus as bipartite. Sequence Demarcation Tool (SDT) analysis of the DNA A component of the WM1 isolate showed the maximum nt identity of 94.6-97.9% and 85.2-95.8% with ToLCNDV infecting cucurbits. The recombinant analysis showed that the genome was likely to be derived from the recombination of already reported begomoviruses (ToLCNDV, ToLCPalV, and MYMIV) infecting diverse crops. The whitefly cryptic species predominant in the begomovirus-infected watermelon fields were identified as Asia-II-5 group. The LAMP assay developed based on coat protein gene sequence was able to detect the ToLCNDV in the infected samples. Visual detection of the LAMP-amplified products was observed with the hydroxy naphthol blue. LAMP assay was also validated with ToLCNDV infected sponge gourd, spine gourd, ivy gourd, ridge gourd, and cucumber. This is the first report of ToLCNDV association with leaf curl and yellowing disease of watermelon from India and World based on complete genome sequencing.

Molecular and biological characterization of Chilli leaf curl virus and associated betasatellite infecting Cucurbita maxima in Oman.

During a survey in February 2016, leaf curl disease symptoms were witnessed in Cucurbita maxima plants in Al-Batina commercial farm in Oman. Symptoms exhibited were characteristic of begomovirus infection as leaf curling, yellowing, and color breaking followed by mosaic pattern. The transmission electron microscopy confirmed the presence of typical twinned geminate typical of Geminate virus particles. Rolling circle amplification (RCA) was employed to characterize the unknown causal agent of C. maxima disease. In molecular identification RCA produced nearly 2.8 and 1.4kb DNA molecules corresponding to begomovirus and satellite molecules, cloned and sequenced them. In Blast, species demarcation tool and phylogenetic analysis revealed the begomovirus and satellite isolates were determined as Chilli leaf curl virus (ChLCV) and tomato leaf curl betasatellite (ToLCB). In biological analysis by agrobacterium mediated inoculation, ChLCV displayed upward leaf curling and vein swelling symptoms in Nicotiana benthamiana plants; however, in presence of ToLCB enhanced downward leaf curling and crumpling symptoms were revealed. This study provides the first evidence that ChLCV and ToLCB caused leaf curl disease of C. maxima in Oman.

Molecular detection of Euphorbia yellow mosaic virus infecting chili pepper

Begomoviruses (family Geminiviridae, genus Begomovirus) are responsible for considerable losses in several tropical crops, more commonly in bean and tomato. Begomoviruses comprise the most numerous and economically destructive group within Geminiviridae. Characteristic symptoms of begomovirus infection in Capsicum chinense (chili pepper, Solanaceae) were observed in smallholder farms in the state of Amazonas. The molecular detection and identification of begomoviruses affecting chili pepper were conducted from symptomatic foliar samples collected at 14 farms in the municipalities of Presidente Figueiredo, Iranduba and Manaus (Amazonas state). Begomovirus was detected in five out of the 69 samples, all of the cultivar Lupita. Pairwise sequence comparisons and phylogenetic analysis based on the complete sequence of the DNA-A component identified the isolates as members of the species Euphorbia yellow mosaic virus (EuYMV). Comparative analysis with 36 EuYMV isolates grouped the Amazonian isolates in a single clade, indicating a low degree of genetic variability for this begomovirus.

The threat of seed-transmissible pepper yellow leaf curl Indonesia virus in chili pepper

Recently, chili pepper (Capsicum annuum) plants in Indonesia have been devastated by a notorious bipartite begomovirus infection named Pepper yellow leaf curl Indonesia virus (PepYLCIV), which causes a distinct decrease in chili pepper production. Pepper yellow diseases have been known since early 2000; however, the spread of this virus thus far is distressing. These diseases can reduce chili yields by 20–100% in Indonesia. As previously known, begomovirus can be transmitted through whitefly to several host plants from the families Solanaceae, Compositae, and Leguminosae. In the field, a single plant was observed with severe symptoms of pepper yellow leaf curl disease, while other plants in the same field were asymptomatic and healthy. The observation leads to the possibility that the virus can be transmitted from previously infected chili pepper plants through seeds, as begomovirus transmission through seeds has been reported before. This study was conducted using seeds from chili peppers infected with viruses from different places in Indonesia. Whole seeds, embryos, and seedlings from PepYLCIV infected seeds were investigated in this study by performing viral genome DNA extraction, uracil DNA glycosylase-PCR, and sequencing analysis. Results revealed that both DNA-A and DNA-B of PepYLCIV in seeds and embryos of infected chili pepper plants were detected. The results also showed that 25–67% of PepYLCIV DNA-A and 50–100% of DNA-B were detected from seedlings grown from infected chili pepper seed collected from different location, thus confirming PepYLCIV as a seed-transmissible virus in chili pepper plants.

Association of Tomato leaf curl New Delhi virus with mosaic and leaf curl disease of Chrysanthemum and its whitefly cryptic species

Leaf samples of chrysanthemum plants showing typical begomovirus symptoms of mosaic, mottling and downward leaf curl were collected from famer’s field in Karnataka state of India. The begomovirus infection was confirmed by PCR using specific primers in twenty two infected chrysanthemum samples and no amplification was observed in twenty two healthy chrysanthemum samples. The partial begomovirus genome (1.2 kb) sequencing indicates, twenty two infected chrysanthemum samples are infected with begomovirus (tomato leaf curl New Delhi virus, ToLCNDV). Therefore one representative sample was selected for complete genome amplification using RCA method. The complete genome amplified was cloned and sequenced. The Species Demarcation Tool (SDT) analysis of full genome of ToLCNDV showed maximum nucleotide (nt) identity of 89.2–97.6% (DNA A) and 87.7–96.3% (DNA B) percent with ToLCNDV infecting cucurbits. Based begomoviruses species demarcation criteria (91% nucleotide sequence identity), the virus infecting chrysanthemum is considered as a strain of ToLCNDV. The betasatellite amplified from the begomovirus infected chrysanthemum showed maximum nt identity of 89% with Tomato leaf curl Bangladesh betasatellite (ToLCBDB) infecting papaya in India. Further, break point recombination analysis showed that genome and betasatellite of ToLCNDV infecting chrysanthemum is recombinant with already know begomoviruses. The whitefly cryptic species predominantly present in the begomovirus infected chrysanthemum fields was identified as Asia-II-5 group. The significance of these findings is discussed.

Reconstruction and Characterization of Full-Length Begomovirus and Alphasatellite Genomes Infecting Pepper through Metagenomics

In northwestern Argentina (NWA), pepper crops are threatened by the emergence of begomoviruses due to the spread of its vector, Bemisia tabaci (Gennadius). The genus Begomovirus includes pathogens that can have a monopartite or bipartite genome and are occasionally associated with sub-viral particles called satellites. This study characterized the diversity of begomovirus and alphasatellite species infecting pepper in NWA using a metagenomic approach. Using RCA-NGS (rolling circle amplification-next generation sequencing), 19 full-length begomovirus genomes (DNA-A and DNA-B) and one alphasatellite were assembled. This ecogenomic approach revealed six begomoviruses in single infections: soybean blistering mosaic virus (SbBMV), tomato yellow spot virus (ToYSV), tomato yellow vein streak virus (ToYVSV), tomato dwarf leaf virus (ToDfLV), sida golden mosaic Brazil virus (SiGMBRV), and a new proposed species, named pepper blistering leaf virus (PepBLV). SbBMV was the most frequently detected species, followed by ToYSV. Moreover, a new alphasatellite associated with ToYSV, named tomato yellow spot alphasatellite 2 (ToYSA-2), was reported for the first time in Argentina. For the Americas, this was the first report of an alphasatellite found in a crop (pepper) and in a weed (Leonurus japonicus). We also detected intra-species and inter-species recombination.

Mixed infection of phytoplasma and begomovirus associated with leaf curling and witches’ broom disease of Zinnia elegans in India

Zinnia elegans belonging to the asteraceae family is a flowering annual summer plant and is native to Mexico. Symptoms of excessive leaf curling, little leaf, stunting and witches’ broom were observed on Z. elegans in Tripura state of India during 2016–2017. PCR assays using phytoplasma universal primer pairs P1/P7 and R16F2n/R16R2 and tomato leaf curl virus coat protein specific primers yielded specific amplifications of ~ 1.25 kbp and ~ 800 bp, respectively from the symptomatic zinnia leaf samples. Sequence comparison, phylogenetic and virtual RFLP analysis of 16S rRNA gene sequences of zinnia isolate confirmed the presence of ‘Candidatus Phytoplasma asteris’ (16SrI-B subgroup). Further coat protein sequence comparison and phylogenetic tree analysis of coat protein gene confirmed association of begomovirus with zinnia symptomatic samples and identified it as a strain of tomato leaf curl virus. Our results confirmed a mixed infection of ‘Ca. P. asteris’ and begomovirus with zinnia leaf curl and witches’ broom symptoms.

Characterization of recombinant tomato leaf curl Palampur virus causing leaf curl disease of Basella alba L. in India

Malabar spinach (Basella alba L.), a leafy vegetable native to the Asian and African continents, was observed with rosetting and curling of leaves associated with begomovirus infection. PCR assay involving the universal begomovirus specific primer pair was used to detect the viral infection. Subsequently, the circular DNA molecules associated with the infected samples were enhanced through rolling circle amplification (RCA). After restriction analysis, the desired viral DNA molecules were cloned, sequenced and analyzed. Based on sequencing analysis, the bipartite tomato leaf curl Palampur virus (ToLCPalV) was found to be associated with leaf curl disease of Basella. DNA-A and DNA-B of the ToLCPalV isolate infecting basella (VNS Bas1) revealed 95.65 and 98.64% identity, respectively, with that of the ToLCPalV reported earlier from India. No evidence was found for the association of alpha- and beta-satellites through testing by PCR assay. Interestingly, DNA-A sequences of ToLCPalV infecting Basella appear to be a recombinant of sequences closely related to tomato leaf curl New Delhi virus (ToLCNDV-KY780202). To the best of our knowledge, this is the first confirmed report on the association of ToLCPalV with a leaf curl disease in a new host Basella from India.

First report of naturally occurring recombinant non-coding DNA satellite associated with Tomato yellow leaf curl Kanchanaburi virus on eggplant in Indonesia

Abstract. Kandito A, Hartono S, Sulandari S, Somowiyarjo S, Widyasari YA. 2019. First report of naturally occurring recombinant non-coding DNA satellite associated with Tomato yellow leaf curl Kanchanaburi virus on eggplant in Indonesia. Biodiversitas 20: 129-136. Begomovirus is a viral genus which is a major impediment in the cultivation of plants, especially Solanaceae. Tomato yellow leaf curl Kanchanaburi virus (TYLCKaV) is a member of the Begomovirus genus that is widely reported to infect eggplant and results in severe symptoms of chlorosis. In addition to the type of virus that infects, the severity of symptoms can also be caused by the presence of betasatellite associated with several begomovirus species. The presence of betasatellite that is related to several begomovirus species can lead to more severe symptoms. The aim of this study was to identify satellites associated with begomovirus in Indonesia. Eggplant samples from Bantul, Special Region of Yogyakarta show with mosaic and severe chlorotic symptoms due to Begomovirus infection. Total DNA samples were subject to PCR amplification using universal primer for begomovirus PALIV1978/PAR1C715 and specific primer for betasatellite β01/β02. The PCR amplification produced a DNA band measuring ± 1600bp, identical size of begomovirus. The amplification of the betasatellite specific primer produced a DNA band featuring ± 1300bp, identical size of betasatellites that were associated with begomovirus in the sample. The results of DNA sequencing, suggested that begomovirus in this study had a close relationship with TYLCKaV from Thailand and Indonesia. Characterization of the satellite-based on nucleotide sequence revealed the presence of stem-loop structures, satellite conservative regions, and adenine-rich regions that resembled structures present in the betasatellite, which were associated with Tomato leaf curl virus and Ageratum yellow vein virus, with no ORF found. These results indicated that the TYLCKaV could be associated with a non-coding satellite. This result is the first report regarding the TYLCKaV association with a non-coding satellite in Indonesia.

Identification of “Malvastrum yellow vein Lahore virus” a proposed new species of begomovirus in association with cotton leaf curl Multan betasatellite infecting green bean (Phaseolus vulgaris) in Pakistan

Begomoviruses; have devastating impact on the yield of many economically important crops around the globe. During field survey in 2016, typical symptoms of begomovirus infections were observed in green beans in district Hazara, Pakistan. We identified a new strain of Malvastrum yellow vein Lahore virus in green beans for the first time in Pakistan.

Association of cotton leaf curl Multan betasatellite and Ageratum conyzoides symptomless alphasatellite with tomato leaf curl New Delhi virus in Luffa cylindrica in Pakistan

Luffa plants showing symptoms of begomovirus infection were collected from Vehari, Punjab in 2016. RCA and PCR amplifications of begomovirus genomic components showed the presence of a typical Old World bipartite begomovirus Tomato leaf curl New Delhi virus (ToLCNDV). Interestingly, Ageratum conyzoides symptomless alphasatellite and CLCuMB were also found associated with bipartite begomoviruses in the same host, however, no monopartite begomovirus was detected. To the best of our knowledge, this is the first report of ToLCNDV with associated satellites infecting L. cylindrica in Pakistan. The importance of begomovirus extended host range with different combinations of associated satellites has been discussed.

First Report of Tomato Leaf Curl New Delhi Virus Infecting Crossandra infundibuliformis in India

Crossandra infundibuliformis (L.), commonly known as firecracker flower, is a common flowering plant belonging to the family Acanthaceae. It is commonly cultivated in southern India for its attractive flowers. Symptoms of leaf yellowing, leaf mottling, leaf curling, and distortion as well as stunted growth and flower abortion were observed in C. infundibuliformis plants grown in Dharmapuri district (Tamil Nadu, India). In 2011, we observed the first symptom of leaf curl disease, and a partial sequence of tomato leaf curl New Delhi virus (ToLCNDV) was deposited in GenBank (JN410650). Further, in a survey conducted from 2013 to 2015, a total of 30 symptomatic samples suspected of begomovirus infection were collected along with a nonsymptomatic sample. In particular, intact twin icosahedral geminivirus particles (∼18 × 30 nm) were observed by high-resolution transmission electron microscopy in a partially purified extract from symptomatic leaves. Total DNA was extracted and subjected to PCR using coat protein gene-specific degenerate primers (FP/RP) (Riyaz et al. 2013) and replication-associated protein gene-specific degenerate primers ToLCNDVRepFP (5′-ATGAAGTAWGAACAGCCRCAC-3′) and ToLCNDVRepRP (5′-CCATCCGAACATTCAGGGAG-3′). PCR products of the expected sizes of ∼500 and ∼850 bp were obtained from 25 out of 30 plants, and respective PCR products were eluted from gels and sequenced. In sequence analysis, the isolate from C. infundibuliformis (CI1) showed 89 to 92% nucleotide identity with the ToLCNDV isolates reported earlier from India (KM269361, KM269355, and KP868764). Rolling circle amplification (RCA) was performed using a TempliPhi kit (GE Healthcare). RCA products were subjected to PCR amplification employing abutting primers AKKCI1F (5′-ACCTAATGGCAGAATTGTAATTTTG-3′) and AKKCI1R (5′-AATATTATACGAATGGCCGCTTTAG-3′) for DNA-A and BKKCI1F (5′-ACCGAAAGGCCGCGAAAATTTATCCTG-3′) and BKKCI1R (5′-AATATTAGACGAAAGGCCGCTATGA-3′) for DNA-B. The amplified PCR products were ligated into pJET1.2/blunt vector and cloned using a CloneJet PCR cloning kit (Thermo Scientific, U.S.A.). The clones were sequenced by primer walking, and the corresponding sequences assembled to DNA-A and DNA-B fragments were deposited under GenBank accessions MK395548 and MK395549, respectively. DNA-A (2,742 bp) showed 96% sequence identity with the DNA-A component of a ToLCNDV isolate infecting the bitter gourd crop in India (KP868764), whereas DNA-B (2,694 bp) showed 87% identity with the DNA-B component of a ToLCNDV isolate from India infecting chrysanthemum (MG758147). In addition, association of a betasatellite was also confirmed through PCR assay using universal primer pair Beta01/Beta02 (Briddon et al. 2002), followed by sequencing. The nucleotide sequence of the associated betasatellite (1,352 bp) shared 93.7% identity with the betasatellite component of tomato leaf curl New Delhi betasatellite (ToLCuNDB) reported from New Delhi (JX679002) on tomato. The betasatellite sequence was deposited in GenBank (MK040727). This study records the coexistence of bipartite ToLCNDV and ToLCuNDB in C. infundibuliformis with the evidence of their full-length genome for the first time from India.

Occurrence and distribution of begomoviruses infecting tomatoes, peppers and cucurbits in Thailand

Incidence of begomoviruses infecting tomatoes, peppers and cucurbits was investigated in major production fields in the north, northeastern, central and southern regions of Thailand between January 2015 and June 2017. Of 585 samples subjected to begomovirus detection by polymerase chain reaction (PCR), 57% were positive for begomovirus infection. Nucleotide sequence analysis of coat protein (CP) and replication initiation protein (Rep) genes identified 15 previously known begomovirus species, 7 of which were reported infecting these crops in Thailand for the first time. A candidate novel bipartite begomovirus designated Melon yellow mosaic virus was also identified in melons from a northeastern province of Thailand. The predominant begomovirus species were different depending on the host plant (Tomato yellow leaf curl Thailand virus [TYLCTHV] for tomato, Pepper yellow leaf curl Thailand virus [PepYLCTHV] for pepper, Tomato leaf curl New Delhi virus [ToLCNDV] for loofah, bitter gourd, cucumber and melon, and Squash leaf curl China virus [SLCCNV] for pumpkin and wax gourd) and location. The update on diversity and distribution of begomoviruses obtained from this study provides useful information for further epidemiological study and support development of effective disease management through selective use of resistant cultivars suitable for particular plants and locations. Additionally, these data can be applied for development of diagnostic assays to specifically detect begomoviruses for continuous monitoring of viral diversity in these economic crops.