Begomovirus and DNA-satellites association with mosaic and leaf curl disease of Solanum nigrum and Physalis minima: the new hosts for chilli leaf curl virus.

The numerous plants of Solanum nigrum L, and Physalis minima L, well-known weeds with medicinal properties in agriculture and horticulture crops exhibiting severe mosaic, enation and leaf curl symptoms, were collected from the Varanasi and Mirzapur districts of Uttar Pradesh, India. The begomovirus infection in S. nigrum and P. minima was validated by PCR using virus-specific primers. The whole genome of the represented isolate of S. nigrum (SN1), P. minima (PM1), and beta satellite was amplified, cloned and sequenced. The SDT analysis showed that the DNA-A of PM1 and SN1 isolate showed the highest nt identity of 87.4 to 99.1%, with several chilli leaf curl virus (ChiLCuV) isolates from India and Oman, respectively. The betasatellite sequence (PM1ß) obtained from the PM1 isolate showed a very low identity of 83.1-84.5%. A demarcation threshold of 91% for betasatellite species delineation has led to identifying a new betasatellite in the PM1 sample. This unique betasatellite has been named "physalis minima leaf curl betasatellite," indicating its novelty with the plant. Whereas, betasatellite sequence (SN1ß) obtained from the SN1 sample showed 86.8-91.2% nucleotide identity with ChiLCB isolates infecting several crops in Indian subcontinents. The RDP analysis of the viral genome and betasatellite of SN1 and PM1 isolates revealed recombination in substantial portions of their genetic makeup, which appeared to have originated from pre-existing begomoviruses known to infect diverse host species. The present research also highlights the potential role of these plants as significant reservoir hosts for ChiLCuV in chili plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00850-x.

Genome sequencing of cucumber mosaic virus (CMV) isolates infecting chilli and its interaction with host ferredoxin protein of different host for causing mosaic symptoms.

Chilli (Capsicum annuum L.) is an important vegetable crop grown in the Indian sub-continent and is prone to viral infections under field conditions. During the field survey, leaf samples from chilli plants showing typical symptoms of disease caused by cucumber mosaic virus (CMV) such as mild mosaic, mottling and leaf distortion were collected. DAC-ELISA analysis confirmed the presence of CMV in 71 out of 100 samples, indicating its widespread prevalence in the region. Five CMV isolates, named Gu1, Gu2, BA, Ho, and Sal were mechanically inoculated onto cucumber and Nicotiana glutinosa plants to study their virulence. Inoculated plants expressed the characteristic symptoms of CMV such as chlorotic spots followed by mild mosaic and leaf distortion. Complete genomes of the five CMV isolates were amplified, cloned, and sequenced, revealing RNA1, RNA2, and RNA3 sequences with 3358, 3045, and 2220 nucleotides, respectively. Phylogenetic analysis classified the isolates as belonging to the CMV-IB subgroup, distinguishing them from subgroup IA and II CMV isolates. Recombination analysis showed intra and interspecific recombination in all the three RNA segments of these isolates. In silico protein-protein docking approach was used to decipher the mechanism behind the production of mosaic symptoms during the CMV-host interaction in 13 host plants. Analysis revealed that the production of mosaic symptoms could be due to the interaction between the coat protein (CP) of CMV and chloroplast ferredoxin proteins. Further, in silico prediction was validated in 13 host plants of CMV by mechanical sap inoculation. Twelve host plants produced systemic symptoms viz., chlorotic spot, chlorotic ringspot, chlorotic local lesion, mosaic and mild mosaic and one host plant, Solanum lycopersicum produced mosaic followed by shoestring symptoms. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03777-8.

Molecular characterization of novel bipartite begomovirus associated with enation leaf disease of Garden croton (Codiaeum variegatum L.).

Garden croton (Codiaeum variegatum L.) plants showing typical begomovirus symptoms of vein twisting, enation and curling were collected from different gardens at Varanasi, Uttar Pradesh state of India ranged from 20 to 30%. All the 10 ten (CR1-CR10) infected samples of garden croton resulted in expected amplicon of 1.2 Kb in PCR specific to begomoviruses. No amplification was obtained for betasatellite and alphasatellite specific primers. The complete genome sequence of DNA-A and DNA-B for two isolates (CR1 and CR2) was obtained through rolling cycle amplification (RCA) and comparisons were made with other begomoviruses using Sequence Demarcation Tool (SDT) which revealed that, DNA-A of two isolates, CR1 (Acc.No.: MW816855) and CR2 (Acc.No.: MW816856) showed maximum nucleotide (nt) identity of 85.7-85.9% with Tomato leaf curl Karnataka virus, which is below the threshold percentage of begomovirus species demarcation, hence considered as novel begomovirus and proposed the name Garden croton enation leaf curl virus (CroELCuV) [IN: Varanasi: Croton: 18]. Further, DNA-B these isolates shared maximum nt identity of 91.0-92.2% (DNA-B) with Tomato leaf curl New Delhi virus. Recombination and GC plot analysis showed that the recombination occured at in low GC content regions of DNA-A and DNA-B of the CroELCuV and are derived from the previously reported several begomoviruses. This is the first record of novel bipartite begomovirus associated with vein twisting, enation and leaf curling of disease of garden garden croton in India and world. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-022-00772-0.

Transmission, characterization and occurrence of recombination in Indian strain of squash leaf curl China virus associated with yellow mosaic and leaf curl disease of Summer squash.

Summer squash is one of the important vegetable crops and its production is hampered by various abiotic and biotic stresses. Of the different biotic stresses, viral infections are responsible for causing great losses to this crop. Diseases caused by begomoviruses are becoming a major constraint in the cultivation of summer squash. Samples from summer squash plants exhibiting severe yellow mosaic and leaf curl symptoms were collected from the Varanasi district of Uttar Pradesh (India) and begomovirus associated with these plants was transmitted through whiteflies (Bemisia tabaci) to healthy squash plants. The relationship between causal virus and whitefly vector was determined. The minimum acquisition access period (AAP) and inoculation feeding period (IFP) required by B. tabaci to transmit the virus was determined to be 10 min and female insects have greater efficiency in transmitting virus than male insects. The partial genome of the virus was amplified by PCR (1.2 kb), cloned and sequenced from the ten infected plant samples collected from field. Partial genome sequence analysis (1.2 kb) obtained from the ten samples revealed that they are associated with begomovirus species closely related to the Indian strain of Squash leaf curl China virus (SLCCNV). Therefore, one representative sample (Sq-1) was selected and complete genome of the virus was amplified by rolling circle amplification (RCA) method. Sequence analysis by Sequence Demarcation Tool (SDT) showed that the current isolate has maximum nucleotide (nt) identity of 93.7-98.4% and 89-98.1% with respect to DNA A DNA B, respectively with Indian strains of SLCCNV infecting cucurbits in India. Recombination analysis of genomes (DNA A and DNA B components) showed that a major part of genomes likely to be originated from already known begomoviruses (ToLCNDV, SLCCNV-CN and SLCCNV-IN) are infecting cucurbitaceous crops. Serological assays such as triple antibody sandwich-enzyme-linked immune-sorbent (TAS-ELISA) assay, dot blot immunobinding assay (DIBA), immuno-capture polymerase chain reaction (IC-PCR) were developed for the detection of SLCCNV. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02821-9.

In silico docking analysis revealed the potential of phytochemicals present in Phyllanthus amarus and Andrographis paniculata, used in Ayurveda medicine in inhibiting SARS-CoV-2.

The Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in outbreak of global pandemic, fatal pneumonia in human referred as Coronavirus Disease-2019 (Covid-19). Ayurveda, the age old practice of treating human ailments in India, can be considered against SARS-CoV-2. Attempt was made to provide preliminary evidences for interaction of 35 phytochemicals from two plants (Phyllanthus amarus and Andrographis paniculata used in Ayurveda) with SARS-CoV-2 proteins (open & closed state S protein, 3CLpro, PLpro and RdRp) through in silico docking analysis. The nucleotide analogue remdesivir, being used in treatment of SARS-CoV-2, was used as a positive control. The results revealed that 18 phytochemicals from P. amarus and 14 phytochemicals from A. paniculata shown binding energy affinity/dock score < - 6.0 kcal/mol, which is considered as minimum threshold for any compound to be used for drug development. Phytochemicals used for docking studies in the current study from P. amarus and A. paniculata showed binding affinity up to - 9.10 kcal/mol and - 10.60 kcal/mol, respectively. There was no significant difference in the binding affinities of these compounds with closed and open state S protein. Further, flavonoids (astragalin, kaempferol, quercetin, quercetin-3-O-glucoside and quercetin) and tannins (corilagin, furosin and geraniin) present in P. amarus have shown more binding affinity (up to - 10.60 kcal/mol) than remdesivir (up to - 9.50 kcal/mol). The pharmacokinetic predictions suggest that compounds from the two plants species studied in the current study are found to be non-carcinogenic, water soluble and biologically safe. The phytochemicals present in the extracts of P. amarus and A. paniculata might have synergistic effect with action on multiple target sites of SARS-CoV-2. The information generated here might serve as preliminary evidence for anti SARS-CoV-2 activity of phytochemicals present from P. amarus and A. paniculata and the potential of Ayurveda medicine in combating the virus. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02578-7.

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 1.2 kb PCR product indicating the begomovirus association with the watermelon samples. The sequence comparison results of 1.2 kb 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.

Association of Tomato Leaf Curl New Delhi Virus, Betasatellite, and Alphasatellite with Mosaic Disease of Spine Gourd (Momordica dioica Roxb. Willd) in India.

BACKGROUND: Spine gourd (Momordica dioica Roxb. Willd) is one of the important cucurbitaceous crops grown across the world for vegetable and medicinal purposes. Diseases caused by the DNA viruses are becoming the limiting factors for the production of spine gourd reducing its potential yield. For the commercial cultivation of the spine gourd, propagation material used by most of the growers is tuberous roots and stem cuttings, which in turn results in an increased occurrence of the mosaic disease. There is a need for understanding the causal agent; through characterization of which will lead to the designing management strategies for the spine gourd mosaic disease control. OBJECTIVES: Characterization of a begomovirus and its satellites associated with mosaic disease on spine gourd. MATERIALS AND METHODS: Total DNA was extracted from spine gourd samples exhibiting symptoms typical to the begomoviruses infection (mosaic mottling, leaf curl) and was tested by PCR using begomovirus specific primers. Furthermore, the complete genome of begomo viruses (DNA A, DNA B, alpha satellite, and beta satellite) was amplified by rolling circle amplification (RCA) method. RESULTS: The full-length sequences of DNA A, DNA B, alpha satellite, and beta satellite isolated from symptomatic spine gourd were determined. The full length genomes (DNA A and DNA B) of the Tomato leaf curl New Delhi Virus (ToLCNDV) infecting spine gourd were compared with the other begomovirus genomes available in the data base. The sequence analysis has revealed that DNA A and DNA B components of the begomovirus infecting spine gourd share 95.4-96.2 and 86.7-91.2% identical sequence (i.e., nucleotide (nt) identity) with that of ToLCNDV infecting potato and cucurbits in the Indian subcontinent isolates reported earlier (available in GenBank), respectively. Further, alpha satellite and beta satellite were also detected in the begomovirus infected spine gourd samples. The recombination analysis of the DNA A, DNA B, beta satellite, and alpha satellite of the begomovirus infecting spine gourd showed the associated begomovirus and satellite DNAs were driven from the different begomoviruses, leading to emergence as a new variant of the begomovirus infecting spine gourd. CONCLUSIONS: The commercial cultivation of the spine gourd by most growers depends on the tuberous roots and stem cutting. The occurrence of begomovirus in spine gourd gives an alarming signal against utilization of such infected plant materials in the crop breeding and improvement programs. Using the clean virus-free vegetative propagation material is considered as one of the most important methods for controlling viral diseases. The study is highly useful for detection of the begomovirus infecting spine gourd in the detection of the virus infection in the clonally propagated planting material.

Comparative transmission of Bhendi yellow vein mosaic virus by two cryptic species of the whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae).

The leaf sample from okra plants showing prominent yellow vein mosaic symptoms and healthy plant without any virus symptoms were collected from farmer's field. The presence of begomovirus in the infected sample was confirmed by polymerase chain reaction (PCR) and the amplicons were cloned and sequenced. The genome analysis showed that the isolate in the present study had 99% nucleotide identity with Bhendi yellow vein mosaic virus (BYVMV) revealing it as BYVMV variant. The genetic species of Bemisia tabaci collected from fields were identified as Asia-1 and MEAM-1 genetic species based on silver leaf assay, sequence characterized amplified region marker, and mtCOI gene sequence. The comparative virus-vector relationship of both genetic species of B. tabaci indicates a minimum of two and three B. tabaci in MEAM-1 and Asia-1 genetic species, respectively, per plant were required to transmit the disease. The minimum acquisition access period and inoculation access period of 15 (MEAM-1) and 20 min (Asia-1) were required to transmit the YVMD; it was further confirmed by nucleic acid hybridization using coat protein gene-specific probe of BYVMV. With respect to the sex, the female B. tabaci were more efficient in transmitting the disease as compared to male ones in both the genetic species of B. tabaci. The MEAM-1 to transmit the BYVMV more efficiently than Asia-1 genetic species of B. tabaci.

Survey, symptomatology, transmission, host range and characterization of begomovirus associated with yellow mosaic disease of ridge gourd in southern India.

Ridge gourd is an important vegetable crop and is affected by several biotic and abiotic factors. Among the different biotic factors, ridge gourd yellow mosaic disease (RgYMD) is new emerging threat for the production of ridge gourd. The incidence of the RgYMD varied from 30 to 100% in southern India with highest disease incidence of 100% observed in Belagavi district of Karnataka state. The infected plants showed chlorosis, mosaic, cupping of leaves, blistering, reduction in leaf size and stunted growth. The varieties/hybrids grown in the farmer's fields were found to be susceptible to the disease. Begomovirus was detected in 61 out 64 samples collected from different areas of southern India. Further, all the samples failed to give amplification for beta and alpha satellites. The transmission studies revealed that single whitefly (Bemisia tabaci) is enough to transmit the virus, however, 100% transmission was observed with 10 whiteflies. The minimum acquisition access period and inoculation access period for transmission of virus by whitefly was 15 min. Among the 56 host plants belonging to diversified families tested for host range, sponge gourd, ash gourd, bottle gourd, pumpkin, cucumber, summer squash, cluster bean, tobacco and datura were shown to be susceptible. Seventy six varieties/hybrids evaluated for identifying the resistance source for RgYMD, all were found highly susceptible. Sequence analysis of DNA-A revealed that the causal virus shared highest nucleotide sequence identity (92.3%) with Tomato leaf curl New Delhi virus (ToLCNDV) infecting sponge gourd from northern India. Sequence and phylogenetic analysis of both DNA-A and DNA-B components showed that the begomovirus associated with RgYMD is found to be strain of ToLCNDV. This is first report of ToLCNDV association with RgYMD from southern India.

Molecular characterization of banana bunchy top virus isolate from Sri Lanka and its genetic relationship with other isolates.

Bunchy top disease of banana caused by Banana bunchy top virus (BBTV, genus Babuvirus family Nanoviridae) is one of the most important constraints in production of banana in the different parts of the world. Six genomic DNA components of BBTV isolate from Kandy, Sri Lanka (BBTV-K) were amplified by polymerase chain reaction (PCR) with specific primers using total DNA extracted from banana tissues showing typical symptoms of bunchy top disease. The amplicons were of expected size of 1.0-1.1 kb, which were cloned and sequenced. Analysis of sequence data revealed the presence of six DNA components; DNA-R, DNA-U3, DNA-S, DNA-N, DNA-M and DNA-C for Sri Lanka isolate. Comparisons of sequence data of DNA components followed by the phylogenetic analysis, grouped Sri Lanka-(Kandy) isolate in the Pacific Indian Oceans (PIO) group. Sri Lanka-(Kandy) isolate of BBTV is classified a new member of PIO group based on analysis of six components of the virus.

Development of silverleaf assay, protein and nucleic acid-based diagnostic techniques for the quick and reliable detection and monitoring of biotype B of the whitefly, Bemisia tabaci (Gennadius).

The aim of this study was to develop and optimize silverleaf bioassay, esterase analysis and PCR-based techniques to distinguish quickly and reliably biotype B of the whitefly, Bemisia tabaci (Gennadius), from Indian indigenous biotypes. Zucchini and squash readily develop silverleaf symptoms upon feeding by the B biotype, but they are not readily available in Indian markets. A local pumpkin variety 'Big' was, therefore, used in silverleaf assay, which developed symptoms similar to those on zucchini and squash and can be used reliably to detect B biotype. Analysis of non-specific esterases of B and the indigenous biotypes indicated both quantitative and qualitative differences in esterase patterns. Two high molecular weight bands were unique to B biotype and they occurred in abundance. These esterases were used to develop quick and field-based novel detection methods for differentiating B from the indigenous biotypes. Development of these simple and cost-effective protocols has wider application as they can be potentially used to identify other agricultural pests. Mitochondrial cytochrome oxidase I gene sequences and randomly amplified polymorphic DNA (RAPD) polymorphisms, generated using the primer OpB11, were also found useful for detecting B. tabaci biotypes. A B biotype-specific RAPD band of 800 bp was sequenced, which was used to a develop sequence characterized amplified region (SCAR) marker. The SCAR marker involved the development of B biotype-specific primers that amplified 550 bp PCR products only from B biotype genomic DNA. Silverleaf assay, esterases, RAPDs or a SCAR marker were used in combination to analyse whitefly samples collected from selected locations in India, and it was found that any of these techniques can be used singly or in combination to detect B biotype reliably. The B biotype was found in southern parts of India but not in the north in 2004-06.