Complete genome characterization of chilli veinal mottle virus associated with mosaic and mottling disease of tomato and development of LAMP assay for quick detection.

Chilli veinal mottle virus (ChiVMV) is a potyvirus known to cause havoc in many solanaceous crops. Samples from tomato plants exhibiting typical mosaic and mottling symptoms in two locations from farmers' fields were collected and tested using DAC ELISA for the presence of ChiVMV and other viruses known to infect tomato. ChiVMV Gauribidanur isolate from infected tomato was mechanically inoculated to Datura metel, Nicotiana tabacum, Nicotiana benthamiana, Nicotiana glutinosa, chilli, and tomato plants which exhibited systemic mosaic and mottling symptoms 10 days post-inoculation. This results were further confirmed by RT-PCR and DAC ELISA using CP gene-specific primers and ChiVMV antisera, respectively. Transmission electron microscopy revealed the presence of long filamentous particles (800 × 11 nm) resembling viruses in the Potyviridae family. The complete genome of ChiVMV comprised 9716 nucleotides except for poly A tail, with a predicted open reading frame spanning 9270 nucleotides encoding polyproteins of 3089 amino acids. Comparative analysis revealed that ChiVMV-tomato isolates reported across the world shared maximum nucleotide identity (93-96.7%) with chilli isolates from India and Pakistan. These results were well supported by sequence demarcation analysis. Further, the Neibhor-Net network analysis of the complete genome of ChiVMV-tomato, along with other host isolates, formed a reticular network phylogenetic tree suggesting recombination events. Subsequently, RDP5 detected intra-specific recombination breakpoints at the positions 1656-5666 nucleotides with major parent ChiVMV (MN508960) Uravakonda and minor parent ChiVMV (MN508956) with a significant average p value of 1.905 × 10-22. The LAMP assay using ChiVMV-specific primers resulted in ladder-like amplified products on electrophoresed gel and a distinct red colour pattern with hydroxy naphthalene blue, indicating a positive reaction for the presence of ChiVMV in infected tomato samples. To validate LAMP-designed primers, RNA extracted from ChiVMV-infected tomato, chilli, datura, and tobacco samples were subjected to LAMP assay and it accurately detected the presence of ChiVMV in infected plant samples. Overall, this study provides holistic information of ChiVMV infecting tomato, spanning diagnosis, transmission, genetic characterization, and detection of recombination events, which collectively contribute to effective disease management, crop protection, and informed decision-making in agricultural practices.

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 characterization and host range studies of Cucumber mosaic virus belonging to the Subgroup IB infecting chilli in India and screening of chilli genotypes for identification of resistance.

Chilli pepper is an important vegetable and spice crop grown worldwide. Chilli is susceptible to various pathogens, among them mosaic disease caused by Cucumber mosaic virus (CMV) is a major constraint for its production. Roving survey was carried out for mosaic disease assessment in chilli at 35 locations comprising five districts of south eastern Karnataka, which was later confirmed for the presence of different viruses in random samples by DAC-ELISA. Results revealed the prevalence of the disease caused by CMV up to 43.00% based on visual assessment. However, only in 64 samples out of 140 infected chilli samples showed CMV infection in DAC-ELISA and revealed the mixed infection of viruses. Mechanical sap inoculation of CMV-Ko isolate induced symptoms on chilli plants, which were similar to the symptoms observed in field. Complete genome sequence of CMV-Ko (RNA1, RNA2 and RNA3) isolate was amplified, cloned and sequenced. Sequence analysis revealed that it shared 83.7-99.1% nucleotide (nt) identity with CMV subgroup IB isolates infecting different crops in India. Recombination analysis of CMV-Ko genome showed that, RNA1 and RNA2 had recombinant origin and not RNA3. Host range studies for CMV-Ko isolate showed its potential of infecting nine host plants out of 21 used for transmission. Fifty advanced chilli lines were screened against CMV-Ko isolate and 27 immune lines to CMV were identified, which can be utilized for management of disease caused by CMV in chilli. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13337-021-00713-3.

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.

Association of tomato leaf curl New Delhi virus DNA-B with bhendi yellow vein mosaic virus in okra showing yellow vein mosaic disease symptoms.

Okra samples showing yellow vein mosaic, vein twisting and bushy appearance were collected from different locations of India during the surveys conducted between years 2005-2009. The dot blot and PCR detection revealed that 75.14% of the samples were associated with monopartite begomovirus and remaining samples with bipartite virus. Whitefly transmission was established for three samples representing widely separated geographical locations which are negative to betasatellites and associated with DNA-B. Genome components of these three representative isolates were cloned and sequenced. The analysis of DNA-A-like sequence revealed that three begomovirus isolates shared more than 93% nucleotide sequence identity with bhendi yellow vein mosaic virus from India (BYVMV), a monopartite begomovirus species that was reported previously as causative agent of bhendi yellow mosaic disease in association of bhendi yellow vein mosaic betasatellite. Further, the DNA-B-like sequences associated with the three virus isolates shared no more than 90% sequence identity with tomato leaf curl New Delhi virus (ToLCNDV). Analyses of putative iteron-binding sequence required for trans-replication suggests that begomovirus sequences shared compatible rep-binding iterons with DNA-B of ToLCNDV. Our data suggest that the monopartite begomovirus associated with okra yellow vein disease has captured DNA-B of ToLCNDV to infect okra. Widespread distribution of the complex shows the increasing trend of the capturing of DNA-B of ToLCNDV by monopartite begomoviruses in the Indian subcontinent. The recombination analysis showed that the DNA-A might have been derived from the inter-specific recombination of begomoviruses, while DNA-B was derived from the ToLCNDV infecting different hosts.

Begomovirus characterization, and development of phenotypic and DNA-based diagnostics for screening of okra genotype resistance against Bhendi yellow vein mosaic virus.

The leaf sample from okra plants showing the yellow vein mosaic disease symptoms was collected in Karnataka state, India. The genome of the virus was amplified, cloned and sequenced. Sequence analysis revealed that the viral genome (GU112065) is 2,741 bp in length and genome is similar to that of monopartite begomoviruses originating from the Old World, with seven conserved ORFs. Further nucleotide (nts) sequence comparisons showed that the genome has the highest sequence identities of 96.1 % with Bhendi yellow vein mosaic virus (BYVMV) (GU112057) and 89.7 % with okra yellow vein mosaic virus (OYVMV) (AJ002451) infecting okra in India and Indian subcontinent. These results suggested that the isolate is a new strain of BYVMV. To identify the resistance source to BYVMV, the okra genotypes were screened under both artificial and natural conditions. None of the genotypes showed immunity to the disease. However, the genotypes Nun 1145 and Nun 1144 showed moderate resistance and genotypes M10, Nun 1142, Nun 1140 showed moderately susceptible reactions under both glass house and field conditions. Further, dot-blot hybridization using nonradioactive (digoxigenin) DNA probe showed that the virus was also detected in the symptomless plants.

Association of a recombinant Cotton leaf curl Bangalore virus with yellow vein and leaf curl disease of okra in India.

A begomovirus isolate (OY136A) collected from okra plants showing upward leaf curling, vein clearing, vein thickening and yellowing symptoms from Bangalore rural district, Karnataka, India was characterized. The sequence comparisons revealed that, this virus isolate share highest nucleotide identity with isolates of Cotton leaf curl Bangalore virus (CLCuBV) (AY705380) (92.8 %) and Okra enation leaf curl virus (81.1-86.2 %). This is well supported by phylogentic analysis showing, close clustering of the virus isolate with CLCuBV. With this data, based on the current taxonomic criteria for the genus Begomovirus, the present virus isolate is classified as a new strain of CLCuBV, for which CLCuBV-[India: Bangalore: okra: 2006] additional descriptor is proposed. The betasatellite (KC608158) associated with the virus is having more than 95 % sequence similarity with the cotton leaf curl betasatellites (CLCuB) available in the GenBank.The recombination analysis suggested, emergence of this new strain of okra infecting begomovirus might have been from the exchange of genetic material between BYVMV and CLCuMuV. The virus was successfully transmitted by whitefly and grafting. The host range of the virus was shown to be very narrow and limited to two species in the family Malvaceae, okra (Abelmoschus esculentus) and hollyhock (Althaea rosea), and four in the family Solanaceae.

Molecular characterization of distinct bipartite begomovirus infecting bhendi (Abelmoschus esculentus L.) in India.

Yellow vein mosaic disease of okra is a whitefly transmitted begomovirus causing heavy economic loss in different parts of India. The okra isolate (OY131) of this virus from a bhendi plant [(Abelmoschus esculentus L.) Moench] showing yellow vein mosaic, vein twisting, reduced leaves, and a bushy appearance in the Palem region, New Delhi, India, was characterized in the present study. The complete DNA-A and DNA-B sequences have been determined and are comprised of 2,746 and 2,703 nucleotides, respectively. The betasatellite (DNA-ß) component was absent in the sample. The genome organization was typically of biparite begomoviruses, which were characterized earlier. Comparison of DNA-A component with other known begomoviruses suggest that this virus, being only distantly related (<85.9% similarity with its nearest relative, BYVMV) to other known begomoviruses, is a new species. We have tentatively assigned the genome to a novel geminivirus species Bhendi yellow vein mosaic Delhi virus [BYVDV-IN (India: Delhi: okra)]. DNA-B showed highest sequence identity (87.8% identical) to that of a ToLCNDV (AY158080). The phylogenetic analysis of the present isolate is distinct from all other viruses; however clusters with ToLCNDV group infect different crops. The recombination analysis revealed that this isolate has sequences originated from ToLCNDV. This is the first known bhendi yellow vein mosaic disease associated bipartite begomovirus from India.

Down-regulation of the global regulator SATB1 by statins in COLO205 colon cancer cells.

Special AT-rich sequence binding protein 1 (SATB1) regulates the expression of more than 1,000 genes in tumor cells. SATB1 expression has been implicated in metastasis, and its silencing results in reduced cancer progression and the reversion of metastatic cells to normal appearance. Therefore, any compound causing down-regulation of SATB1 expression or activity may be exploited for its therapeutic potential in terms of cancer regression. Earlier studies showed that the 3-hydroxy-3-methylglutaryl coenzymeA (HMG-CoA) reductase inhibitors (statin drugs), which are widely used to treat hypercholesterolemia, possess other pleotropic activities. These are now increasingly gaining attention for their cancer prevention abilities. However, the downstream interplay of the molecular mechanisms of such anti-cancer activities is unclear. Here, we show that SATB1 is down-regulated by statins in a time- and dose-dependent manner in COLO205 cells. This effect was statin-specific as the down-regulation of SATB1 was brought about by hydrophobic statins, such as simvastatin and fluvastatin, but not by hydrophilic pravastatin. Notably, treatment with mevalonate, an intermediate in the cholesterol and isoprenoid biosynthetic pathways, led to the inhibition of SATB1 down-regulation and cytotoxicity mediated by statins. Treatment with the proteasome inhibitors lactacystine and MG-132 inhibited the statin-mediated down-regulation of SATB1, suggesting that regulation occurs at the post-translational level. Thus, our results demonstrate a novel molecular mechanism for the anti-cancer activity of statin drugs in colon cancer cells, without invoking significant cytotoxicity.

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.