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.

Screening of a multi-virus resistant RNAi construct in cowpea through transient vacuum infiltration method.

Plant viruses are the most devastating pathogens causing substantial economic losses in many crops. Current viral disease management relies on prophylactics, roguing and insect vector control, since in most crops resistant gene pools for resistance breeding are unavailable. RNA interference, a sequence dependent gene silencing mechanism holds great potential in imparting virus resistance. In this study, the efficacy of a RNAi gene construct developed against four viruses commonly infesting tomato and chilli viz., capsicum chlorosis virus, groundnut bud necrosis virus, cucumber mosaic virus and chilli veinal mottle virus was evaluated. A 3546 bp dsRNA-forming construct comprising sense-intron-antisense fragments in binary vector pBI121 (hpRNAi-MVR) was mobilized into Agrobacterium tumefaciens. Cowpea (Vigna unguiculata) was used as an indicator plant for GBNV agroinfiltration to evaluate the efficacy of hpRNAi-MVR construct in conferring GBNV resistance. The type of agroinfiltration, bacterial concentration and incubation-temperatures were optimized. Vacuum infiltration of three pulses of 20-30 s at 66.66 kPa were effective than syringe infiltration. Of the five Agrobacterial concentrations, OD600 0.5 was more efficient. Incubation temperature of 31 ± 1 °C was favorable for development of disease symptoms than 20 ± 1 °C and 26 ± 1 °C. ELISA revealed a 35% decline in viral load in hpRNAi-MVR infiltrated plants compared to vector control plants. Quantitative real time PCR results have shown a viral gene silencing to the extent of 930-990 folds in hpRNAi-MVR infiltrated plants compared to vector control. This approach is simple, rapid and efficient to screen the efficacy of RNAi constructs developed for the RNAi mediated plant virus management.

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.

Isolation, characterization and genetic diversity of NBS-LRR class disease-resistant gene analogs in multiple virus resistant line of chilli (Capsicum annuum L.).

Viruses are serious threat to chilli crop production worldwide. Resistance screening against several viruses resulted in identifying a multiple virus resistant genotype 'IHR 2451'. Degenerate primers based on the conserved regions between P-Loop and GLPL of Resistance genes (R-genes) were used to amplify nucleotide binding sites (NBS)-encoding regions from genotype 'IHR 2451'. Alignment of deduced amino acid sequences and phylogenetic analyses of isolated sequences distinguished into two groups representing toll interleukin-1 receptor (TIR) and non-TIR, and different families within the group confirming the hypotheses that dicots have both the types of NBS-LRR genes. The alignment of deduced amino acid sequences revealed conservation of subdomains P-loop, RNBS-A, kinase2, RNBS-B, and GLPL. The distinctive five RGAs showing specific conserved motifs were subjected to BLASTp and indicated high homology at deduced amino acid level with R genes identified such as Pvr9 gene for potyvirus resistance, putative late blight resistance protein homolog R1B-23 and other disease resistance genes suggesting high correlation with resistance to different pathogens. These pepper RGAs could be regarded as candidate sequences of resistant genes for marker development.

Rapid detection of tomato leaf curl Bengaluru virus through loop mediated isothermal amplification assay.

A loop-mediated isothermal amplification (LAMP) technique was employed to develop a simple and rapid method for the detection of tomato leaf curl Bangalore virus (ToLCBaV) in diseased plants of tomato (Solanum lycopersicum). Six sets of primers were designed for LAMP technique targeting the conserved AC1 region and successfully detected ToLCBaV. No reaction was detected in the tissues of healthy plants by either the LAMP or the polymerase chain reaction (PCR). The LAMP products can be visualized by presence or absence of turbidity and staining (0.2 µL for 25 µL LAMP product) directly in the tube with nucleic acid stain dye which allowed easy detection. Sensitivity of LAMP assay is 100 times of conventional PCR technique. Although, both the LAMP and the PCR methods were capable of detecting ToLCBaV in infected tissues of tomato, the LAMP method would be more useful than the PCR method for detection of ToLCBaV infection in tomato plants because it is more rapid, simple and accurate method.

Phlegmasia cerulean dolens: complication of femoral vein catheterization.

There are three less frequent manifestations of acute massive venous thrombosis and obstruction of the venous drainage of an extremity. They are phlegmasia alba dolens, phlegmasia cerulean dolens (PCD), and venous gangrene. The term PCD differentiates ischemia-associated massive venous thrombosis from phlegmasia alba dolens, which describes fulminant venous thrombosis without ischemia. We present a 55-year-old hypertensive, who presented with paedal oedema and breathlessness at rest. About a month prior to this admission, she suffered dislocation of left patella. She was treated with a plaster cast and immobilization for 3 weeks. Her serum creatinine was 8.8 mg/dL. She was initiated on haemodialysis via two single-lumen catheters placed in left femoral vein. The femoral vein catheters were removed after third session of haemodialysis. On fourth day, the patient complained pain and blue discolouration of left toes. On examination, the left lower limb was swollen, discoloured, and cold with blebs up to upper one-third of left leg. The left dorsalis pedis and posterior tibial arteries were not palpable. A Doppler of veins of lower limb revealed, thrombosis of deep, and superficial venous system of left lower limb. As there was no response to anticoagulation below, knee amputation was performed.

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.

The N-terminal region containing the zinc finger domain of tobacco streak virus coat protein is essential for the formation of virus-like particles.

Tobacco streak virus (TSV), a member of the genus Ilarvirus (family Bromoviridae), has a tripartite genome and forms quasi-isometric virions. All three viral capsids, encapsidating RNA 1, RNA 2 or RNA 3 and subgenomic RNA 4, are constituted of a single species of coat protein (CP). Formation of virus-like particles (VLPs) could be observed when the TSV CP gene was cloned and the recombinant CP (rCP) was expressed in E. coli. TSV VLPs were found to be stabilized by Zn(2+) ions and could be disassembled in the presence of 500 mM CaCl2. Mutational analysis corroborated previous studies that showed that an N-terminal arginine-rich motif was crucial for RNA binding; however, the results presented here demonstrate that the presence of RNA is not a prerequisite for assembly of TSV VLPs. Instead, the N-terminal region containing the zinc finger domain preceding the arginine-rich motif is essential for assembly of these VLPs.

Characterization of a potyvirus associated with yellow mosaic disease of jasmine (Jasminum sambac L.) in Andhra Pradesh, India.

A virus isolate associated with yellow mosaic disease was purified from commercially cultivated jasmine (Jasminum sambac) from Andhra Pradesh, India and it contained flexuous filamentous particles of ~720 × 13 nm. The denatured purified virus had single major polypeptide of molecular weight 32 kDa. Complementary DNA representing 1678 nucleotides (nt) of the 3' terminus of viral RNA was cloned and sequenced. Comparisons of complete coat protein (CP) gene nucleotide and amino acid sequences of the present virus isolate with certain reported potyviruses revealed 86.1 and 92.7 % identity, respectively with jasmine potyvirus T (JaVT) reported from Taiwan and less than 70 % with other potyviruses. Based on the phylogenetic analysis of 3' UTR and CP gene, the present virus isolate was identified as an isolate of JaVT that belongs to the genus Potyvirus and the name Jasmine yellow mosaic virus-Andhra Pradesh (JaYMV-AP) is proposed.

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.

Isolation and characterization of bacterial strains Paenibacillus sp. and Bacillus sp. for kraft lignin decolorization from pulp paper mill waste.

Eight aerobic bacterial strains were isolated from pulp paper mill waste and screened for tolerance of kraft lignin (KL) using the nutrient enrichment technique in mineral salt media (MSM) agar plate (15 g/L) amended with different concentrations of KL (100, 200, 300, 400, 500, 600 ppm) along with 1% glucose and 0.5% peptone (w/v) as additional carbon and nitrogen sources. The strains ITRC S6 and ITRC S8 were found to have the most potential for tolerance of the highest concentration of KL. These organisms were characterized by biochemical tests and further 16S rRNA gene (rDNA) sequencing, which showed 96.5% and 95% sequence similarity of ITRC S(6) and ITRC S(8) and confirmed them as Paenibacillus sp. and Bacillus sp., respectively. KL decolorization was routinely monitored with a spectrophotometer and further confirmed by HPLC analysis. Among eight strains, ITRC S(6) and ITRC S(8) were found to degrade 500 mg/L of KL up to 47.97% and 65.58%, respectively, within 144 h of incubation in the presence of 1% glucose and 0.5% (w/v) peptone as a supplementary source of carbon and nitrogen. In the absence of glucose and peptone, these bacteria were unable to utilize KL. The analysis of lignin degradation products by GC-MS analysis revealed the formation of various acids as lignin monomers which resulted in a decrease in pH and a major change in the chromatographic profile of the bacterial degraded sample as compared to the control clear indications of biochemical modification of KL due to the bacterial ligninolytic system by ITRC S(6), namely, acetic acid, propanoic acid, butanoic acid, guaiacol, hexanoic acid, and ITRC S(8), namely acetic acid, propanoic acid, ethanedioic acid, furan carboxylic acid, 2-propanoic acid, butanoic acid, 3-acetoxybutyric acid, propanedioic acid, acetoguiacone, 1,2,3-thiadiazole, 5-carboxaldixime, 4-hydroxy-3,5-dimethoxyphenol, and dibutyl phthalate, indicating the bacterium characteristic to degrade G and S units of lignin polymer.

First Report of a Tospovirus on Sunflower (Helianthus annus L.) from India.

Virus-like symptoms were observed on sunflower in and around Tirupati during January 1998. Infected plants exhibited severe mosaic, systemic necrosis along the stem and floral heads, leaf distortion, and ringspots on leaves. The causal virus, mechanically transmissible from sunflower to sunflower cvs. Morden, MHSF8, MHSF18, KBHS1, and Cargil, developed symptoms like those in the original plant. The virus caused chlorotic and necrotic spots on Chenopodium amaranticolor, chlorotic and necrotic rings on cowpea cv. C-152, chlorotic spots on Datura metal and Petunia hybrida, chlorotic rings and systemic infection on Gomphrena globosa, tarlike symptoms on Catharanthus roseus, and local brown lesions on Cassia tora (1). Virus was isolated from infected sunflower leaves (2), and particles in negatively stained preparations were enveloped and 80 to 90 nm in diameter. Cytopathic effects included accumulation of virus particles in the endoplasmic reticulum, the formation of viroplasm, and aggregates consisting of nonenveloped viral nucleocapsids in the cytoplasm of ultrathin sections of infected sunflower leaves. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of capsid protein resolved as a single band of 31 kD. The dsRNA isolated from infected sunflower leaves resolved as three bands of 9, 4.9, and 3 kb in 2% agarose gel electrophoresis. In enzyme-linked immunosorbent assay, the purified virus reacted with homologous and Peanut bud necrosis virus (PBNV-ICRISAT, India) antisera and not to polyclonal antibodies to Iris yellow spot virus (Netherlands), Tomato spotted wilt virus-T (Georgia), Impatiens necrotic spot virus, and several isolates of Cucumber mosaic virus (CMV-B, CMV-C, CMV-To). In western blotting analysis, the virus coat protein reacted with homologous and PBNV antisera corresponding to coat protein band of 31 kD. In reverse transcription polymerase chain reaction, the viral RNA was amplified by using primers derived from NP gene sequence of PBNV and Watermelon silver mottle virus (WSMV). Based on these properties, the virus causing sunflower mosaic followed by necrosis in India was identified as a tospovirus, which may be as a distinct isolate of sero group IV. References: (1) A. A. Brunt et al. Viruses of Plants Online. 1996. Australian National University, Canberra, 1996. (2) D. V. R. Reddy et al. Bud necrosis virus: A disease of peanut caused by Tomato spotted wilt virus. ICRISAT Inf. Bull. No. 31, 1991.

Changes in levels of substrates and enzymes in osme organs of female rats in response to plant extract.

Levels of glycogen, lactate, pyruvate, total proteins, free amino acids andenzymes (LDH & SDH) have been recorded in brain, heart, kidney and liver of rats in response to treatment with crude drug combination. The study indicates increase in the levels of glycogen, pyruvate, total proteins, LDH and SDH and decrease in levels of lactate and free amino acids.