Lakhmir Singh - Academia.edu (original) (raw)

Papers by Lakhmir Singh

PLANT CELL BIOTECHNOLOGY AND MOLECULAR BIOLOGY

Urdbean leaf crinkle virus (ULCV) is a widespread and most destructive virus that causes leaf cri... more Urdbean leaf crinkle virus (ULCV) is a widespread and most destructive virus that causes leaf crinkle disease in black gram. Virus infection causes alteration in physiological and biochemical processes within the plant. In present study, effect of viral infection was investigated on chlorophyll content, total soluble sugar, total protein, phenolic contents, sodium dismutase (SOD) and nitrate reductase activities in the leaves of susceptible and resistant genotypes of urdbean at 30 days after virus inoculation. Disease symptoms were mild (6.7% to 10%) in resistant genotypes and severe (36% to 43%) in susceptible genotypes. ULCV infection resulted in a significant decrease in chlorophyll content in infected plants of all genotypes over control plants; however, magnitude of reduction was more in susceptible genotypes (41%) as compared to the resistant genotypes (14.35%). The total sugar and protein contents were significantly higher in the inoculated susceptible (210.26 % and 68.67%, r...

Journal of Obstetrics and Gynaecology

Scientia Horticulturae, 2007

... cultivars from India. The methods have also been standardized to eradicate these viruses from... more ... cultivars from India. The methods have also been standardized to eradicate these viruses from Chrysanthemum cv. Regol Time using tissue culture technique (Verma et al., 2004 and Ram et al., 2005). A number of chrysanthemum ...

Scientia Horticulturae, 2005

In a survey of lily growing fields in various parts of Himachal Pradesh, India, three viruses, Cu... more In a survey of lily growing fields in various parts of Himachal Pradesh, India, three viruses, Cucumber mosaic (CMV), Lily symptomless (LSV) and Lily mottle virus (LMoV) lily strain, were found quite prevalent in Asiatic (12 cultivars) and Oriental hybrids (4 cultivars) of lily, Lilium longiflorum and L. tigrinum. Apart from these viruses, Strawberry latent ringspot virus (SLRSV) was also found to infect Oriental hybrid lily. The disease incidence on the basis of symptoms observed ranged between minimum (40.7%) in cv. Alaska and maximum (83.7%) in cv. America, both Asiatic hybrid lily types. These viruses were indexed by testing outer and inner scales of bulbs and the leaves at early and flowering stages using ELISA and reverse transcription polymerase chain reaction (RT-PCR). Mechanical and insect transmission, purification, electron microscopy and sequencing of the PCR fragments were carried out for these viruses. Cloning and sequence analysis confirmed the viruses as CMV-subgroup II, LSVand LMoV. There were 96-98% nucleotide and 93-97% amino acid homology with CMV subgroup II sequences, 97-99% nucleotide and 91-92% amino acid homology with LSV sequences and 92-99% nucleotide and 85-98% amino acid homology with LMoV sequences.

Indian Journal of Virology, 2011

Chrysanthemum virus B coat protein constitutes the viral capsid which, besides other functions, e... more Chrysanthemum virus B coat protein constitutes the viral capsid which, besides other functions, encapsulates and protects the viral nucleic acid. We have demonstrated homotypic interaction of the coat protein subunits, essentially important for dimer formation, which is the first step during capsid assembly in vivo and in vitro. Interaction capacity of full length and truncated protein has been investigated and important regions have been identified through protein-protein interaction in yeast and by coimmunoprecipitation assays. Complete coat protein was found to interact strongly with similar subunits. Constructs with 102 amino acids from the N-terminal and 64 amino acids from C-terminal were found to be inconsequential for dimer formation as they did not show any interaction with similar subunits or with full length protein when analysed for b-galactosidase or histidine prototrophy. Results suggest that the region of 98-184 amino acids from the middle plays an important role in the process, probably without the involvement of N-and C-terminals.

Indian Journal of Virology, 2013

During a survey conducted in the grapevine orchards of Himachal Pradesh, variety of symptoms rang... more During a survey conducted in the grapevine orchards of Himachal Pradesh, variety of symptoms ranging from leaf yellowing, vein greening, reduced leaf size, downward rolling/cup shaped leaves to reduced fruit bearing were observed. Symptomatic leaf samples were collected and analyzed by serological (DAS-ELISA) and molecular methods (RT-PCR, PCR) for viruses and phytoplasma known worldwide on grapevine. DAS-ELISA was used for detection of Grapevine leafroll associated virus 1, 2 and 3 (GLRaV-1, 2 & 3), Grapevine virus A (GVA), Grapevine fan leaf virus (GFLV), Grapevine fleck virus (GFkV) and successfully detected GLRaV-1 & 3 and GFkV. All these samples were complemented with RT-PCR along with GVb and phytoplasma (additional to ELISA) using specific primers. Specific amplification in RT-PCR for GLRaV-1 (*232 bp), GLRaV-3 (*300 bp), GFkV (*179 bp) and GVB (*440 bp) confirmed the presence of these pathogens. Overall, ELISA and RT-PCR results confirmed the presence GLRaV-3 (66.7 %), GLRaV-1& GFkV (50 %), and Grapevine virus B (GVB) (12.5 %) in symptomatic plants. None of the samples were found positive for GFLV, GLRaV-2 and phytoplasma. Mixed infection was common and none of the plants were found virus free. To the best of our knowledge this is the first report of detection of GFkV and GVB in India.

Indian Journal of Virology, 2013

Cucumber mosaic virus (CMV) has a wide host range causing severe damage in many important agricul... more Cucumber mosaic virus (CMV) has a wide host range causing severe damage in many important agricultural and ornamental crops. Earlier reports showed the prevalence of CMV subgroup I isolates in India. However, some recent reports point towards increasing incidence of subgroup II isolates in the country. The complete genome of a CMV isolate causing severe mosaic in cucumber was characterized and its phylogenetic analysis with other 21 CMV isolates reported worldwide clustered it with subgroup II strains. The genome comprised of RNA 1 (3,379 nucleotides), RNA 2 (3,038 nucleotides) and RNA 3 (2,206 nucleotides). The isolate showed highest homology with subgroup II isolates: 95.1-98.7, 87.7-98.0, and 85.4-97.1 % within RNA1, RNA2, and RNA3, respectively. RNA1 and RNA2 were closely related to the Japanese isolate while RNA3 clustered with an American isolate. Host range studies revealed that isolate showed severe mosaic symptoms on Nicotiana spp. and Cucumis spp. The isolate induced leaf deformation and mild filiform type symptoms in tomato. To best of our knowledge this is the first report of complete genome of CMV subgroup II isolate from India.

Journal of Plant Biochemistry and Biotechnology, 2013

ABSTRACT Cherry necrotic rusty mottle virus (CNRMV) and Cherry virus A (CVA) are important graft ... more ABSTRACT Cherry necrotic rusty mottle virus (CNRMV) and Cherry virus A (CVA) are important graft transmitted viruses in the family Betaflexiviridae, infecting cherry. It is believed that CVA causes severe symptoms and disease in combination with other stone fruit viruses in both cherry and non-cherry hosts while CNRMV infected tree show reduced growth, significant yield loss and early death. Detection of these viruses is crucial for their sanitation, indexing and certificate programmes. In this study, a polyclonal antibody was produced against recombinant coat protein (CP) of CVA and CNRMV expressed in Escherichia coli for the development serological based diagnostics. The coat protein gene of CVA and CNRMV was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using specifically designed primer pair with restriction sites at both ends and further cloned and expressed in bacterial expression vector (pET32a). CP coding region of both the viruses was expressed separately and successfully as a fusion protein. The fusion proteins were purified and directly used for rabbit immunizations. Antibodies were purified, conjugated with alkaline phosphatase and used in DAS-ELISA. A total of 74 and 43 samples were checked for CVA and CNRMV, respectively. In this analysis, 40/74 samples were found positive for CVA and 20/43 tested samples were positive for CNRMV by DAS-ELISA, further confirmed by RT-PCR. Antibodies raised against recombinant CNRMV CP also detected the virus consistently in western blot analysis with high sensitivity and specificity. IC-RT-PCR was also developed for the detection of CVA using the produced antibody. To the best of our knowledge, this is the first report of DAS-ELISA and IC-RT-PCR based diagnostics for CVA, and first report of DAS-ELISA based diagnostics for CNRMV from India.

Journal of Plant Biochemistry and Biotechnology, 2011

Chrysanthemum virus B (CVB genus Carlavirus, family Flexiviridae) is one of the major viral patho... more Chrysanthemum virus B (CVB genus Carlavirus, family Flexiviridae) is one of the major viral pathogens of chrysanthemum. This virus occurs worldwide, is a potential threat to the floriculture industry and hence is a quarantine pathogen. CVB has a positive sense single-stranded RNA (ssRNA) genome~8.8 kb. The coat protein gene of CVB was amplified by RT-PCR, cloned and over expressed in E. coli BL21. The protein (CP) was expressed as a fusion protein with Glutathione S-Transferase (GST). Fused protein with GST was purified by GST tagged affinity chromatography and polyclonal (but monospecific) antibodies from rabbits immunized with the fusion protein was used for formulation of ELISA based diagnostic kit for CVB detection. The antisera produced showed specific reaction to CVB from infected chrysanthemums, Nicotiana glutinosa, Nicotiana clevelandii and Chenopodium quinoa at dilution of 1:1000 in ELISA. Results obtained were comparable (some times better) than commercial kit. The IgG against CVB performed favorably in specificity and sensitivity to the virus. Findings present a procedure for production of antibodies for CVB indexing of chrysanthemums propagative and mother stock materials to provide the disease free planting material.

Indian Journal of Microbiology, 2013

A rapid and sensitive two-step RT-PCR protocol for simultaneous detection of major apple viruses,... more A rapid and sensitive two-step RT-PCR protocol for simultaneous detection of major apple viruses, namely Apple mosaic virus (ApMV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple scar skin viroid (ASSVd), was developed. Five specific primer pairs were tested and confirmed for these viruses and viroid together in a single tube, giving amplicons of *198, *330, *370, *547 and *645 bp corresponding to ASGV, ASSVd, ASPV, ApMV and ACLSV, respectively. Using a guanidinium-based extraction buffer along with a commercial kit resulted in better quality RNA as compared to kit, suited for multiplex RT-PCR. A rapid CTAB method for RNA isolation from apple tissue was developed, which produce good yield and saves time. To the best of our knowledge, this is the first report on the simultaneous detection of five pathogens (four viruses and a viroid) from apple with NADH dehydrogenase subunit 5 (nad5) as an internal control.

Archives of Virology, 2011

American Journal of Animal and Veterinary Sciences, 2009

Problem statement: Gohilwari breed of goat is a multipurpose goat mainly for milk and meat purpos... more Problem statement: Gohilwari breed of goat is a multipurpose goat mainly for milk and meat purposes and best suited in its harsh climatic condition. This breed is inadequately characterized till now at DNA level. So the present study was undertaken for population genetic analysis at molecular level to exploit the breed for planning sustainable improvement, conservation and utilization, which subsequently can improve the livelihood of its stake holders. Approach: The experiment was conducted on 50 genomic DNA samples of unrelated goat using 25 microsatellite markers selected from the list suggested by International Society for Animal Genetics (ISAG) and FAO's (DAD-IS). Results: All of the 25 microsatellites were well amplified. The observed number of alleles detected per locus ranged from 4-24 with an overall mean of 10.12±5.46. Overall mean observed heterozygosity of 0.505 was lower than the overall mean expected heterozygosity of 0.684. Most of the loci showed the heterozygote deficit as also depicted by F is value. There was substantial genetic variation and polymorphism across studied loci in the Gohilwari breed of goat. And this population was not in Hardy-Weinberg equilibrium at most of the studied loci. This population was also receiving new genetic materials through introduction of immigrants. Conclusion: The strong inference that the Gohilwari breed of goat has not undergone bottleneck is also important for goat breeders and conservationists, as it suggests that any unique alleles present in this breed may not have been lost. Therefore, it can be recommended that within-breed diversity is actively maintained to enable these extensively unmanaged stocks to adapt to future demands and conditions and there is ample scope for further improvement in its productivity through appropriate breeding strategies. Though, microsatellites are neutral to selection with Ewens-Watterson test for neutrality some microsatellites were found not neutral or linked to some selective trait that must be further investigated for association to selective traits.

Indian Journal of Virology, 2010

The viral diseases have been a constraint in improving productivity of a variety of crops since t... more The viral diseases have been a constraint in improving productivity of a variety of crops since the time of their discovery just over a century ago. During the pre-Green Revolution period only a handful viruses were of concern. The development of high yielding and fertilizer responsive crop varieties ushered the famous Green Revolution (GR) in the mid 1960s. The GR was accompanied by the introduction of intensive cropping systems, introduction of new crops, large scale exchange of germplasm and unprecedented movement of seeds and planting materials with increasing international travel and trade. These developments have played important role in the emergence and spread of new viruses including the evolution of variants of viruses, changes in vector biology, movement of viruses with the infected planting materials, and the introduction of host susceptibility genes through the exchange of germplasm. Lately, the global climate change is also contributing to the emergence and spread of viruses by creating favourable environment for the viruses and their vectors. In the post-GR period the number of viruses affecting crop plants, particularly in the tropics and semi-tropics, has swelled enormously resulting in enormous economic losses and in many cases threatened food and nutrition security. The tropics and semi-tropics provide ideal environmental conditions for the perpetuation of both the viruses and their vectors. India is no exception; with its diverse agro-climate zones and cropping systems, it has its own share of threatening viral diseases caused by many emerging and reemerging viruses. The most destructive of these are the whitefly-transmitted begomoviruses and thrips-transmitted tospo-and ilarviruses. Begomoviruses have been known to cause serious diseases even in the pre-GR period, but during the post-GR period they seem to have undergone rapid mutations and re-combinations, resulting in the fast emergence of new viruses and strains. In contrast, tospoviruses have evolved gradually, as they are less prone to rapid mutation and recombination, but have spread widely. Both these groups of viruses have evolved phylogeographically. Vector transmission and human interventions have played important roles in the evolution and spread of these viruses. The changing scenario and some major epidemics of these viruses will be discussed.

The climate change and ever-increasing population has stressed the plant scientists to devise eco... more The climate change and ever-increasing population has stressed the plant scientists to devise economically viable, ecologically safe and socially acceptable agricultural practices that can keep the menace of plant disease losses under economic threshold levels. In the recent past, the philosophy of crop protection has shifted from the use of environmentally unsafe chemical pesticides to the eco-friendly approaches. The conventional methods of plant disease management like cultural practices, biological control, chemical control, and natural resistance are still in practice, but are not adequate to control many destructive diseases. The sustenance of disease resistant varieties under variable eco-systems remained a challenge to the breeders due to the fast-evolving nature of many plant pathogens resulting in the breakdown of the R-genes. This emphasized the plant breeders to have a wide range of genetic options to diversify the resistance traits having potential to reduce the pressur...

Paro verificar a importAncia da meta-análise de representações sociais. observamos representações... more Paro verificar a importAncia da meta-análise de representações sociais. observamos representações de familiares (RF) e suas relações com representações de grupos minoritários (RGM). Comparamos grupos segundo auto-identificaçlo tlnica/religiosa e situaçllo socioeconÕmica. Os sujeitos foram cristllos/católic05 e judeusdeclassemédia(N-148)e .. negros .. ,"mcstiços ..... brancos"e .. indefinidos"demciopopular(N-179) Solicitamos aos sujeitos que relatassem suas lembranças de familiarcs e que fizC5sem uma aulo-apresemaçllo paro um muçulmano ou budista. Assim, enquanto as Rf em situações de família (relações mterpessoais. autoridade) estavam associadas a indif«rençalhostilidadequantoàs RGM. as Rf como individuosautônomos ou identificados ao grupo émico estavam dissociadas, wbretudo quando O próprio grupo era minontárin, e independente da siruaçlo socioeeonômica. Os resultados demonstraram a importância da meta-an~Jisc de representaçOes sociais, incluindo a observação de múltiplos objetos de represcntaçllo, simultaneamente, sobretudo daquclcs relacionados à vida individual, grupal sociocultural e coletiva. hlarrn•thn:representaç6essociais,meta-análiseeidentidadesocial.

PLOS ONE, 2016

Plant viral movement proteins facilitate virion movement mainly through interaction with a number... more Plant viral movement proteins facilitate virion movement mainly through interaction with a number of factors from the host. We report the association of a cell wall localized ascorbate oxidase (CsAO4) from Cucumis sativus with the movement protein (MP) of Cucumber mosaic virus (CMV). This was identified first in a yeast two-hybrid screen and validated by in vivo pull down and bimolecular fluorescence complementation (BiFC) assays. The BiFC assay showed localization of the bimolecular complexes of these proteins around the cell wall periphery as punctate spots. The expression of CsAO4 was induced during the initial infection period (up to 72 h) in CMV infected Nicotiana benthamiana plants. To functionally validate its role in viral spread, we analyzed the virus accumulation in CsAO4 overexpressing Arabidopsis thaliana and transiently silenced N. benthamiana plants (through a Tobacco rattle virus vector). Overexpression had no evident effect on virus accumulation in upper non-inoculated leaves of transgenic lines in comparison to WT plants at 7 days post inoculation (dpi). However, knockdown resulted in reduced CMV accumulation in systemic (non-inoculated) leaves of NbΔAO-pTRV2 silenced plants as compared to TRV inoculated control plants at 5 dpi (up to 1.3 fold difference). In addition, functional validation supported the importance of AO in plant development. These findings suggest that AO and viral MP interaction helps in early viral movement; however, it had no major effect on viral accumulation after 7 dpi. This study suggests that initial induction of expression of AO on virus infection and its association with viral MP helps both towards targeting of the MP to the apoplast and disrupting formation of functional AO dimers for spread of virus to nearby cells, reducing the redox defense of the plant during initial stages of infection.

Journal of Phytopathology, 2015

ABSTRACT During 2011, leaf crumpling, yellowing and stunting were observed on soya bean (Glycine ... more ABSTRACT During 2011, leaf crumpling, yellowing and stunting were observed on soya bean (Glycine max) in Himachal Pradesh, India. PCR-based detection confirmed the presence of a begomovirus. The viral genome was amplified by rolling circle amplification, cloned and sequenced. The complete nucleotide sequence of DNA-A showed highest nucleotide identity to an isolate of Ageratum enation virus infecting a weed Ageratum conyzoides. In addition, a DNA molecule was found which shared 95% nucleotide identity with an alphasatellite infecting ageratum. Neither beta satellite nor DNA-B was detected in the infected samples.

PLANT CELL BIOTECHNOLOGY AND MOLECULAR BIOLOGY

Urdbean leaf crinkle virus (ULCV) is a widespread and most destructive virus that causes leaf cri... more Urdbean leaf crinkle virus (ULCV) is a widespread and most destructive virus that causes leaf crinkle disease in black gram. Virus infection causes alteration in physiological and biochemical processes within the plant. In present study, effect of viral infection was investigated on chlorophyll content, total soluble sugar, total protein, phenolic contents, sodium dismutase (SOD) and nitrate reductase activities in the leaves of susceptible and resistant genotypes of urdbean at 30 days after virus inoculation. Disease symptoms were mild (6.7% to 10%) in resistant genotypes and severe (36% to 43%) in susceptible genotypes. ULCV infection resulted in a significant decrease in chlorophyll content in infected plants of all genotypes over control plants; however, magnitude of reduction was more in susceptible genotypes (41%) as compared to the resistant genotypes (14.35%). The total sugar and protein contents were significantly higher in the inoculated susceptible (210.26 % and 68.67%, r...

Journal of Obstetrics and Gynaecology

Scientia Horticulturae, 2007

... cultivars from India. The methods have also been standardized to eradicate these viruses from... more ... cultivars from India. The methods have also been standardized to eradicate these viruses from Chrysanthemum cv. Regol Time using tissue culture technique (Verma et al., 2004 and Ram et al., 2005). A number of chrysanthemum ...

Scientia Horticulturae, 2005

In a survey of lily growing fields in various parts of Himachal Pradesh, India, three viruses, Cu... more In a survey of lily growing fields in various parts of Himachal Pradesh, India, three viruses, Cucumber mosaic (CMV), Lily symptomless (LSV) and Lily mottle virus (LMoV) lily strain, were found quite prevalent in Asiatic (12 cultivars) and Oriental hybrids (4 cultivars) of lily, Lilium longiflorum and L. tigrinum. Apart from these viruses, Strawberry latent ringspot virus (SLRSV) was also found to infect Oriental hybrid lily. The disease incidence on the basis of symptoms observed ranged between minimum (40.7%) in cv. Alaska and maximum (83.7%) in cv. America, both Asiatic hybrid lily types. These viruses were indexed by testing outer and inner scales of bulbs and the leaves at early and flowering stages using ELISA and reverse transcription polymerase chain reaction (RT-PCR). Mechanical and insect transmission, purification, electron microscopy and sequencing of the PCR fragments were carried out for these viruses. Cloning and sequence analysis confirmed the viruses as CMV-subgroup II, LSVand LMoV. There were 96-98% nucleotide and 93-97% amino acid homology with CMV subgroup II sequences, 97-99% nucleotide and 91-92% amino acid homology with LSV sequences and 92-99% nucleotide and 85-98% amino acid homology with LMoV sequences.

Indian Journal of Virology, 2011

Chrysanthemum virus B coat protein constitutes the viral capsid which, besides other functions, e... more Chrysanthemum virus B coat protein constitutes the viral capsid which, besides other functions, encapsulates and protects the viral nucleic acid. We have demonstrated homotypic interaction of the coat protein subunits, essentially important for dimer formation, which is the first step during capsid assembly in vivo and in vitro. Interaction capacity of full length and truncated protein has been investigated and important regions have been identified through protein-protein interaction in yeast and by coimmunoprecipitation assays. Complete coat protein was found to interact strongly with similar subunits. Constructs with 102 amino acids from the N-terminal and 64 amino acids from C-terminal were found to be inconsequential for dimer formation as they did not show any interaction with similar subunits or with full length protein when analysed for b-galactosidase or histidine prototrophy. Results suggest that the region of 98-184 amino acids from the middle plays an important role in the process, probably without the involvement of N-and C-terminals.

Indian Journal of Virology, 2013

During a survey conducted in the grapevine orchards of Himachal Pradesh, variety of symptoms rang... more During a survey conducted in the grapevine orchards of Himachal Pradesh, variety of symptoms ranging from leaf yellowing, vein greening, reduced leaf size, downward rolling/cup shaped leaves to reduced fruit bearing were observed. Symptomatic leaf samples were collected and analyzed by serological (DAS-ELISA) and molecular methods (RT-PCR, PCR) for viruses and phytoplasma known worldwide on grapevine. DAS-ELISA was used for detection of Grapevine leafroll associated virus 1, 2 and 3 (GLRaV-1, 2 & 3), Grapevine virus A (GVA), Grapevine fan leaf virus (GFLV), Grapevine fleck virus (GFkV) and successfully detected GLRaV-1 & 3 and GFkV. All these samples were complemented with RT-PCR along with GVb and phytoplasma (additional to ELISA) using specific primers. Specific amplification in RT-PCR for GLRaV-1 (*232 bp), GLRaV-3 (*300 bp), GFkV (*179 bp) and GVB (*440 bp) confirmed the presence of these pathogens. Overall, ELISA and RT-PCR results confirmed the presence GLRaV-3 (66.7 %), GLRaV-1& GFkV (50 %), and Grapevine virus B (GVB) (12.5 %) in symptomatic plants. None of the samples were found positive for GFLV, GLRaV-2 and phytoplasma. Mixed infection was common and none of the plants were found virus free. To the best of our knowledge this is the first report of detection of GFkV and GVB in India.

Indian Journal of Virology, 2013

Cucumber mosaic virus (CMV) has a wide host range causing severe damage in many important agricul... more Cucumber mosaic virus (CMV) has a wide host range causing severe damage in many important agricultural and ornamental crops. Earlier reports showed the prevalence of CMV subgroup I isolates in India. However, some recent reports point towards increasing incidence of subgroup II isolates in the country. The complete genome of a CMV isolate causing severe mosaic in cucumber was characterized and its phylogenetic analysis with other 21 CMV isolates reported worldwide clustered it with subgroup II strains. The genome comprised of RNA 1 (3,379 nucleotides), RNA 2 (3,038 nucleotides) and RNA 3 (2,206 nucleotides). The isolate showed highest homology with subgroup II isolates: 95.1-98.7, 87.7-98.0, and 85.4-97.1 % within RNA1, RNA2, and RNA3, respectively. RNA1 and RNA2 were closely related to the Japanese isolate while RNA3 clustered with an American isolate. Host range studies revealed that isolate showed severe mosaic symptoms on Nicotiana spp. and Cucumis spp. The isolate induced leaf deformation and mild filiform type symptoms in tomato. To best of our knowledge this is the first report of complete genome of CMV subgroup II isolate from India.

Journal of Plant Biochemistry and Biotechnology, 2013

ABSTRACT Cherry necrotic rusty mottle virus (CNRMV) and Cherry virus A (CVA) are important graft ... more ABSTRACT Cherry necrotic rusty mottle virus (CNRMV) and Cherry virus A (CVA) are important graft transmitted viruses in the family Betaflexiviridae, infecting cherry. It is believed that CVA causes severe symptoms and disease in combination with other stone fruit viruses in both cherry and non-cherry hosts while CNRMV infected tree show reduced growth, significant yield loss and early death. Detection of these viruses is crucial for their sanitation, indexing and certificate programmes. In this study, a polyclonal antibody was produced against recombinant coat protein (CP) of CVA and CNRMV expressed in Escherichia coli for the development serological based diagnostics. The coat protein gene of CVA and CNRMV was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using specifically designed primer pair with restriction sites at both ends and further cloned and expressed in bacterial expression vector (pET32a). CP coding region of both the viruses was expressed separately and successfully as a fusion protein. The fusion proteins were purified and directly used for rabbit immunizations. Antibodies were purified, conjugated with alkaline phosphatase and used in DAS-ELISA. A total of 74 and 43 samples were checked for CVA and CNRMV, respectively. In this analysis, 40/74 samples were found positive for CVA and 20/43 tested samples were positive for CNRMV by DAS-ELISA, further confirmed by RT-PCR. Antibodies raised against recombinant CNRMV CP also detected the virus consistently in western blot analysis with high sensitivity and specificity. IC-RT-PCR was also developed for the detection of CVA using the produced antibody. To the best of our knowledge, this is the first report of DAS-ELISA and IC-RT-PCR based diagnostics for CVA, and first report of DAS-ELISA based diagnostics for CNRMV from India.

Journal of Plant Biochemistry and Biotechnology, 2011

Chrysanthemum virus B (CVB genus Carlavirus, family Flexiviridae) is one of the major viral patho... more Chrysanthemum virus B (CVB genus Carlavirus, family Flexiviridae) is one of the major viral pathogens of chrysanthemum. This virus occurs worldwide, is a potential threat to the floriculture industry and hence is a quarantine pathogen. CVB has a positive sense single-stranded RNA (ssRNA) genome~8.8 kb. The coat protein gene of CVB was amplified by RT-PCR, cloned and over expressed in E. coli BL21. The protein (CP) was expressed as a fusion protein with Glutathione S-Transferase (GST). Fused protein with GST was purified by GST tagged affinity chromatography and polyclonal (but monospecific) antibodies from rabbits immunized with the fusion protein was used for formulation of ELISA based diagnostic kit for CVB detection. The antisera produced showed specific reaction to CVB from infected chrysanthemums, Nicotiana glutinosa, Nicotiana clevelandii and Chenopodium quinoa at dilution of 1:1000 in ELISA. Results obtained were comparable (some times better) than commercial kit. The IgG against CVB performed favorably in specificity and sensitivity to the virus. Findings present a procedure for production of antibodies for CVB indexing of chrysanthemums propagative and mother stock materials to provide the disease free planting material.

Indian Journal of Microbiology, 2013

A rapid and sensitive two-step RT-PCR protocol for simultaneous detection of major apple viruses,... more A rapid and sensitive two-step RT-PCR protocol for simultaneous detection of major apple viruses, namely Apple mosaic virus (ApMV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple scar skin viroid (ASSVd), was developed. Five specific primer pairs were tested and confirmed for these viruses and viroid together in a single tube, giving amplicons of *198, *330, *370, *547 and *645 bp corresponding to ASGV, ASSVd, ASPV, ApMV and ACLSV, respectively. Using a guanidinium-based extraction buffer along with a commercial kit resulted in better quality RNA as compared to kit, suited for multiplex RT-PCR. A rapid CTAB method for RNA isolation from apple tissue was developed, which produce good yield and saves time. To the best of our knowledge, this is the first report on the simultaneous detection of five pathogens (four viruses and a viroid) from apple with NADH dehydrogenase subunit 5 (nad5) as an internal control.

Archives of Virology, 2011

American Journal of Animal and Veterinary Sciences, 2009

Problem statement: Gohilwari breed of goat is a multipurpose goat mainly for milk and meat purpos... more Problem statement: Gohilwari breed of goat is a multipurpose goat mainly for milk and meat purposes and best suited in its harsh climatic condition. This breed is inadequately characterized till now at DNA level. So the present study was undertaken for population genetic analysis at molecular level to exploit the breed for planning sustainable improvement, conservation and utilization, which subsequently can improve the livelihood of its stake holders. Approach: The experiment was conducted on 50 genomic DNA samples of unrelated goat using 25 microsatellite markers selected from the list suggested by International Society for Animal Genetics (ISAG) and FAO's (DAD-IS). Results: All of the 25 microsatellites were well amplified. The observed number of alleles detected per locus ranged from 4-24 with an overall mean of 10.12±5.46. Overall mean observed heterozygosity of 0.505 was lower than the overall mean expected heterozygosity of 0.684. Most of the loci showed the heterozygote deficit as also depicted by F is value. There was substantial genetic variation and polymorphism across studied loci in the Gohilwari breed of goat. And this population was not in Hardy-Weinberg equilibrium at most of the studied loci. This population was also receiving new genetic materials through introduction of immigrants. Conclusion: The strong inference that the Gohilwari breed of goat has not undergone bottleneck is also important for goat breeders and conservationists, as it suggests that any unique alleles present in this breed may not have been lost. Therefore, it can be recommended that within-breed diversity is actively maintained to enable these extensively unmanaged stocks to adapt to future demands and conditions and there is ample scope for further improvement in its productivity through appropriate breeding strategies. Though, microsatellites are neutral to selection with Ewens-Watterson test for neutrality some microsatellites were found not neutral or linked to some selective trait that must be further investigated for association to selective traits.

Indian Journal of Virology, 2010

The viral diseases have been a constraint in improving productivity of a variety of crops since t... more The viral diseases have been a constraint in improving productivity of a variety of crops since the time of their discovery just over a century ago. During the pre-Green Revolution period only a handful viruses were of concern. The development of high yielding and fertilizer responsive crop varieties ushered the famous Green Revolution (GR) in the mid 1960s. The GR was accompanied by the introduction of intensive cropping systems, introduction of new crops, large scale exchange of germplasm and unprecedented movement of seeds and planting materials with increasing international travel and trade. These developments have played important role in the emergence and spread of new viruses including the evolution of variants of viruses, changes in vector biology, movement of viruses with the infected planting materials, and the introduction of host susceptibility genes through the exchange of germplasm. Lately, the global climate change is also contributing to the emergence and spread of viruses by creating favourable environment for the viruses and their vectors. In the post-GR period the number of viruses affecting crop plants, particularly in the tropics and semi-tropics, has swelled enormously resulting in enormous economic losses and in many cases threatened food and nutrition security. The tropics and semi-tropics provide ideal environmental conditions for the perpetuation of both the viruses and their vectors. India is no exception; with its diverse agro-climate zones and cropping systems, it has its own share of threatening viral diseases caused by many emerging and reemerging viruses. The most destructive of these are the whitefly-transmitted begomoviruses and thrips-transmitted tospo-and ilarviruses. Begomoviruses have been known to cause serious diseases even in the pre-GR period, but during the post-GR period they seem to have undergone rapid mutations and re-combinations, resulting in the fast emergence of new viruses and strains. In contrast, tospoviruses have evolved gradually, as they are less prone to rapid mutation and recombination, but have spread widely. Both these groups of viruses have evolved phylogeographically. Vector transmission and human interventions have played important roles in the evolution and spread of these viruses. The changing scenario and some major epidemics of these viruses will be discussed.

The climate change and ever-increasing population has stressed the plant scientists to devise eco... more The climate change and ever-increasing population has stressed the plant scientists to devise economically viable, ecologically safe and socially acceptable agricultural practices that can keep the menace of plant disease losses under economic threshold levels. In the recent past, the philosophy of crop protection has shifted from the use of environmentally unsafe chemical pesticides to the eco-friendly approaches. The conventional methods of plant disease management like cultural practices, biological control, chemical control, and natural resistance are still in practice, but are not adequate to control many destructive diseases. The sustenance of disease resistant varieties under variable eco-systems remained a challenge to the breeders due to the fast-evolving nature of many plant pathogens resulting in the breakdown of the R-genes. This emphasized the plant breeders to have a wide range of genetic options to diversify the resistance traits having potential to reduce the pressur...

Paro verificar a importAncia da meta-análise de representações sociais. observamos representações... more Paro verificar a importAncia da meta-análise de representações sociais. observamos representações de familiares (RF) e suas relações com representações de grupos minoritários (RGM). Comparamos grupos segundo auto-identificaçlo tlnica/religiosa e situaçllo socioeconÕmica. Os sujeitos foram cristllos/católic05 e judeusdeclassemédia(N-148)e .. negros .. ,"mcstiços ..... brancos"e .. indefinidos"demciopopular(N-179) Solicitamos aos sujeitos que relatassem suas lembranças de familiarcs e que fizC5sem uma aulo-apresemaçllo paro um muçulmano ou budista. Assim, enquanto as Rf em situações de família (relações mterpessoais. autoridade) estavam associadas a indif«rençalhostilidadequantoàs RGM. as Rf como individuosautônomos ou identificados ao grupo émico estavam dissociadas, wbretudo quando O próprio grupo era minontárin, e independente da siruaçlo socioeeonômica. Os resultados demonstraram a importância da meta-an~Jisc de representaçOes sociais, incluindo a observação de múltiplos objetos de represcntaçllo, simultaneamente, sobretudo daquclcs relacionados à vida individual, grupal sociocultural e coletiva. hlarrn•thn:representaç6essociais,meta-análiseeidentidadesocial.

PLOS ONE, 2016

Plant viral movement proteins facilitate virion movement mainly through interaction with a number... more Plant viral movement proteins facilitate virion movement mainly through interaction with a number of factors from the host. We report the association of a cell wall localized ascorbate oxidase (CsAO4) from Cucumis sativus with the movement protein (MP) of Cucumber mosaic virus (CMV). This was identified first in a yeast two-hybrid screen and validated by in vivo pull down and bimolecular fluorescence complementation (BiFC) assays. The BiFC assay showed localization of the bimolecular complexes of these proteins around the cell wall periphery as punctate spots. The expression of CsAO4 was induced during the initial infection period (up to 72 h) in CMV infected Nicotiana benthamiana plants. To functionally validate its role in viral spread, we analyzed the virus accumulation in CsAO4 overexpressing Arabidopsis thaliana and transiently silenced N. benthamiana plants (through a Tobacco rattle virus vector). Overexpression had no evident effect on virus accumulation in upper non-inoculated leaves of transgenic lines in comparison to WT plants at 7 days post inoculation (dpi). However, knockdown resulted in reduced CMV accumulation in systemic (non-inoculated) leaves of NbΔAO-pTRV2 silenced plants as compared to TRV inoculated control plants at 5 dpi (up to 1.3 fold difference). In addition, functional validation supported the importance of AO in plant development. These findings suggest that AO and viral MP interaction helps in early viral movement; however, it had no major effect on viral accumulation after 7 dpi. This study suggests that initial induction of expression of AO on virus infection and its association with viral MP helps both towards targeting of the MP to the apoplast and disrupting formation of functional AO dimers for spread of virus to nearby cells, reducing the redox defense of the plant during initial stages of infection.

Journal of Phytopathology, 2015

ABSTRACT During 2011, leaf crumpling, yellowing and stunting were observed on soya bean (Glycine ... more ABSTRACT During 2011, leaf crumpling, yellowing and stunting were observed on soya bean (Glycine max) in Himachal Pradesh, India. PCR-based detection confirmed the presence of a begomovirus. The viral genome was amplified by rolling circle amplification, cloned and sequenced. The complete nucleotide sequence of DNA-A showed highest nucleotide identity to an isolate of Ageratum enation virus infecting a weed Ageratum conyzoides. In addition, a DNA molecule was found which shared 95% nucleotide identity with an alphasatellite infecting ageratum. Neither beta satellite nor DNA-B was detected in the infected samples.