Browsing by Author "Sharma, Sushil K"

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    Bacillus spp. for suppression of eggplant bacterial wilt pathogen in Andaman Islands: Isolation and characterization
    (NISCAIR-CSIR, India, 2019-02) Sakthivel, K; Manigundan, K; Gautam, RK; Singh, PK; Nakkeeran, S; Sharma, Sushil K
    Bacillus spp. isolated from different soils of Andaman Islands was characterized for antimicrobial potential by different methods. Among 65 strains tested, six Bacillus strains showed best in vitro antagonistic potential against solanaceous bacterial wilt pathogen Ralstonia solanacearum. The identity of the Bacillus strains were confirmed using 16S rRNA gene sequencing and biolog based phenotypic fingerprinting. The functional analysis revealed that most of the strains were positive for the production of IAA, siderophore and phosphate solubilization. In glass house and field studies, Bacillus amyloliquifaceans strain (Bs_Abi) showed significant biocontrol efficacy (92.1 and 73.7%, respectively) against brinjal bacterial wilt disease. The antimicrobial potential of Bs_Abi were further ascertained by presence of nine antimicrobial peptide (AMP) gene biosynthesis in PCR amplifications which confirmed the presence of peptide genes for six antibiotics (iturin, bacillomycin D, mycosubtilin, surfactin, bacilysin and subtilin). Our study revealed that the utilization of such antagonistic Bacillus spp. with multi antimicrobial peptide genes and more functional traits from tropical/ extreme soils would play a vital role in management of eggplant bacterial wilt disease and in formulation of new bioinoculants for improving the cropping system in Andaman and Nicobar Islands
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    Characterization of differentially expressed stress-associated proteins in starch granule development under heat stress in wheat (Triticum aestivum L.).
    (2013-04) Kumar, Ranjeet R; Sharma, Sushil K; Goswami, Suneha; Singh, G P; Singh, Rajendra; Singh, Khushboo; Pathak, Himanshu; Rai, Raj D
    Abiotic stress causes abrupt increase in the expression of stress-associated proteins, which provide tolerance by modulating the defense mechanism of plants. Small heat shock proteins (sHSPs) and anti-oxidant enzymes are important for environmental stress tolerance of the plants. In this study, two full-length cDNAs encoding small heat shock protein (sHSP) and superoxide dismutase (SOD), designated as TasHSP and SODI were identified and characterized from C-306 (thermotolerant) and PBW343 (thermosusceptible) cultivars of wheat (Triticum aestivum L.). An alpha crystalline domain was observed in TasHSP and manganese/iron binding domain in case of SODI. Quantitative real-time PCR showed very high transcript level of TasHSP and SOD in C-306 compared to PBW343 at different stages of growth and against differential heat stress (HS). Under differential HS at milky-dough stage, the fold change in transcript of both TasHSP and SOD was observed maximum in C-306, compared to PBW343. Protein profiling and isoenzymes analysis showed the expression of several heat-stable proteins and prominent isoenzymes of SOD in C-306, compared to PBW343. Scanning electron microscopy (SEM) of starch granules showed globular, well-shaped and more numbers of endospermic cells in C-306, compared to defragmented, irregular shaped and shrunken granules in case of PBW343 under HS treatment (42°C for 2 h). Diurnal change in soluble starch synthase (SSS) activity showed an increase in the activity during afternoon (35°C), compared to morning (29°C) and evening (32°C) in both the cultivars. Under heat stress (42°C for 2 h), a drastic decrease in the SSS activity was observed, due to the thermal denaturation of the enzyme. Thermotolerance capacity analyzed using cell membrane stability (CMS) showed significantly higher CMS in case of C-306, compared to PBW343 at different stages of growth. Findings suggest that abundance of TasHSP and SODI during milky-dough stage plays a very important role in starch granule biosynthesis. The mechanism may be further exploited to develop tolerant wheat cultivar with high quality seeds.
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    Exogenous application of putrescine at pre-anthesis enhances the thermotolerance of wheat (Triticum aestivum L.).
    (NISCAIR CSIR India., 2014-10) Kumar, Ranjeet R; Sharma, Sushil K; Rai, Gyanendra K; Singh, Khushboo; Choudhury, Madhumanthi; Dhawan, Gaurav; Singh, Gyaneshwar P; Goswami, Suneha; Pathak, Himanshu; Rai, Raj D
    Antioxidant enzymes, besides being involved in various developmental processes, are known to be important for environmental stress tolerance in plants. In this study, the effect of treatment of 2.5 mM putrescine (Put), heat stress (HS -42°C for 2 h) and their combination on the expression and activity of antioxidant enzymes was studied at pre-anthesis in the leaves of two wheat (Triticum aestivum L.) cultivars — HDR77 (thermotolerant) and HD2329 (thermosusceptible). We observed that 2.5 mM Put before HS significantly enhanced the transcript levels of superoxide dismutase (SOD), catalase (CAT), cytoplasmic and peroxisomal ascorbate peroxidase (cAPX, pAPX) in both the cultivars. However, the activities of antioxidant enzymes (SOD, CAT, APX and GR), as well as accumulation of antioxidants (ascorbic acid and total thiol content) were higher in HDR77 than in HD2329 in response to the treatment 2.5 mM Put + HS. No significant change was observed in the proline accumulation in response to HS and combined treatment of 2.5 mM Put + HS. A decrease in the H2O2 accumulation, lipid peroxidation and increase in cell membrane stability (CMS) were observed in response to 2.5 mM Put + HS treatment, as compared to HS treatment alone in both the cultivars; HDR77 was, however, more responsive to 2.5 mM Put + HS treatment. Put (2.5 mM) treatment at pre-anthesis thus modulated the defense mechanism responsible for the thermotolerance capacity of wheat under the heat stress. Elicitors like Put, therefore, need to be further studied for temporarily manipulating the thermotolerance capacity of wheat grown under the field conditions in view of the impending global climate change.
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    Genome Wide Identification of Target Heat Shock Protein90 Genes and Their Differential Expression against Heat Stress in Wheat.
    (2012-01) Kumar, Ranjeet R; Goswami, Suneha; Sharma, Sushil K; Pathak, Himanshu; Rai, Gyanendra K; Rai, Raj D
    Aims: To study the genetic and transcript profiling of the genes specifying cytosolic HSP90s in Triticum aestivum. Study Design: Random sampling. Place and Duration of Study: Indian Agricultural Research Institute, New Delhi, India, between August to December, 2011. Methodology: We include C-306 (thermotolerant) and PBW343 (thermosusceptible) cultivars of wheat for the study. Total RNA was isolated using Trizol method and gene was identified and isolated using RT-PCR. In silico characterization was done using different bioinformatic tools. Quantitative real time PCR was carried out using BioRad CFX96 platform and Pfaffl’s method was used for the comparative change in fold expression of the gene. Results: Here, we report cloning of an HSP90 gene from C-306 wheat cultivar having an ORF of 700 amino acids. Genome Blast identified 3 different clusters of reference sequence on chromosome no 4, 8 and 9 having LOC 100125696 and showing maximum homology with HSP90 reported from Triticum aestivum. Pure amino acid composition revealed highest composition of glutamic acid followed by lysine and leucine whereas, cysteine composition was lowest. Protein characterization showed the existence of 10 networks of coevolved amino acids. Quantitative real time PCR showed 1.5, 4.5, 5 & 7.4 fold increase in expression of HSP90 in case of C-306 compared to 2.5, 6.4, 6.9 & 5.6 fold increase in case of PBW343 at vegetative (root & shoot), pollination and milky dough stage. Multiple co-chaperones of HSP90 were observed by immunoblot assay in response to differential heat shock. Conclusion: This investigation proves that HSP90 is one of the key components of defense mechanism in wheat against heat stress which requires the formation of cochaperone complexes with HSP70 for its functional activity. There is a need to exploit the transcription factors associated with HSP90 and its regulation and differential expression in order to use it for developing thermotolerant wheat cultivars.