Quality evaluation of improved and traditionally dried Bombay duck (Harpodon nehereus) through biochemical, microbiological, and organoleptic analysis.

The study was conducted to compare the quality and shelf life of traditionally dried (collected from the local markets) Bombay duck (Harpodon nehereus) with improved dried products (produced using a newly developed fish dryer) to assess its suitability. The quality of these products was evaluated through organoleptic, water reconstitution, nutritional, chemical, and microbiological characteristics. The organoleptic quality of improved dried fish was excellent while those produced traditionally were with grayish and dark brown color, rancid odor, and soft and fragile texture with insect infestation. The water reconstitution properties of the improved dried sample were 75.71% and 89.39% at room temperature and 80 °C, respectively, which were comparatively higher than the traditional dried products. The protein, ash, and contents were significantly higher in improved dried fish products while the lipid and total volatile base nitrogen (TVB-N) content were much lower than those of market-dried samples. The total viable counts (TVC) of bacteria were significantly higher in the traditional products which indicated poor quality. To find out the best storage method, dried fish was kept at three different conditions: in the open air at room temperature, in a sealed pack at room temperature, and a sealed pack at refrigeration temperature (4 °C). The shelf life of the products in different storage conditions was evaluated by estimating their moisture, protein, lipid, ash, TVB-N, and TVC values. The products kept at 4 °C temperature was found almost unaltered in terms of their nutritional properties after 4-months storage period. Results indicated that the newly developed fish dryer produced high-quality dried fish products with longer shelf life can be expected if the dried fish is stored at 4 °C refrigeration temperature. Our findings will be a valuable tool for the fish processors to ease the fish drying process and its storage that will enable them to commercially supply good quality dried Harpodon nehereus in the market chain at a low-cost.

Kisspeptin stimulates oestradiol biosynthesis by upregulating steroidogenic transcripts and proliferation markers in the bubaline granulosa cells in vitro.

Kisspeptin (Kp), an upstream regulator of GnRH release, is essential for the development and function of reproductive axis. Previously, we demonstrated the localization of Kp and its receptor (Kiss1r) in the active follicle in the bubaline ovary. Present study aimed to determine the effect of Kp on granulosa cell (GCs) functions, especially oestradiol (E2 ) and progesterone (P4 ) production, and differential expression of genes regulating the proliferation, apoptosis and steroidogenesis in the buffalo. The ovaries with 6-10 mm size follicles obtained from the cyclic buffaloes after slaughtering were used for isolation of GCs for in vitro study. The primary GCs culture was treated with Kp (0, 10, 50 and 100 nM) and incubated for 48 h. Production of E2 and P4 was estimated in the culture supernatant by ELISA. The expression of gonadotropin receptors (FSHR and LHR), steroidogenic genes (STAR, 3ß-HSD, CYP19A1), proliferation marker (PCNA), apoptotic factors (CASP3 and BCL2) and Kp signalling molecule (extracellular signal-regulated kinase 1/2, ERK1/2 and p-ERK1/2) was studied in the GCs by qPCR. Significant E2 production was found in the Kp 50 and 100 nM groups (p < .05), whereas P4 production was reduced in Kp 100 nM group (p < .05). There was concomitant upregulation of FSHR, ERK1/2, STAR and CYP19A1 in the Kp 100 nM treated GCs. In addition, Kp at 100 nM stimulated the proliferation of GCs by upregulating the expression of BCL2 (5.0 fold) and PCNA (94.9 fold). Further, high immunoreactivity of p-ERK1/2 was observed in the Kp-treated GCs. It was concluded that Kp at 100 nM concentration stimulated E2 production by upregulating the steroidogenic pathway through ERK1/2, STAR and CYP19A1 and modulating PCNA and BCL2 expressions in the GCs. Further experiments are warranted using Kp antagonist in different combinations to establish the signalling pathway in Kp-mediated steroidogenesis in the GCs for developing strategies to control ovarian functions.

Investigating the impact of n-heptane (C7H16) and nanoparticles (TiO2) on diesel-microalgae biodiesel blend in CI diesel engines.

Recent global challenges encompass profound environmental pollution and the depletion of finite fuel resources. In this study, the biodiesel used in the mixture was derived from Azolla pinnata microalgae oil through a trans-esterification reaction chosen for its high oil concentration. During the initial phase of the experiment, varying volumes of biodiesel (5%, 10%, and 15%) and n-heptane (5%, 10%, and 15%) were introduced to diesel to form a ternary fuel blend. The experimental outcome shows that an n-heptane and biodiesel mixture of 10% by volume would produce the best results. Next, experiments were carried out by incorporating 10, 40, and 80 ppm titanium oxide (TiO2) nanoparticles (NPs) in a recommended ternary fuel blend. The experimental investigation showed that D80A10H10TNP40 (diesel 80% + biodiesel 10% + n-heptane 10% + TiO2 40 ppm) caused a 7.21% increase in brake thermal efficiency (BTE) with a decrease in brake specific fuel consumption (BSFC) and brake specific energy consumption (BSEC) by 9.58% and 10%, respectively, compared to (diesel 80% + biodiesel 20%) D80A20. D80A10H10TNP40 exhibits lower emissions, with a significant reduction of 11.29% and 20.96% in carbon monoxide (CO) and unburnt hydrocarbons (UBHC), respectively. Nitrogen oxide (NOX) and smoke emissions were reduced by 3.3% and 11.13%, respectively, compared to D80A10H10. Furthermore, D80A10H10TNP40 demonstrated enhanced combustion properties, comprising a significant rise of 4.39% in-cylinder pressure (CP), 35.29% in heat release rate (HRR), and 25.05% in the rate of pressure rise (RPR). The findings of this investigation indicate that D80A10H10TNP40 exhibits enhanced efficiency, emission, and combustion properties compared to the D80A20 fuel.

Evaluation of sexed semen-based artificial insemination in Tharparkar cattle under organized farm condition.

Sexed semen facilitates additional female calf production for the expansion of a herd at a faster rate and also curtails the surplus production of unwanted male calves. A study was conducted to evaluate the performance of sexed semen in indigenous Tharparkar cows based on 114 artificial inseminations (AI) performed at natural oestrus using two protocols i.e., single AI (n = 48) and double AI (n = 66). Overall, the first service conception rate (CR) was significantly higher in double (53.0%) than single (33.3%) AI protocol. The odds ratio of conception rate in double AI was 2.26 (χ2 = 4.4, df = 1, p = .04) with respect to single AI. The time that elapsed since the detection of oestrus to insemination was also analysed. In a single AI protocol, the CR was higher (p < .05) at 16 h (54.6%) than insemination at 8 h (27.0%) following the onset of oestrus. Yet, the CR using double AI protocol did not differ (p = .73) significantly when AIs were performed either at 8 h and 24 h (51.9%) or 16 h and 24 h (57.1%) post onset of oestrus. Besides, like the single AI protocol, the parity of the animals also influenced the CR, being higher in heifers (n = 22) than those of parous (n = 92) cows (72.73 vs. 40.43%, χ2 = 7.48, df = 1, p = .006) in the present study. The odds ratio of conception in heifers was 3.93 with respect to parous cows. Overall, the birth of female calf was 91.7%. In conclusion, the present study indicates a future promise of the sexed semen for the production of more female offspring from Tharparkar cattle.

An experimental and numerical approach for thermal performance investigation of solar flat plate collector.

The present work aims to investigate thermal performance of a solar flat plate collector using water and Cu-MWCNTs nanoparticle-based hybrid nanofluid both experimentally and numerically. X-ray diffraction and FESEM with EDAX mapping were performed to characterize nanoparticles. The experimental setup was developed for thermal performance of FPC varying flow rates (0.5, 1.0, 1.5 LPM), inclination angle (25°, 30°, 35°, 40°, 45°), volume concentration (0%, 0.1%, 0.2%, 0.3%, 0.4%), and intensity (400 W/m2). The 3D numerical model having similar geometry as of actual flat plate collector was modeled using Fluents 15.0. The SST turbulence model was used to capture the chaotic changes in the velocity, temperature, and pressure fields. The experimental findings revealed 79.74% improvement in instantaneous efficiency at 0.4% vol., 1.5 LPM, 45° inclination angle, and 400 W/m2 intensity. The maximum deviation between the experimental and numerically calculated outlet and inlet temperature difference (ΔT) was 3.5% using a hybrid nanofluid. When numerical data are compared, instantaneous efficiency and heat gain both deviate by 2.8% and 2.9% from experimental values. Because of the numerical simulation analysis, it is possible to observe the temperature and flow pattern in flat plate collectors using nanofluids under a set of operating conditions, which would not be possible without the simulation.

Nano-depletion of morbid spermatozoa up-regulate Ca2+ channel, depolarization of membrane potential and fertility in buffalo.

Despite recent advances in technique of spermatozoa cryopreservation, there are still ejaculates present that fail to meet strict quality standard; mainly due to detrimental effect of imbalance of free radicals. The omnipresence of dead/defective spermatozoa in ejaculates of eutherian species is a major source of excessive free radicals. Though sperm-selection techniques, as well as addition of antioxidants addressed the problem to a certain extent, the major source of free radicals in the semen remained, causing much damage. This study attempts to remove dead/damaged spermatozoa using negative fertility-marker. The effect is unraveled by Hypo-osmotic (HOS), and fluorescein-conjugated Pisum sativum agglutinin (FITC-PSA) assay, further confirmed by Ca2+-regulating mechanisms and depolarization of sperm membrane potential, reduction in concentration of free radicals and finally by in vitro fertility assay. The study involved functionalization of iron oxide nanoparticles (IONPs) with silane followed by bio-conjugation with anti-ubiquitin antibodies. The nano-purification of semen using anti-ubiquitin conjugated iron oxide nanoparticles (IONPs) (antibody concentrations 0.5, 1.0 and 2.0 µg/ml) was attempted. The efficiency of nano-purification was 18.1%-43.8% in the study. The results revealed greater (P ≤ 0.05) spermatozoa population with intact plasma membrane, acrosome integrity, high mitochondrial membrane potential and pattern-F (least intracellular Ca2+), evidence of low lipid peroxidation and higher total antioxidant capacity in nano-purified groups. More number of spermatozoa were bound to zona pellucida of matured oocytes from nano-depleted than non-depleted group. The findings demonstrate antibody concentration of 1.0 µg/ml bio-conjugated with IONPs as most efficient in enriching the ejaculate with functional spermatozoa with the highest percentage of zona binding.

An evidence of Humanin-like peptide and Humanin mediated cryosurvival of spermatozoa in buffalo bulls.

Buffalo spermatozoa are vulnerable to cryo-injuries due to inherent deficiency of endogenous antioxidants, high polyunsaturated fatty acids (PUFA) content in plasma membrane and low cholesterol/phospholipid (C/P) ratio. Humanin is a potent cytoprotective agent that protects the cells against oxidative stress and apoptosis. The present study was designed to establish the presence of Humanin in buffalo and effect of Humanin supplementation on freezability of buffalo spermatozoa. Indirect immunofluorescence test revealed presence of Humanin in ejaculated and epididymal spermatozoa, and, elongated spermatids and interstitial space in the testicular tissue section. Humanin levels in seminal plasma were significantly and positively correlated with sperm concentration and individual progressive motility (IPM) in good (n = 22; IPM >70%) and poor (n = 10; IPM <50%) quality ejaculates. For supplementation studies, a total of 24 ejaculates (IPM ≥70%) were collected and each ejaculate was then divided into four aliquots. First aliquot was diluted with egg yolk-tris-glycerol (EYTG) extender without Humanin and served as control group (Group I). Rest three aliquots were diluted with extender containing 2 (Group II), 5 (Group III) and 10 µM Humanin (Group IV), respectively. Semen was cryopreserved using standard protocol and evaluated at pre-freeze for lipid peroxidation (LPO) and post-thaw stages for spermatozoa kinematics, LPO, mitochondrial membrane potential (MMP), capacitation, apoptotic status and DNA integrity. The treatment group that showed best results (5 µM) was compared with control group for in vitro fertility assessment by homologous zona binding assay. The LPO levels were lower (p < 0.05) in 5 and 10 µM Humanin supplemented group. The MMP and DNA integrity were higher (p < 0.05) in 5 µM group than other groups. F-pattern was higher (p < 0.05) and B-pattern was lower (p < 0.05) in 5 and 10 µM Humanin supplemented groups. Lower apoptotic and higher viable spermatozoa (p < 0.05) were observed in 5 µM Humanin group. The mean number of spermatozoa bound to zona pellucida was higher (p < 0.05) in 5 µM Humanin treated group than the control group. The study established the presence of Humanin in buffalo spermatozoa and seminal plasma for very first time and concluded that Humanin supplementation at 5 µM concentration improves the freezability and in vitro fertility of buffalo spermatozoa.

Microcapillary LAMP for rapid and sensitive detection of pathogen in bovine semen.

A microcapillary-based loop-mediated isothermal amplification (µcLAMP) has been described for specific detection of infectious reproductive pathogens in semen samples of cattle without sophisticated instrumentation. Brucella abortus, Leptospira interrogans serovar Pomona and bovine herpesvirus 1 (BoHV-1) cultures were mixed in bovine semen samples. The µcLAMP assay is portable, user-friendly, cost-effective, and suitable to be performed as a POC diagnostic test. We have demonstrated high sensitivity and specificity of µcLAMP for detection of Brucella, Leptospira, and BoHV-1 in bovine semen samples comparable to PCR and qPCR assays. Thus, µcLAMP would be a promising field-based test for monitoring various infectious pathogens in biological samples.HighlightsDetect infectious organism in bovines semenReduction in carryover contamination is an important attribute, which may reduce the false-positive reaction.µcLAMP is a miniaturized form, which could be performed with a minimum volume of reagents.The µcLAMP assay is portable, user-friendly, and suitable to be performed as a POC diagnostic test.

Optimization of dissolved oxygen levels in extender prevents development of cryocapacitation like changes, oxidative stress and augments zona binding capacity of crossbred bull spermatozoa.

The present study was undertaken to determine the efficacy of partial deoxygenation of extender at constant temperature (35°C) in freezability of crossbred bull semen. The dissolved oxygen (DO) levels were reduced by the use of newly developed technique of nitrogen effervescence at a flow rate of 2-3 bubbles per second. Four different levels of oxygen in semen extender, that is 11.7, 2, 4 and 8 ppm as control (Group-I), Group-II, Group-III and Group-IV, respectively, were used to assess the effect of partial deoxygenation on semen quality parameters. The 4 ppm level of DO resulted in higher (p < 0.05) progressive motility in comparison with non-treated group at post-thaw stage, whereas reduction up to 2 ppm resulted in drastic fall in motility. Oxidative stress status revealed low superoxide dismutase (SOD) and total antioxidant capacity (TAC) in Group-II, whereas higher (p < 0.05) SOD and TAC activities were observed in Group-III in comparison with non-treated group at pre-freeze and post-thaw stages. The sperm-zona binding at 4 ppm level of DO was significantly higher than control group, 2 and 8 ppm levels of DO. In conclusion, reduction of DO in the extender up to 4 ppm reduced oxidative stress and improved in vitro fertility of crossbred bull spermatozoa.

Current scenario and challenges ahead in application of spermatogonial stem cell technology in livestock.

PURPOSE: Spermatogonial stem cells (SSCs) are the source for the mature male gamete. SSC technology in humans is mainly focusing on preserving fertility in cancer patients. Whereas in livestock, it is used for mining the factors associated with male fertility. The review discusses the present status of SSC biology, methodologies developed for in vitro culture, and challenges ahead in establishing SSC technology for the propagation of superior germplasm with special reference to livestock. METHOD: Published literatures from PubMed and Google Scholar on topics of SSCs isolation, purification, characterization, short and long-term culture of SSCs, stemness maintenance, epigenetic modifications of SSCs, growth factors, and SSC cryopreservation and transplantation were used for the study. RESULT: The fine-tuning of SSC isolation and culture conditions with special reference to feeder cells, growth factors, and additives need to be refined for livestock. An insight into the molecular mechanisms involved in maintaining stemness and proliferation of SSCs could facilitate the dissemination of superior germplasm through transplantation and transgenesis. The epigenetic influence on the composition and expression of the biomolecules during in vitro differentiation of cultured cells is essential for sustaining fertility. The development of surrogate males through gene-editing will be historic achievement for the foothold of the SSCs technology. CONCLUSION: Detailed studies on the species-specific factors regulating the stemness and differentiation of the SSCs are required for the development of a long-term culture system and in vitro spermatogenesis in livestock. Epigenetic changes in the SSCs during in vitro culture have to be elucidated for the successful application of SSCs for improving the productivity of the animals.

Improved quality and fertilizability of cryopreserved buffalo spermatozoa with the supplementation of methionine sulfoxide reductase A.

BACKGROUND: The excessive reactive oxygen species produced during semen-freezing and -thawing damage the macromolecules resulting in impairment of cellular functions. Proteins are the primary targets of oxidative damage, wherein methionine residues are more prone to oxidation and get converted into methionine sulfoxide, thus affecting the protein function. The methionine sulfoxide reductase A (MsrA) catalyzes the conversion of methionine sulfoxide to methionine and restores the functionality of defective proteins. OBJECTIVES: To establish the expression of MsrA in male reproductive organs, including semen and its effect on quality of cryopreserved semen upon exogenous supplementation, taking buffalo semen as a model. MATERIALS AND METHODS: The expression of MsrA was established by immunohistochemistry, PCR, and Western blots. Further, the effect of recombinant MsrA (rMsrA) supplementation on the quality of cryopreserved spermatozoa was assessed in three treatment groups containing 1.0, 1.5, and 2.0 µg of rMsrA/50 million spermatozoa in egg yolk glycerol extender along with a control group; wherein the post-thaw progressive motility, viability, membrane integrity, and zona binding ability of cryopreserved spermatozoa were studied. RESULTS: The MsrA was expressed in buffalo testis, epididymis, accessory sex glands, and spermatozoa except in seminal plasma. In group 2, the supplementation has resulted in a significant (p < 0.05) improvement as compared to the control group in mean progressive motility (47.50 ± 2.50 vs. 36.25 ± 2.63), viability (56.47 ± 1.85 vs. 48.05 ± 2.42), HOST (50.76 ± 1.73 vs. 44.29 ± 1.29), and zona binding ability of spermatozoa (149.50 ± 8.39 vs. 29.50 ± 2.85). DISCUSSION AND CONCLUSION: In the absence of native MsrA of seminal plasma, the supplementations of rMsrA may repair the oxidatively damaged seminal plasma proteins and exposed sperm plasma membrane proteins resulting in better quality with a fivefold increase in fertilizability of frozen-thawed spermatozoa. The findings can be extended to other species to improve the semen quality with the variation in the amounts of rMsrA supplementation.

Exogenous cholesterol prevents cryocapacitation-like changes, membrane fluidity, and enhances in vitro fertility in bubaline spermatozoa.

A study was conducted to determine the optimum dosage of the exogenous cholesterol-loaded cyclodextrins (CLC) to get maximum cryoprotection for bubaline spermatozoa. In the present study, 120 × 106 spermatozoa were incubated in 2, 3 and 4 mg of CLC as grouped as Gr II, III and IV, respectively, and sperm progressive motility, intracellular Ca2+ , capacitation status by protein tyrosine phosphorylation (PTP) assay and zona binding per cent (ZBP) and cleavage rate (CR) of the cryopreserved buffalo spermatozoa by in vitro fertility assay were assessed in comparison with an untreated control group (Gr I). Results revealed that there was a significant (p < .05) linear decrease in percentage of sperm population with higher intracellular Ca2+ and percentage of sperm population with medium or high capacitated by PTP in CLC treated from 2 to 3 mg and then increased to 4 mg/120 × 106 spermatozoa whereas sperm progressive motility, percentage of sperm population with low capacitated, ZBP and CR were increased significantly (p < .05) in sperm population treated from 2 to 3 mg CLC and then decreased to 4 mg/120 × 106 spermatozoa. The study has clearly indicated that CLC at 3 mg/120 × 106 spermatozoa has maximum beneficial effects in protection of sperm progressive motility, membrane fluidity (low intracellular Ca2+ ); prevention of cryocapacitation (low capacitation pattern in immunolocalization) and enhancement of in vitro ZBP and CR. Post-thaw motility of the CLC-treated sperm has shown positively significant (p < .05) correlation with sperm population with low intracellular Ca2+ , low capacitated sperm population, ZBP and CR, whereas it was negatively (p < .05) correlated with sperm population with high intracellular Ca2+ , medium or high capacitated sperm. The present study has revealed for the first time that incubation of spermatozoa with CLC of higher dose (>3 mg/120 × 106 spermatozoa) had adverse effects on sperm cryopreservation, although incubation of sperm with 3 mg/120 million prior to processing had minimised the freezing-thawing-associated damages in bubaline species.

Identification of sperm motility markers in bovine transition protein genes.

Transition proteins (TNPs) are essential in chromatin condensation during spermiogenesis, and hence, they are the candidate genes for identifying sperm motility markers. Coding and in silico predicted promoter regions of these genes were investigated in crossbred and purebred cattle, and also, their mRNA quantification was done to explore its use as a diagnostic tool of infertility. PCR-SSCP analysis revealed two band patterns in fragment III of TNP1 and fragment II of TNP2 gene. Sequence analysis revealed a deletion of "G" nucleotide in 3'UTR region of TNP1 and C>T SNP in intronic region of TNP2 gene. Least square analysis of variance did not reveal any significant influence of nucleotide deletion on any sperm motility parameters in both crossbred and purebred cattle. However, C>T SNP had a significant effect on initial progressive motility (p < 0.05) in purebred cattle and post-thaw motility in overall cattle population. RT-qPCR analysis did not reveal any significant variation in TNP1 and TNP2 gene expression among poorly motile and good quality spermatozoa of Vrindavani bulls.

Isolation and enrichment of putative spermatogonial stem cells from ram (Ovis aries) testis.

The present study aimed to isolate and enrich putative SSCs from ram testes, which are positive for promyelocytic leukaemia zinc-finger protein (PLZF). The putative SSCs were isolated using a combination of enzymes with different concentrations, collagenase (1 and 2 mg/ml), hyaluronidase (1 mg/ml) and trypsin (0.25 and 0.5 mg/ml). The isolated SSCs were purified using an extracellular matrix such as laminin (20 µg/ml), DSA-lectin (5 µg/ml) and gelatin (0.2%) in combination with BSA (0.5 mg/ml). The number of putative SSCs/ tubule was significantly (p < 0.05) higher in prepubertal (3.1 ±â€¯0.51) and adult (3.45 ±â€¯0.58) than the number of gonocytes/tubule in neonatal (0.59 ±â€¯0.03) testis. Optimum enzyme combinations required for isolation of putative SSCs from prepubertal testis (collagenase; 2 mg/ml and trypsin; 0.5 mg/ml) were different from adult testis (collagenase; 1 mg/ml, trypsin; 0.25 mg/ml and hyaluronidase; 1 mg/ml). Though the number of putative SSCs/tubule was comparable in prepubertal and adult animals, a significantly (p < 0.05) higher percentage of putative SSCs (7.33 Vs 0.47%) were isolated from prepubertal testis than the adult. Differential plating using laminin along with BSA resulted in a significantly (p < 0.05) higher number of putative SSCs. The enzyme combinations suitable for isolation of putative SSCs from prepubertal testis are different from adult ram testis and the laminin has been found to be effective for purification of putative SSCs from testicular cells isolates.

Nitric oxide production by necrotrophic pathogen Macrophomina phaseolina and the host plant in charcoal rot disease of jute: complexity of the interplay between necrotroph-host plant interactions.

M. phaseolina, a global devastating necrotrophic fungal pathogen causes charcoal rot disease in more than 500 host plants. With the aim of understanding the plant-necrotrophic pathogen interaction associated with charcoal rot disease of jute, biochemical approach was attempted to study cellular nitric oxide production under diseased condition. This is the first report on M. phaseolina infection in Corchorus capsularis (jute) plants which resulted in elevated nitric oxide, reactive nitrogen species and S nitrosothiols production in infected tissues. Time dependent nitric oxide production was also assessed with 4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate using single leaf experiment both in presence of M. phaseolina and xylanases obtained from fungal secretome. Cellular redox status and redox active enzymes were also assessed during plant fungal interaction. Interestingly, M. phaseolina was found to produce nitric oxide which was detected in vitro inside the mycelium and in the surrounding medium. Addition of mammalian nitric oxide synthase inhibitor could block the nitric oxide production in M. phaseolina. Bioinformatics analysis revealed nitric oxide synthase like sequence with conserved amino acid sequences in M. phaseolina genome sequence. In conclusion, the production of nitric oxide and reactive nitrogen species may have important physiological significance in necrotrophic host pathogen interaction.

Detection of Corchorus golden mosaic virus Associated with Yellow Mosaic Disease of Jute (Corchorus capsularis).

Yellow mosaic disease, caused by a whitefly transmitted New World Begomovirus, named Corchorus golden mosaic virus (CoGMV), is emerging as a serious biotic constraint for jute fibre production in Asia. For rapid and sensitive diagnosis of the Begomovirus associated with this disease, a non-radiolabelled diagnostic probe, developed against the DNA A component of the east Indian isolate of CoGMV, detected the presence of the virus in infected plants and viruliferous whiteflies following Southern hybridization and nucleic acid spot hybridization tests. Presence of the virus was also confirmed when polymerase chain reaction amplification was performed using virus-specific primers on DNA templates isolated from infected plants and viruliferous whiteflies.

Biochemical characterization of compatible plant-viral interaction: a case study with a begomovirus-kenaf host-pathosystem.

Yellow vein mosaic disease of mesta, a compatible plant virus interaction poses a serious threat to mesta cultivation in India. Plants respond to invasion by pathogens with multi component defense responses particularly in incompatible interaction. With the aim of understanding, a biochemical approach was attempted to study the cellular redox status in early stages of yellow vein mosaic virus infection associated with different age's plant of Hibiscus cannabinus. Comparative analysis of GSH and GSSG content in infected and control plant of different ages indicated that infected plants are under oxidative or nitrosative stress condition. A significant change was observed in Glutathione Reductase, Catalase and Ascorbate Peroxidase level in early stage of infection. We also showed microscopic evidence of nitrosylated thiols in infected leaves, stems and roots of H. cannabinus. Furthermore, we identified few defense related proteins in infected plant using MALDI TOF mass spectrometric analysis.

Production of nitric oxide in host-virus interaction: a case study with a compatible Begomovirus-Kenaf host-pathosystem.

Nitric oxide (NO) plays a key role in plant diseases resistance. Here we have first time demonstrated that begomovirus infection in susceptible H. cannabinus plants, results in elevated NO and reactive nitrogen species production during early infection stage not only in infected leaf but also in root and shoot. Production of NO was further confirmed by oxyhemoglobin assay. Furthermore, we used Phenyl alanine ammonia lyase as marker of pathogenesis related enzyme. In addition evidence for protein tyrosine nitration during the early stage of viral infection clearly showed the involvement of nitrosative stress.

An improved method of DNA isolation suitable for PCR-based detection of begomoviruses from jute and other mucilaginous plants.

A relatively quick and inexpensive modified cetyl trimethylammonium bromide method for extraction of DNA from leaf materials containing large quantities of mucilage is described. The modification including use of more volume of extraction buffer and dissolving crude nucleic acid pellet in 1 M NaCl, reduced markedly the viscosity of the mucilage and thus in the final purification step yielded a larger quantity of mucilage-free DNA suitable for subsequent PCR-based detection of begomoviruses. The method was standardized with jute samples with yellow mosaic disease and validated with different other mucilaginous-hosts with low titre of begomoviruses. DNA isolated using this method showed consistency in yield and compatibility with PCR for detection of begomoviruses from different mucilaginous plant species. The method was compared for efficacy with other reported methods and it was found to be superior over the existing methods described for isolation of DNA from mucilaginous hosts. Thus the method described could be used on a wider scale for reliable and consistent detection of begomoviruses from mucilaginous hosts for characterization and variability study.

Distribution, epidemiology and molecular variability of the begomovirus complexes associated with yellow vein mosaic disease of mesta in India.

Yellow vein mosaic disease of mesta (Hibiscus spp.) poses a serious threat to the cultivation of this crop in India. The disease was found to be associated with two different whitefly-transmitted monopartite begomoviruses, Mesta yellow vein mosaic virus and Mesta yellow vein mosaic Bahraich virus, together with two betasatellite species, Cotton leaf curl Multan betasatellite and Ludwigia leaf distortion betasatellite. These begomovirus complexes were detected in different combinations throughout the mesta growing regions of India. All the eight cultivars tested were highly susceptible to the disease. The effect of the disease in terms of loss in fibre yield was greatest (around 70%) in plants that were inoculated at an early stage of growth. A regression approach was adopted to consider the relationship of whitefly vector populations with weather conditions and disease spread which explained that different conducive weather factors facilitated the build up of whitefly populations and contributed to the spread of the disease.