National Research Center for Grapes

Pune, India

National Research Center for Grapes

Pune, India
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Sawant S.D.,National Research Center for Grapes | Ghule M.R.,Shivaji University | Sawant I.S.,National Research Center for Grapes
Plant Disease | Year: 2017

Downy mildew, caused by Plasmopara viticola (Berk. & Curt.) Berl. & de Toni, is a destructive disease of grape (Vitis spp.), and it is controlled by fungicides including those from quinone outside inhibitors (QoI) and carboxylic acid amide (CAA) groups. Resistance to QoI fungicides in P. viticola was recently reported in India (Sawant et al. 2016). Downy mildew control failures were also observed from vineyards in Sangli, Maharashtra, India, where CAA fungicides were used for disease management. This study was conducted to detect the possible development of CAA resistance in P. viticola isolates in commercial vineyards in Sangli using biological and molecular methods. Four field isolates of P. viticola were collected in October 2014 from three vineyards of Sangli district that were reported with failure of downy mildew control. These vineyards had a history of six to 13 applications of CAA fungicides in previous growing (fruiting) seasons but no CAA applications during the subsequent growing (vegetative) season. Infected leaf samples were brought to the lab in individual polypropylene bags (pp) and kept overnight in pp bags inserted with a wet paper at 22°C for sporulation. A sensitive isolate of P. viticola, collected from ICAR-National Research Centre for Grapes, was included for the study. Sensitivity to mandipropamid (Revus 23.4% SC, Syngenta, India) and dimethomorph (97%, BASF, India) was determined by leaf disc bio-assay in 24 well plates (FRAC 2013). Working solutions of 100, 50, 10, 1, 0.1, and 0 µg/ml concentrations were prepared by appropriate dilutions of the 1,000 µg/ml stock solutions using sterile distilled water. The experiment was conducted in completely randomized design (CRD) with four replicates. Fungicides were applied by camel hair brush to the abaxial surface of 15 mm leaf discs 24 h before inoculation. Discs were centrally inoculated with a 10 µl suspension of P. viticola containing 50,000 sporangia/ml and incubated at 22°C with alternating periods of 12 h light and dark. After 6 to 8 days, the lesion diameter was measured and converted to area. Then the percent area of infection was calculated, considering the area in control as 100%, and plotted against the log10 fungicide concentration to obtain the EC50 value. The glycine to serine amino acid point mutation at position 1105 (G1105S) in the cellulose synthase gene (PvCesA3) (Blum et al. 2010) was detected by the PCR-RFLP method (Aoki et al. 2011). The P. viticola isolate from this center (ICAR-NRC for grapes) recorded less than 0.1 µg/ml EC50 values for both dimethomorph and mandipropamid, showing its sensitivity to CAA fungicides. The EC50 values of the four isolates from the commercial vineyards were 54.28 to >100 for dimethomorph and 42.54 to >100 for mandipropamid, showing their insensitivity to the CAA fungicides. Furthermore, the 144-bp PCR product of PvCesA3 gene was not cleaved by AluI restriction enzyme in the sensitive isolate from this center, while it was cleaved into a 107-bp and a 37-bp product in the insensitive isolates, confirming development of resistance. The frequent reports of failure of disease control and detection of resistance to CAA fungicides in P. viticola from three vineyards in Sangli, Maharashtra, India, requires concerted efforts for monitoring and mitigating the problem for successful management of downy mildew in commercial vineyards. © The American Phytopathological Society.

Banerjee K.,National Research Center for Grapes | Utture S.,National Research Center for Grapes | Dasgupta S.,National Research Center for Grapes | Kandaswamy C.,Agilent Technologies | And 3 more authors.
Journal of Chromatography A | Year: 2012

A residue analysis method for the simultaneous estimation of 349 pesticides, 11 PCBs and 15 PAHs extracted from grape, pomegranate, okra, tomato and onion matrices, was established by using a gas chromatograph coupled to an electron impact ionization triple quadrupole mass spectrometer (GC-EI-MS/MS). The samples were extracted by ethyl acetate and cleaned by dispersive solid phase extraction with PSA and/or GCB/C18 by the methods reported earlier. The GC-EI-MS/MS parameters were optimized for analysis of all the 375 compounds within a 40min run time with limit of quantification for most of the compounds at <10μg/L, which is well below their respective European Union-Maximum Residue Levels. The coefficient of determination (r2) was >0.99 within the calibration linearity range of <5-250ng/mL for compounds with LOQs<5ng/mL. While for the compounds with LOQs within 5-10μg/kg, the lowest calibration level was 5 and 10μg/kg as applicable. The recoveries at 10, 25 and 50ng/mL were within 70-110% (n=6) with associated RSDs<20% indicating satisfactory precision. The information generated from the single laboratory validation was further utilized for building a semi-quantitative approach. The accuracies in quantification obtained via individual calibration standards vis-à-vis semi-quantification approach were comparable. For incurred samples, the concentrations estimated by the semi-quantification approach were within ±10% of the values obtained by direct quantification. This approach complements the existing GC-EI-MS/MS methods by offering targeted screening and quantification capabilities. © 2012 Elsevier B.V.

Oulkar D.P.,National Research Center for Grapes | Banerjee K.,National Research Center for Grapes | Kulkarni S.,Agilent Technologies
Journal of AOAC International | Year: 2011

A selective and sensitive LC-MS/MS method is presented for simultaneous determination of 12 plant growth regulators, viz., indol- 3-acetic acid, indol-3-butyric acid, kinetin, zeatin, 6-benzyl aminopurine, gibberellic acid, abscisic acid, chlormequat chloride, forchlorfenuron, paclobutrazole, daminozide, and 2,4-dichlorophenoxy acetic acid, in bud sprouts and grape berries. The sample preparation method involved extraction of homogenized sample (5 g) with 40 mL methanol (80%), and final determination was by LC-MS/MS in the multiple reaction monitoring (MRM) mode with time segmentation for quantification supported by complementary analysis by quadrupole-time of flight (Q-TOF) MS with targeted high-resolution MS/MS scanning for confirmatory identification based on accurate mass measurements. The recovery of the test compounds ranged within 90-107% with precision RSD less than 5% (n = 6). The method could be successfully applied in analyzing incurred residue samples, and the strength of accurate mass analysis could be utilized in identifying the compounds in cases where the qualifier MRM ions were absent or at an S/N less than 3:1 due to low concentrations. © 2012 Publishing Technology.

Fontana A.R.,CONICET | Patil S.H.,National Research Center for Grapes | Banerjee K.,National Research Center for Grapes | Altamirano J.C.,CONICET | Altamirano J.C.,National University of Cuyo
Journal of Agricultural and Food Chemistry | Year: 2010

A fast and effective microextraction technique is proposed for preconcentration of 2,4,6-trichloroanisole (2,4,6-TCA) from wine samples prior gas chromatography tandem mass spectrometric (GC-MS/MS) analysis. The proposed technique is based on ultrasonication (US) for favoring the emulsification phenomenon during the extraction stage. Several variables influencing the relative response of the target analyte were studied and optimized. Under optimal experimental conditions, 2,4,6-TCA was quantitatively extracted achieving enhancement factors (EF) ≥ 400 and limits of detection (LODs) 0.6-0.7 ng L -1 with relative standard deviations (RSDs) -11.3%, when 10 ng L -1 2,4,6-TCA standard-wine sample blend was analyzed. The calibration graphs for white and red wine were linear within the range of 5-1000 ng L -1, and estimation coefficients (r 2) were ≥0.9995. Validation of the methodology was carried out by standard addition method at two concentrations (10 and 50 ng L -1) achieving recoveries >80% indicating satisfactory robustness of the method. The methodology was successfully applied for determination of 2,4,6-TCA in different wine samples. © 2010 American Chemical Society.

Mujawar S.,National Research Center for Grapes | Utture S.C.,National Research Center for Grapes | Fonseca E.,University of Costa Rica | Matarrita J.,University of Costa Rica | Banerjee K.,National Research Center for Grapes
Food Chemistry | Year: 2014

A sensitive and rugged residue analysis method was validated for the estimation of dithiocarbamate fungicides in a variety of fruit and vegetable matrices. The sample preparation method involved reaction of dithiocarbamates with Tin(II) chloride in aqueous HCl. The CS2 produced was absorbed into an isooctane layer and estimated by GC-MS selected ion monitoring. Limit of quantification (LOQ) was ≤40 μg kg-1 for grape, green chilli, tomato, potato, brinjal, pineapple and chayote and the recoveries were within 75-104% (RSD < 15% at LOQ). The method could be satisfactorily applied for analysis of real world samples. Dissipation of mancozeb, the most-used dithiocarbamate fungicide, in field followed first + first order kinetics with pre-harvest intervals of 2 and 4 days in brinjal, 7 and 10 days in grapes and 0 day in chilli at single and double dose of agricultural applications. Cooking practices were effective for removal of mancozeb residues from vegetables. © 2013 Elsevier Ltd. All rights reserved.

Khar A.,Directorate of Onion and Garlic Research ICAR | Banerjee K.,National Research Center for Grapes | Jadhav M.R.,National Research Center for Grapes | Lawande K.E.,Directorate of Onion and Garlic Research ICAR
Food Chemistry | Year: 2011

An aqueous extraction method coupled with ultra performance liquid chromatography with photo diode array detection was optimised and validated for screening of 93 garlic ecotypes for allicin and other allyl thiosulphinate contents. Variability in allicin, allyl methyl thiosulphinate and allyl trans-1-propenyl thiosulphinate was observed both on fresh and dry weight basis. Bulb to bulb variation for allicin levels was less in clonal selections followed by commercial varieties, exotic line and landraces, respectively. Five ecotypes on fresh weight basis and all the ecotypes on dry weight basis had allicin contents more than the recommended pharmacological values (≥4.5 mg g-1). Cluster analysis and principal component analysis results revealed that geographical origin, breeding status and colour did not play any role in ecotype clustering. This study is useful as a base for selection of garlic ecotypes as a dependable raw material source for pharmaceutical and food processing industries. © 2011 Elsevier Ltd. All rights reserved.

Dasgupta S.,University of Pune | Banerjee K.,National Research Center for Grapes | Dhumal K.N.,University of Pune | Adsule P.G.,National Research Center for Grapes
Journal of AOAC International | Year: 2011

This paper describes single-laboratory validation of a multiresidue method for the determination of 135 pesticides, 12 dioxin-like polychlorinated biphenyls, 12 polyaromatic hydrocarbons, and bisphenol A in grapes and wine by GC/time-of-flight MS in a total run time of 48 min. The method is based on extraction with ethyl acetate in a sample-to-solvent ratio of 1:1, followed by selective dispersive SPE cleanup for grapes and wine. The GC/MS conditions were optimized for the chromatographic separation and to achieve highest S/N for all 160 target analytes, including the temperature-sensitive compounds, like captan and captafol, that are prone to degradation during analysis. An average recovery of 80-120% with RSD <10% could be attained for all analytes except 17, for which the average recoveries were 70-80%. LOQ ranged within 10-50 ng/g, with <25% expanded uncertainties, for 155 compounds in grapes and 151 in wine. In the incurred grape and wine samples, the residues of buprofezin, chlorpyriphos, metalaxyl, and myclobutanil were detected, with an RSD of <5% (n = 6); the results were statistically similar to previously reported validated methods.

Banerjee K.,National Research Center for Grapes | Mujawar S.,National Research Center for Grapes | Utture S.C.,National Research Center for Grapes | Dasgupta S.,National Research Center for Grapes | Adsule P.G.,National Research Center for Grapes
Food Chemistry | Year: 2013

A single quadrupole GC-MS method was optimized for multiresidue determination of 47 pesticides in grapes with limit of quantifications of each compound in compliance with the EU-MRL requirements. Sample preparation involved extraction of 10 g sample with 10 ml ethyl acetate (+10 g sodium sulphate) by homogenization at 15,000 rpm followed by centrifugation at 3000 rpm. The supernatant was cleaned by dispersive solid phase extraction with primary secondary amine and acidified with 0.1% formic acid. Residues were estimated in selected ion monitoring mode with programmable temperature vaporizer-large volume injection (8 μl). All the GC and MS parameters were thoroughly optimized to achieve satisfactory linearity (R2 > 0.99) within 0.01-0.25 mg kg-1 with minimum matrix interferences. Recoveries at 0.01 and 0.02 mg kg-1 were within 67-120% with associated precision RSD below 19%. The method was successfully applied for analysis of the real world samples for incurred residues. © 2012 Elsevier Ltd. All rights reserved.

Upadhyay A.,National Research Center for Grapes | Upadhyay A.K.,National Research Center for Grapes | Bhirangi R.A.,National Research Center for Grapes
Biologia Plantarum | Year: 2012

Grapevine rootstocks belonging to different species of Vitis differ in their response to stress. Vines of rootstocks 110R and 1613C were subjected to different salt and water stresses individually and in combination. Expression of VvNHX1, a Na +/H + antiporter gene, was analyzed at 7 and 21 d of stress treatment. In 110R, the expression of VvNHX1 gene increased in response to both salinity and water stress already after 7 d. Under salinity and combined stress enhanced expression of this gene was observed also after 21 d whereas expression decreased under water stress. In 1613C, expression of this gene did not increase under salinity stress. There was delayed response to water and combined stress and expression increased several fold after 21 d of stress. The stressed vines of 110R maintained lower sodium content and higher K +/Na + ratio as compared to rootstock 1613C. © 2012 Springer Science+Business Media B.V.

Jadhav M.R.,National Research Center for Grapes | Oulkar D.P.,National Research Center for Grapes | Banerjee K.,National Research Center for Grapes
Journal of Agricultural and Food Chemistry | Year: 2015

A buffered ethyl acetate extraction method is proposed for the simultaneous analysis of 296 agrochemicals in a wide range of fruit and vegetable matrices by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The optimized quantity of acetate buffer (1% acetic acid + 0.5 g of sodium acetate per 10 g of sample) adjusted the pH of each test matrix to 5-6, which in turn significantly improved recoveries of acidic and basic compounds. The role of diethylene glycol (used in the evaporation step) on signal suppression of certain compounds was evaluated, and its quantity was optimized to minimize such an effect. The method was validated in grape, mango, drumstick, bitter gourd, capsicum, curry leaf, and okra as per the DG-SANCO/12571/2013 guidelines. Recoveries in the fortification range of 1-40 μg/kg were within 70-120% with associated relative standard deviations below 20% for most of the compounds. The method has potential for regulatory and commercial applications with a generic approach. © 2015 American Chemical Society.

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