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Dr. Y. S. Parmar University of Horticulture & Forestry, Solan
Dr. Yashwant Singh Parmar University of Horticulture and Forestry, Solan, was established on 1st December, 1985 with the objective to promote education, research and extension education in the fields of Horticulture, Forestry and allied disciplines. Late Dr. Yashwant Singh Parmar, the first Chief Minister and the architect of Himachal Pradesh perceived the importance of Horticulture and Forestry to develop and improve the State economy which led to the establishment of this University. Its history lies in erstwhile Himachal Agricultural College, Solan, established in 1962 and affiliated to the Panjab University. It became one of the campuses of Agriculture Complex of Himachal Pradesh University on its formation in 1970. Consequent upon the establishment of Himachal Pradesh Krishi Vishvavidyalaya in 1978, this campus became its Horticulture Complex and finally in 1985, assumed the status of a State University, being the only University in the country engaged exclusively in teaching, research and extension in Horticulture and Forestry.
The University is located at Nauni in Solan District of Himachal Pradesh, 13 km from Solan on Solan-Rajgarh Road, at an elevation of 1300 metres above mean sea level. Solan town is situated on national highway (NH-22) and is well connected by train and bus services.
The University has four constituent colleges, out of which, two are located at the main campus Nauni, one for horticulture and the other for forestry, having 9 and 7 departments, respectively. The third College i.e., College of Horticulture & Forestry is located at Neri in Hamirpur District on Nadaun-Hamirpur state highway, about 6 Km from Hamirpur town and is well connected with bus service. The college offers three Undergraduate Degree Programmes i.e. BSc (Hons.) Horticulture, BSc (Hons.) Forestry and B. Tech. Biotechnology and MSc degree programme in a few subjects. The fourth college i.e. College of Horticulture and Forestry, Thunag (Mandi) is located at Thunag District Mandi. This college offer BSc (Hons.) Horticulture and BSc (Hons.) Forestry degree programme. In addition, there are five Regional Research Stations, 12 Satellite Stations and five Krishi Vigyan Kendras (KVKs) situated in different zones of the State.
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ThesisItem Open Access Studies on production and characterization of tannase (Tannin acyl hydrolase)(YSPU, 2015) Nisha; Nath, A.K.Penicillium crustosum AN3 isolated from apple orchard soil is the first report from Penicillium genera to produce maximum tannase which is an important industrial enzyme used in biodegradation and food industry. A total of eleven fungal isolates were obtained from apple orchard soil, pine forest soil and botanical garden soil, respectively. Out of these eleven isolates five isolates viz., three isolates from apple orchard soil, one isolate from botanical garden soil and one isolate from pine forest soil were showed zone of degradation on Czapdox minimal medium supplemented with 0.5% tannic acid confirming tannase activity. These isolates were rescreened qualitatively for maximum tannase production in submerged fermentation isolate A3 exhibited maximum tannase activity of 2.48Uml-1. Molecular characterization of these isolates was carried out using 18S rrna gene technology and in silico analysis of 18S rrna gene sequences lead to identification of these fungal isolates as Fusarium redolens AN1, Aspergillus fumigatus AN2, Penicillium crustosum AN3, P.restrictum AN4 and P. commune AN5. Tannase producing bacterial isolates were also obtained on nutrient agar (NA) medium supplemented with 0.5% tannic acid. A total of four bacterial isolates from tea garden soil, pine forest soil, ruminial fluid and sheep excreta were screened on the basis of their ability to utilize tannic acid and were characterized morphologically, biochemically and for tannase activity leads to identification as Klebsiella sp, Enterobacter sp, Staphylococcus sp and E.coli respectively. Molecular characterization two bacterial isolates was carried out using 16S rrna gene technology and in silico analysis of 16S rrna gene sequences lead to confirmation of these isolates as Klebsiella variicola AN1 and Enterobacter hormaechei AN2. Penicillium crustosum AN3 produced maximum tannase (2.45Uml-1) in submerged fermentation was selected for tannase production in SSF. The enzyme was partially purified from culture extract of enzyme. Cultural conditions and process parameters i.e. type of substrate, incubation time, temperature, initial pH, moisture level, substrate concentration, tannic acid concentration, carbon sources, peptone, yeast extract, urea, NH4NO3 were optimized using one factor at a time approach and activity obtained was 39.5Ug-1. Further optimization was carried out using central composite design following response surface methodology with three independent variables which resulted in increased in tannase production to 55.55 Ug-1. The enzyme was partially purified by acetone precipitation and gel filteration chromatography which showed 46.48 yield with 3.94 fold purification and had a molecular mass of 205kDa. Gallic acid the hydrolytic product in crude enzyme was detected by TLC, FTIR and HPLC using gallic acid as standard. Crude enzyme obtained was studied for its ability in pine needle degradation, tea colour decolourization, dye decolourization and removal of apple browning.ThesisItem Open Access In vitro selection of Punica granatum L. cv. Kandhari Kabuli against bacterial blight and pomegranate wilt(YSPU, 2015) Soni, Madhvi; Kanwar, KamleshThe present investigation aims at “In vitro selection of Punica granatum L cv. Kandhari Kabuli against bacterial blight and pomegranate wilt”. A valuable plant regeneration and in vitro selection protocol against bacterial blight and pomegranate wilt disease was developed for Punica granatum L. cv. Kandhari Kabuli. Indirect organogenesis form juvenile (cotyledon and hypocotyl) explants excised from 14 to 15 days old in vitro germinated seedlings and mature explant (leaf) was carried out. Callus was induced from cotyledon explant on MS medium supplemented with 4.0 mg/l NAA and 3.0 mg/l BA while in case of hypocotyl explant the best treatment for callus induction was found out to be solid MS medium supplemented with 4.0 mg/l NAA and 2.0 mg/l BA. Callus was induced from leaf explant on solid MS medium supplemented with 4.0 mg/l NAA and 2.0 mg/l Kinetin. Highest percentage of callus was obtained from cotyledon (83.33%) explants followed by hypocotyl (72.21%) and leaf (68.25%) explants. Shoots were induced from hypocotyl and cotyledon derived calli on MS medium supplemented with 1.0 mg/l BA, 0.50 mg/l Kinetin and 0.25 mg/l NAA with 76.39 and 62.50 % shoot bud induction. Leaf derived calli responded best on MS medium supplemented with 1.5 mg/l BA, 0.50 mg/l Kinetin and 0.25 mg/l NAA with 59.72 per cent of shoot bud induction. The regenerated shoots were rooted in half strength MS medium containing 500 mg/l activated charcoal. Cotyledon explants was found to be most responsive explants for indirect regeneration method. To carry out in vitro selection for resistance development the pathogen causing bacterial blight and pomegranate wilt disease were isolated. On the basis of morphological, biochemical features and by BLASTn analysis of sequenced 16S rRNA/ITS region of the pathogen, the pathogen causing bacterial blight was identified as Xanthomonas axonopodis pv. punicae and pathogen causing wilt disease was identified as Ceratocystis fimbriata. Cell line selection against bacterial blight was done by using culture filtrate of Xanthomonas axonopodis pv. punicae while selection against wilt was done by using culture filtrate of Ceratocystis fimbriata as a selective agent. The optimum concentration of bacterial culture filtrate at which calli selected was 25.0 per cent resulting in 12.96 per cent survival of calli. However, the optimum concentration of fungal culture filtrate for selection against pomegranate wilt disease was 40.0 per cent resulting in 18.52 per cent survival of calli. Shoots were regenerated from the selected calli after two cycles of selection. The calli selected against BCF showed shoot regeneration only after 3rd subculture passage and increased up to fourth subculture passage resulting in 44.45 per cent shoot regeneration and thereafter start decreasing. Similarly, in calli selected against FCF shoot regeneration was observed only after 2nd subculture passage and increased up to fourth subculture passage resulting in 52.78 per cent shoot regeneration and thereafter start decreasing. In vitro selected shoots were rooted in half strength MS medium supplemented with 500 mg/l activated charcoal followed by hardening. The plants selected against BCF showed resistance development against Xanthomonas axonopodis pv. punicae and plants regenerated from callus selected against FCF showed resistance against Ceratocystis fimbriata during in vitro and ex vitro testing. Dendrogram generated using RAPD and ISSR marker separated in two clusters where mother plant and control plant always fall in one cluster showing maximum similarity with each other while the selected variants clustered separately suggesting genetic variation with mother plant, control plant and within the variants.ThesisItem Open Access Evaluation of antimicrobial compounds from Juniperus communis L. and Vitex negundo L. against Xanthomonas sp. and Pseudomonas sp.(YSPU, 2015) Digvijay; Bhardwaj, Satya VratEssential oils from the shade dried leaves of Juniperus communis L. and Vitex negundo L. from H.P. (India) were examined for their antimicrobial activities against eight human and plant pathogenic bacteria. Essential oils were obtained by using Soxhlet extraction apparatus. Chemical analysis of the oils was done by using gas chromatography and mass spectrometry (GC/MS). The antimicrobial effects were tested by inhibition zone diameter (IZD)/ zone of inhibition test. Essential oil from methanol extract showed very good activity at 300 ppm concentration after 72 hours and was more effective as compared to others, however, all the plant extracts showed inhibitory effect on linear growth of selected microorganism. Higher concentrations of 1-Phenanthrene carboxylic acid (11.44 %) and Propylparaben, Benzoic acid, 4-hydroxy, Benzoic acid, 3-hydroxy and Benzoic acid, 3- hydroxy-2-methylpropyl ester (23.23%) content released from Juniperus communis L. and Vitex negundo L. H.P. (India) respectively, might be assigned to be responsible for such excellent antimicrobial activity against the tested pathogens. Docking studies were also performed for sixteen compounds in order to evaluate their affinity to bacterial proteins that are known targets for some antibiotics with different mechanisms of action. The purpose of the present studies was to encourage use of Juniperus communis L. and Vitex negundo L. essential oils against bacterial infections so as to provide an approach to develop herbal formulations as potential disease management strategies in place of chemicals. However, the results are yet to be authenticated through wet lab experimentations for each biomolecules before going ahead for potential drug development.ThesisItem Open Access Studies on Agrobacteriummediated insect resistance gene [cry1A(a)] transfer in cauliflower (Brassica oleracea L. var. botrytis)(YSPU, 2015) Gaur, Ayesh; Srivastava, D.K.Genetic transformation studies were carried out to standardize a protocol for insect resistance gene (cryIAa) transfer in cauliflower (Brassica oleracea L. var. botrytis cv. Pusa Snowball K1). Agrobacterium tumefaciens strain containing npt-II and cryIAa genes in binary vector pBin-1Aa was used for genetic transformation studies. Plant regeneration studies were carried out using four different types of explants viz. hypocotyl, cotyledon, petiole and leaf. High efficiency shoot regeneration was obtained in hypocotyl (96.00%), cotyledon (76.00%), petiole (76.00%) and leaf (56.00%) explants on MS medium supplemented with 0.44mg/l TDZ + 0.08mg/l IAA, 0.30mg/l BAP + 0.10mg/l NAA, 0.22mg/l TDZ and 0.77mg/l TDZ + 79.7mg/l Adenine, respectively. Hypocotyl explants showed better shoot regeneration as compared to other explants. MS medium supplemented with 0.10mg/l IBA was found best for root regeneration from in vitro developed shoots. The regenerated plantlets were acclimatized successfully on cocopeat and were morphologically similar and normal. Kanamycin sensitivity (10-60mg/l) was checked by observing fresh weight of the leaf and petiole explants which was measured at the interval of 7 days. From the relative growth of the explants it was found that the concentration as low as 10mg/l is toxic to the explants. Effect of different concentrations of cefotaxime was studied on the regeneration potential in leaf and petiole explants of cauliflower and found not much effect of cefotaxime on regeneration potential. Effect of different concentrations of cefotaxime and kanamycin (20mg/l) were studied on the growth of agrobacterial cells and regeneration potential of hypocotyl and petiole tissues after cocultivation. In both the explants the growth of agrobacterial cells were controlled at concentration of 300mg/l cefotaxime and maximum per cent shoot regeneration in hypocotyl (22.60%) and petiole (13.30 %) was obtained on MS medium supplemented with 300mg/l cefotaxime, respectively. Effect of preculturing and co-cultivation was studied on the transformation frequency. Preculturing of hypocotyl and petiole explants for 48 hours and co-cultivation with agrobacterial cells for 96 hours worked out to be the best treatment as it gave the highest transformation frequency (4.28 %) and (3.28 %) in respective explants. Effect of different concentrations of acetosyringone was studied in hypocotyl and petiole explants to enhance the transformation frequency. The maximum percent shoot regeneration was obtained from hypocotyl (9.23%) and petiole (10.13%) explants cultured on shoot regeneration medium containing 100μM acetosyringone and 125μM acetosyringone respectively at standardized preculturing and cocultivation time interval. The presence/integration of transgene (cryIAa) into the genome of cauliflower was confirmed by PCR using gene specific primers and Southern blot analysis using radioactive labelled DNA probe. The expression of the transgene (cryIAa) in cauliflower at transcriptional level was confirmed by reverse transcriptase-PCR and Real Time-PCR and at translational level by Bioassay. A protocol for high frequency plant regeneration and insect resistance gene transfer in cauliflower (Brassica oleracea L. var. botrytis cv. Pusa Snowball K1) has been standardized.ThesisItem Open Access STUDIES ON PROTEASE INHIBITOR IN GERMINATED HORSEGRAM (Dolichos biflorus L.) SEEDS(2015) SHARMA, ANKITA; NATH, AMARJEET K.ABSTRACT Protease inhibitors present in seeds of leguminous plants possess strong inhibitory activity against trypsin and confer resistance against pests and microbes. Trypsin inhibitor from cotyledons of 24 hours soaked seeds of Dolichos biflorus L. cultivar HPK5 showed highest activity in distilled water (pH 7.0) in 3 hours of extraction time. The inhibitor activity was found to decrease gradually with increase in germination period. Decrease in the intensity and number of bands on native PAGE at different stages of seed germination indicated the reduction of soluble protein content with increasing germination period. Partially purified inhibitor protein from Sephadex G-50 was characterized. Linear increase in per cent inhibition with increase in inhibitor concentration was observed till 72 %. The inhibitor protein was found to be thermostable and retained 98 % activity of control at 100 оC. It had pH optima of 7.5. The inhibitor lost its activity on incubation with varying concentrations of DTT thereby indicating the role of disulfide linkages in maintaining its stability and three dimensional structures. Trypsin inhibitor was further partially purified to 3.83 fold with 78.5 per cent recovery by ammonium sulfate precipitation and gel filtration chromatography on Sephadex G-100 column. It exhibited competitive inhibition pattern and showed stability on incubation with urea even at high temperature (97 оC). However, it lost its stability on incubation with 0.6 N HCl at 37 оC and 97 оC. Partially purified inhibitor protein (6.17mg) inhibited the growth of fungus Alternaria spp. to 70 %. It also inhibited the growth of bacterium Ralstonia solanacerum. This study signifies the antimicrobial potential of Dolichos biflorus L. cultivar HPK5 which might be exploited for crop protection programmes.ThesisItem Open Access BIOPROSPECTING OF THERMOPHILIC BACTERIA FROM HOT WATER SPRINGS OF HIMACHAL PRADESH FOR LACCASE ENZYME PRODUCTION(2015) SHARMA, RUCHIKA; SHIRKOT, POONAMABSTRACT Laccase enzyme has acquired the status of ‘green catalyst’ as it possesses remarkable bioremediation potential along with numerous applications in effluent detoxification, degradation of textile dyes, herbicide and insecticide degradation, wine clarification, enzymatic conversion of chemical intermediates, biosensors and organic synthesis. In the present study, significantly high diversity of laccase producing bacteria from hot water springs of Himachal Pradesh was assessed. A total of 118 laccase producing thermophilic bacterial isolates were obtained from 200 hot water spring samples using TYM containing 5 mM guaiacol which were morphologically characterized. These were rescreened on the basis of their ability to oxidise tannic acid, dimethoxyphenol and syringaldazine leading to selection of 50 laccase producing thermophilic bacterial isolates, which were characterized biochemically. Eighteen thermophilic bacterial isolates exhibiting maximum laccase activity of 0.0007-0.0038 U/l were selected for further, molecular characterization using RAPD-PCR and 16S rrna gene technology. In silico analysis of 16S rrna gene sequences led to identification of these bacterial isolates and they were found to belong to genus Bacillus, Aneurinibacillus and Pseudomonas, as Bacillus licheniformis strain RSV20, Bacillus licheniformis strain RSM8, Bacillus licheniformis strain RSV10, Bacillus sonerensis strain RSM17, Bacillus sonerensis strain RSV8, Bacillus licheniformis strain RSP1, Bacillus licheniformis strain RSP2, Bacillus licheniformis strain RSP3, Bacillus licheniformis strain RSP7, Bacillus sonerensis strain RSP5, Bacillus sonerensis strain RSP11, Aneurinibacillus thermophilus strain RSP13, Bacillus aerius strain RSP4, Bacillus aerius strain RSP9, Bacillus subtilis strain RSP8, Bacillus amyloliquifacience strain RSP10, Bacillus pumilis strain RSP12 and Pseudomonas taiwanensis strain RSP6. On the basis of maximum laccase enzyme activity Bacillus licheniformis strain RSM8 was selected for production and purification of the laccase enzyme. Maximum extracellular enzyme production was achieved at 60°C, pH 9.0 and 24 hrs incubation with 5 mM guaiacol, 5 % tryptone and 3 % yeast extract in combination with nitrogen source. Crude extracellular thermolaccase enzyme preparation was purified by ammonium salt precipitation (50-90%) followed by gel filtration and ion exchange chromatography which showed 15.21 yield and 10.5 fold purification. The purified enzyme had optimal activity at pH 9.0 and 60 °C, and 16.22 μM Km value. The molecular weight of thermolaccase in the present study was found to be 72.5 kDa. However activity was inhibited by sodium azide and DTT. Bacillus licheniformis strain RSM8 as well as its enzyme preparations were investigated for their ability to decolourize dyes which are the potential contributors of water pollution. Six different synthetic dyes were decolourized RBBR (68 %), congo red (86 %), indigo carmine (73 %), brilliant blue (40 %), bromophenol blue (51 %) and aniline blue (54 %) when treated with the crude enzyme preparation of Bacillus licheniformis strain RSM8. And partially purified enzyme preparation of Bacillus licheniformis strain RSM8 showed greater decolourization of dyes comparatively RBBR (74 %), congo red (91 %), indigo carmine (80 %), brilliant blue (60 %), bromophenol blue (64 %) and aniline blue (67 %). The purified enzyme was successfully immobolized using adsorption method in calcium alginate beads with 76% immobolization percentage and immobolized laccase enzyme beads were studied for their ability to degrade dyes. The stability and reusability of the immobilized enzyme system has the potential to make the entire treatment process inexpensive. Bacillus licheniformis strain RSM8 enzyme preparations was investigated for phytotoxicity evaluation of three dyes viz., Congo red, RBBR and Indigo carmine and each of enzyme treated dyes for Phaseolus mungo and Calendula officinalis and Tagetes patula plant species respectively, under in vitro conditions and Phaseolus mungo with Congo red dye under in vivo conditions. Significant germination inhibition, a slower rate of plumule and radicle seedlings growth was observed for Congo red, RBBR and Indigo carmine dyes as compared to enzyme treated dyes. An extracellular laccase producing gene has been isolated using degenerate primer based on the copper I and II conserved site of laccase enzyme, from the hot water spring bacteria, Bacillus licheniformis strain RSM8 followed by determination of the amino acid which were translated from nucleotide sequence and encodes a polypeptide comprised of 50 amino acids showeng 97 % identity with the amino acid sequences of bacterial laccases i.e. copper oxidase [Bacillus licheniformis]. Further multiple sequence alignment using MULTALIN and structure prediction using Phyre1 & 2 revealed conserved histidine residues.ThesisItem Open Access STUDIES ON PRODUCTION AND CHARACTERIZATION OF TANNASE (TANNIN ACYL HYDROLASE)(2015) NISHA; NATH, A.K.ABSTRACT Penicillium crustosum AN3 isolated from apple orchard soil is the first report from Penicillium genera to produce maximum tannase which is an important industrial enzyme used in biodegradation and food industry. A total of eleven fungal isolates were obtained from apple orchard soil, pine forest soil and botanical garden soil, respectively. Out of these eleven isolates five isolates viz., three isolates from apple orchard soil, one isolate from botanical garden soil and one isolate from pine forest soil were showed zone of degradation on Czapdox minimal medium supplemented with 0.5% tannic acid confirming tannase activity. These isolates were rescreened qualitatively for maximum tannase production in submerged fermentation isolate A3 exhibited maximum tannase activity of 2.48Uml-1. Molecular characterization of these isolates was carried out using 18S rrna gene technology and in silico analysis of 18S rrna gene sequences lead to identification of these fungal isolates as Fusarium redolens AN1, Aspergillus fumigatus AN2, Penicillium crustosum AN3, P.restrictum AN4 and P. commune AN5. Tannase producing bacterial isolates were also obtained on nutrient agar (NA) medium supplemented with 0.5% tannic acid. A total of four bacterial isolates from tea garden soil, pine forest soil, ruminial fluid and sheep excreta were screened on the basis of their ability to utilize tannic acid and were characterized morphologically, biochemically and for tannase activity leads to identification as Klebsiella sp, Enterobacter sp, Staphylococcus sp and E.coli respectively. Molecular characterization two bacterial isolates was carried out using 16S rrna gene technology and in silico analysis of 16S rrna gene sequences lead to confirmation of these isolates as Klebsiella variicola AN1 and Enterobacter hormaechei AN2. Penicillium crustosum AN3 produced maximum tannase (2.45Uml-1) in submerged fermentation was selected for tannase production in SSF. The enzyme was partially purified from culture extract of enzyme. Cultural conditions and process parameters i.e. type of substrate, incubation time, temperature, initial pH, moisture level, substrate concentration, tannic acid concentration, carbon sources, peptone, yeast extract, urea, NH4NO3 were optimized using one factor at a time approach and activity obtained was 39.5Ug-1. Further optimization was carried out using central composite design following response surface methodology with three independent variables which resulted in increased in tannase production to 55.55 Ug-1. The enzyme was partially purified by acetone precipitation and gel filteration chromatography which showed 46.48 yield with 3.94 fold purification and had a molecular mass of 205kDa. Gallic acid the hydrolytic product in crude enzyme was detected by TLC, FTIR and HPLC using gallic acid as standard. Crude enzyme obtained was studied for its ability in pine needle degradation, tea colour decolourization, dye decolourization and removal of apple browningThesisItem Open Access EVALUATION OF ANTIMICROBIAL COMPOUNDS FROM Juniperus communis L. AND Vitex negundo L. AGAINST Xanthomonas sp. AND Pseudomonas sp.(2015) DIGVIJAY; BHARDWAJ, Satya VratABSTRACT Essential oils from the shade dried leaves of Juniperus communis L. and Vitex negundo L. from H.P. (India) were examined for their antimicrobial activities against eight human and plant pathogenic bacteria. Essential oils were obtained by using Soxhlet extraction apparatus. Chemical analysis of the oils was done by using gas chromatography and mass spectrometry (GC/MS). The antimicrobial effects were tested by inhibition zone diameter (IZD)/ zone of inhibition test. Essential oil from methanol extract showed very good activity at 300 ppm concentration after 72 hours and was more effective as compared to others, however, all the plant extracts showed inhibitory effect on linear growth of selected microorganism. Higher concentrations of 1-Phenanthrene carboxylic acid (11.44 %) and Propylparaben, Benzoic acid, 4-hydroxy, Benzoic acid, 3-hydroxy and Benzoic acid, 3- hydroxy-2-methylpropyl ester (23.23%) content released from Juniperus communis L. and Vitex negundo L. H.P. (India) respectively, might be assigned to be responsible for such excellent antimicrobial activity against the tested pathogens. Docking studies were also performed for sixteen compounds in order to evaluate their affinity to bacterial proteins that are known targets for some antibiotics with different mechanisms of action. The purpose of the present studies was to encourage use of Juniperus communis L. and Vitex negundo L. essential oils against bacterial infections so as to provide an approach to develop herbal formulations as potential disease management strategies in place of chemicals. However, the results are yet to be authenticated through wet lab experimentations for each biomolecules before going ahead for potential drug development.ThesisItem Open Access BIOPROSPECTING OF CHLORPYRIFOS DEGRADING BACTERIA FOR BIOREMEDIATION OF APPLE ORCHARD SOILS OF HIMACHAL PRADESH(2015) SHARMA, ANISH KUMAR; SHIRKOT, POONAMABSTRACT Organophosphorus compounds are the most widely used insecticides, accounting for an estimated 34% of worldwide insecticide sales. Chlorpyrifos is a non-systemic organophosphorus insecticide, with broad spectrum insecticidal activity. Contamination of soil from pesticides as a result of their bulk handling at the farmyard or following application in the field or accidental release may lead occasionally to contamination of surface and ground water. The present study was carried out to explore the status of indigenous chlorpyrifos degrading bacteria in the apple orchard soils in the state of Himachal Pradesh, which harbours plenty of diverse forms of microbial life, which has remained largely unexplored till date. The pH and electrical conductivity (EC) of the thirty soil samples from apple orchards were recorded and ranged from 6.55- 7.58 and 0.24-0.39 dSm-1 respectively. The percent organic carbon (% OC), nitrogen (N), phosphorus (P) and potassium (K) content ranged from 0.66-0.116%, 302-486 Kg/ha, 12.8-23.6 Kg/ha and 255-371 Kg/ha respectively. A total of 72 bacterial isolates were isolated from 30 soil samples using enrichment technique and mineral salt medium. These 72 isolates were rescreened on selective medium i.e. eosin-methylene blue agar (EMBA) containing 50 mg/l chlorpyrifos and only 15 isolates were able to change the colour of colonies from creamish/yellowish/whitish/transparent to red, which was observed as an indication of chlorpyrifos degradative ability of these bacterial isolates. These 15 bacterial isolates were characterized biochemically alongwith organophosphorus hydrolase activity and 6 bacterial isolates exhibiting highest extracellular OPH activity of 0.012-0.165 U/ml were selected. Molecular characterization of selected isolates was carried out using RAPD-PCR and 16S rrna gene technology. Only 5 bacterial isolates were amplified by universal primers and in silico analysis of 16S rrna gene sequence led to identification of these bacterial isolates as Pseudomonas indoloxydans ASK3.2, Pseudomonas aeruginosa ASR1.1, Pseudomonas resinovorans AST2.2, Pseudomonas otitidis AST3.2 and Pseudomonas stutzeri ASM1.1. Two bacterial strains Pseudomonas indoloxydans strain ASK3.2 and Pseudomonas resinovorans strain AST2.2 exhibited mottled growth in form of colonies on agar plates fortified with 1600 mg/l concentration of chlorpyrifos and these two bacterial strains were selected for optimization and biodegradation experiments. In both strains highest OPH activity was observed at pH 8.0, temperature 37°C, after 48hrs in presence of glucose as carbon source. Activity of extracellular OPH enzyme in both the strains was found to be enhanced in presence of Co+2 metal ions, while Cu+2, Mg+2 and Fe+2 ions had showed lesser effect on OPH activity. GC-FID results confirmed that Pseudomonas indoloxydans strain ASK3.2 is a more efficient degrader as 82.72% chlorpyrifos was degraded successfully as compared to 43.90% by Pseudomonas resinovorans strain AST2.2. GC-MS analysis results confirm that peak area/height in both test samples was found to be lower than in control sample, indicating decreasing amount of chlorpyrifos due to its degradation into smaller intermediate compounds. Only one metabolite i.e. phosphoric acid, diethyl 3,5,6-trichloro-2-pyridyl ester (C9H11Cl3NO4P) with CAS no. 5598-15-2 was identified by NIST library search in both test samples at RT 11.10 and RT 11.11 respectively.