Sample collection
Collection of the soil samples were done from pesticide contaminated soil of apple orchards from Kotkhai, Matiyana, Theog, Chopal and Rohru sites of district Shimla, Himachal Pradesh. Soil samples in triplicates using sterile spatula were collected in sterilized polythene bag from the depth of 10 – 15 cm and then were stored at 4°C in refrigerator. Chlorpyrifos (O, O-diethyl O-(3, 5, 6-trichloro-2-pyridyl) phosphorothioate) with 99.6% purity was procured from Sigma – Aldrich, Switzerland.

Isolation of chlorpyrifos degrading bacteria by enrichment technique
The soil samples were used for enrichment after air drying at room temperature and sieving by using 2mm mesh. The 10 gm of soil mixed with 50 ml of modified minimal salt medium (MSM) broth¹⁹ (gl^-1: K₂HPO₄ 0.5g, KH₂PO₄ 0.5 g, (NH₄)₂SO₄ 0.5 g, NaCl 0.5 g, MgSO₄ 0.2 g, CaCl₂ 0.05g, FeSO₄ 0.02)¹⁹containing 10 mg/l of chlorpyrifos and incubated at 37°C under dark condition on orbital shaker for a week duration. Following incubation period, transferred 10 ml of this one-week old culture in fresh 50 ml of MSM broth containing 30mg/l of chlorpyrifos and again incubated for a week on shaker. Further transfer 10 ml of this culture into the fresh 50 ml MSM containing 50mg/l chlorpyrifos and incubated in same conditions for a week. Chlorpyrifos degrading and resistant bacteria were isolated by spread plate and streak plate technique by using this enriched culture.^6,20

Screening of chlorpyrifos degrading bacteria
High chlorpyrifos tolerance and degradation
Isolates were screened on eosin-methylene blue agar (EMBA) indicator medium fortified with50mg/l chlorpyrifosand incubated at 37°C for 48 hrsto observe color change in of the colony into red²¹. Further, each bacterial isolate with chlorpyrifos degrading activity was streaked on MSM agar with varying concentration of chlorpyrifos (50-800 mg/l).^22,23

Organophosphorus hydrolase (OPH) activity
Bacterial isolates with high chlorpyrifos tolerance and degrading abilities were then screened for Organophosphorus hydrolase activity. Overnight culture of bacterial isolates in Luria Bertani (LB) medium was centrifuged at 8,000 x g, 4°C for 10 minutes and further supernatant was treated as crude extracellular enzyme. Resuspend cell pellet in 50 mmol/l Tris-Cl (pH 8.0) buffer containing 0.1 mmol/l phenylmethylsulphonyl fluoride (PMSF) after washing twice. Cell disruption was done using sonication 10 times for 10 seconds with interval of 15 seconds (Digital Sonifier-MP Biomedical). Intact cells were separated by centrifugation at 15000 × g for 30 minutes following collection of supernatant which further was used for quantitative enzyme assay. For quantitative enzyme assay, standard method was used²⁴.

Assessing molecular genetic diversity of chlorpyrifos degrading bacteria
Genomic DNA extraction and quantification
Standard method using phenol-chloroform-isoamyl alcohol (25:24:1) was employed for total genomic DNA extraction from the selected isolates and further DNA precipitation was done with isopropanol. This was followed by treating extracted DNA with RNase (0.2 mg/ml) at 37°C for 15 min, and then again phenol-chloroform-isoamyl alcohol was used for extraction followed by isopropanol precipitation. DNA pellet was then re-suspended in Tris EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), stored at -20°C, till further use as template DNA for RAPD-PCR.²⁵Quantification of the extracted DNA samples were done on 1.0% agarose gel, in 1X Tris-acetate EDTA (TAE) buffer (2M Tris base, 57.10 ml acetic acid and 0.5 M EDTA (pH 8.0, 50X) comprising ethidium bromide (0.5 µg/ml). DNA samples were mixed with loading dye and load in a well of gel and allowed to get separated at 80V for 1.5-2 hours using power system. Gel Documentation system was employed for visualization and capturing the gel image.

RAPD – PCR analysis
Genomic DNA from the bacteria were analyzed by modified RAPD method of Williams ²⁶.The RAPD analysis was done with 25 primers (13 OPA, 10 OPB and 02 OPC series) supplied by Xcelris labs, India by using thermocycler. PCR amplification was performed with 20µl reaction mixture comprising 13.5 µl deionized water, 2.0µl of 10 X PCR buffer containing MgSO₄, 0.5µl of 1U thermo tolerant enzyme (Taq DNA polymerase), 2.0µl of 10 mM dNTPs, 1.0µl of random primer and 1.0µl of template DNA. PCR tubes containing mixture were loaded in the thermocycler for amplification. The PCR protocol comprised of initial denaturation at 94°C for 5 minutes; followed by denaturation at 94°C for 45 seconds and then annealing at 35°C for 1minute followed by extension at 72°C for 1.5minutes and final extension at temperature of 72°C for 8 minutes with 35 number of cycles with final holding at 4°C. Usingagarose gel electrophoresis, amplified PCR products were further analyzed where 1.0% agarose gelwas dissolved in TAE buffer containing 242g ofTris base, Tris, 57.1ml of glacial acetic acid, 100ml 0.5M EDTA with pH 8.0, ethidium bromide (0.5 µg/ml) along with a size DNA ladder of 5 kb and gels were further visualized by means of gel documentation system.Scoring for presence and absence of bands on gels were done for the amplified products of selected bacterial isolates. A score of ‘1’ was specified for presence and ‘0’ was marked for absence of an amplified product. Genetic similarity from the data generated was analyzed using un-weighted pair group arithmetic mean (UPGMA) program of NTSYS-Pc (version 2.02) (Numerical Taxonomy System, Exeter Software). The dendrogram was constructed²⁷ and it serves a base for genetic relatedness between chlorpyrifos degrading bacterial isolates.