Microscopic analysis and colonization Plant

roots infected with fungal endophyte were sectioned and treated with sodium hypochlorite (2.5%) for 10 min for clarification. Latter, it was treated with KOH (20%) for 24 h which was extensively rinsed with autoclaved DW. The root pieces were acidified with HCl (10%); stained for 24 h using tryptophan blue (0.8%) and lactic acid (95%). At the end, the root pieces were distained in lactic acid for 24 h. The endophytic colonization in roots pieces was assessed through light microscope (Stemi SV 11 Apo, Carl Zeiss). The rate of colonization was determined according to the method of Kumar and Hyde [21]. Determination of antioxidants To determine reduced glutathione Stem Cells inhibitor (GSH), leaves tissues (100 mg) of all the treated pepper plant samples were ground in 3 ml 5% (v/v) trichloroacetic acid using chilled mortar and pestle. The homogenate was obtained through centrifugation (at 15000 rpm for 15 min at 4°C). The homogenate obtained was analysed for reduced glutathione (GSH) activity as described by Ellman [22]. The reaction mixture comprised of sample supernatant (0.1 ml), monosodium phosphate (3.0 ml; 150 mM this website NaH2PO4; pH 7.4) and Ellman’s reagent (0.5 ml). The mixture was incubated at 30°C for 5 min. Absorbance was determined at 412 nm and the GSH activity was calculated by a standard curve. Total polyphenol

content was determined by the Folin-Ciocalteau method as mentioned by Kumazawa second et al. [23]. Plant tissues (100 mg) were ground with 80% ethanol and the resultant extracts (0.5 ml) were mixed with Folin-Ciocalteau reagent (0.5 ml) and 10% Na2CO3 (0.5 ml). The absorbance of the reaction mixture was measured at 760 nm after 1 h incubation at room temperature. Total polyphenol content was expressed as micro g/mg (gallic acid equivalents). The detection of superoxide anion (O2 -) was based on its ability to reduce nitro blue tetrazolium (NBT) as performed by Doke [24]. Treated plant tissues (100 mg) were cut into 1 mm2 pieces and AR-13324 ic50 immediately immersed in 10 mM phosphate buffer (pH 7.8), containing NBT (0.05% (w/v)) and 10 mM NaN3. The reaction mixture was left for incubation till one hour at room temperature. The reaction

mixture was heated at 85 ± 2°C for 15 min and cooled quickly to 0°C. The absorbance was measured at 580 nm. The O2 – content was expressed as an increase of absorbance / 0.1 g dry weight. The extent of lipid peroxidation was determined by the method of Ohkawa et al. [25]. The optical density of the resulting light pink colour was recorded at 532 nm. Tetramethoxypropane was used as an external standard. The level of lipid peroxides was expressed as micro moles of malondialdehyde (MDA) formed/g tissue weight. Enzymatic analysis All treated plant’s leaves (200 mg) were homogenized in 50 mM Tris–HCl buffer (pH 7.0) composed of 3 mM MgCl2, 1 mM EDTA and 1.0% PVP and then centrifuged (15,000 rpm for 15 min at 2°C). The supernatant was used for enzymatic analysis.