Indian Phytopath. 67 (2) : 187-189 (2014) Biochemical changes in chilli against Colletotrichum capsici KARTAR SINGH 1 *, S.K. KHIRBAT 1 , L.K. CHUGH 2 and VEENA JAIN 2 1 Department of Plant Pathology, 2 Department of Biochemistry, Chaudhary Charan Singh Haryana Agricultural University, Hisar 125 004, Haryana, India Key words: Chilli, Colletotrichum capsici, defense enzymes SHORT COMMUNICATION *Corresponding author: kartar1532@gmail.com Chilli (Capsicum annum L.) is one of the most important spice vegetable as well as cash crop grown throughout the world. Chilli is good source of vitamins A, B, C and minerals like Ca, P, Fe, Na and Cu in trace amounts. Chilli crop is subjected to various diseases and among them fruit rot of chilli caused by Colletotrichum capsici (Syd.) Butler and Bisby is one of the most destructive diseases in India. The disease causes severe damage to red chilli fruits. It has been reported to cause 20-60 per cent losses in Punjab and Haryana (2). In the present study an attempt was made to quantitatively analyze total phenol content and activity of peroxidase and polyphenol oxidase enzymes in red fruits of resistant and susceptible varieties of chilli in artificial disease stress conditions after inoculation with Colletotrichum capsici. Chilli varieties, resistant (Pusa sadabahar) and susceptible (Pusa jwala) to Colletotrichum capsici were raised at CCS Haryana Agricultural University experimental farm in a randomized block design keeping three replications with recommended package of practices during 2012-2013 crop season. Red ripe fruits of chilli were inoculated with 8 days old culture of Colletotrichum capsici having standard spore suspension solution of 3 x 10 4 spores/ml with the help of pin prick method. Uninoculated and inoculated red fruits of both resistant and susceptible varieties were collected from lower and middle portion of the plants at different intervals of 2, 4, 6, 8 and 10 days after inoculation. The fruits were collected at randomly and a composite sample was prepared which was analysed fresh for total phenol content, peroxidase (PO) and polyphenol oxidase (PPO) enzymes activities. The content of total phenols present in the fruits was estimated (8). Fresh fruit sample of 1g were grounded with a mortar and pestle in 10 ml of 80 per cent alcohol. The homogenate was centrifuged at 10,000 rpm for 20 minutes. The clear supernatant was taken and residue was re-extracted thrice with five ml of 80 per cent alcohol. The supernatant was pooled and final volume was made to 20 ml with 80 per cent alcohol. One ml of the supernatant was evaporated to dryness. The dissolved residue in one ml of distilled water. Then 3 ml of distilled water was added to make final volume of 4.0 ml. After 3 minutes of addition of 0.5 ml of Folin-Ciocalteau reagent, 2 ml of saturated Na 2 CO 3 solution was added to each tube. The contents were mixed thoroughly. Placed the tube in boiling water exactly for one minute. Tubes were cooled and absorbance was recorded at 650 nm against a reagent blank. A standard curve prepared by using different concentrations of catechol (0-100 μg/ml) was used to calculate total phenol content. Results were expressed as mg total phenol/g fresh wt. of fruit. For analysis of peroxidase enzyme (9) fruit tissue (1g) was homogenized in 2 ml of 0.1M phosphate buffer (pH 6.1) by grinding in a pre-cooled mortar and pestle. The homogenate was centrifuged at 10,000 rpm at -4 o C for 20 minutes. Supernatant thus obtained was used as enzyme source. The extract was stored in a refrigerator. The activity was estimated within 4 hours of extraction. In preliminary studies, it was first established that under the assay conditions employed, the rate of enzyme catalyzing reaction was proportional both to the amount of enzyme as well as the reaction time. In a clean dry cuvette 2.5 ml of acetate buffer was taken. In this 0.5μl enzyme extract and 0.1ml O-dianisidine (M.W. 244.3) solution was added. Then after adding 0.4 ml H 2 O 2 in the solution mixed the content thoroughly. Place the cuvette in the spectrophotometer set at 430 nm. Then immediately started the stop watch. Read the initial absorbance and at every 15 seconds interval up to 3 minutes. Blank did not contain H 2 O 2 . The enzyme activity was expressed in terms of specific activity (units/g fresh weight). One unit of activity was defined as the amount of enzyme which produce a change of 0.1 in absorbance at 430 nm/min of incubation (1 unit = ∆ 0.1 O.D./min at 430 nm). The Polyphenol oxidase was extracted by earlier described method (8). Fruit tissue (1 g) was homogenized in 2 ml of 0.1M phosphate buffer (pH 6.1) by grinding in a pre-cooled mortar and pestle. The homogenate was centrifuged at 10,000 rpm at -4 o C for 20 minute. Supernatant thus obtained was used as enzyme source. The extract was stored in a refrigerator. The activity was estimated within 4 hours of extraction. The enzyme was assayed by the method of (7) with slight modification using 3, 4-DL-dihydroxy-phenylalanine (DOPA) as substrate. The reaction mixture contains 0.1 ml of enzyme extract, 2.5 ml of substrate (DOPA 0.1% solution prepared in 0.1M sodium phosphate buffer of pH 6.1). The increase in absorbance was determined at 475 nm.