full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
- No tags were found...
Create successful ePaper yourself
Turn your PDF publications into a flip-book with our unique Google optimized e-Paper software.
Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong>Vol. 5 (2) 1098-1103 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1100undiluted <strong>and</strong> diluted samples was done. Dilutionswere carried out using ethanol. The sample wasdiluted with ethanol. Thus, 50ìl <strong>of</strong> sample <strong>and</strong>2ml <strong>of</strong> ethanol was taken in a quartz cuvette. 3ml <strong>of</strong> ethanol was taken as a blank.iv. Detached leaf bioassay: Tomato leaves werewashed in tap water. The leaves were surfacesterilized with a cotton swab dipped in 4% Sodiumhypochlorite solution for 10-15 seconds. Theywere then washed thoroughly in sterile water <strong>and</strong>placed in sterile Petri plates containing WhatmanNo. 1 paper. 100ìl <strong>of</strong> crystals along with waterwas applied gently on leaf surface using amicropipette <strong>and</strong> 100ìl <strong>of</strong> water was applied oncontrol leaves. These plates were allowed toincubate at 25°C±1°C for 1 to 2 days <strong>and</strong> themoisture in dishes was maintained throughout theexperiment. The results were recorded after 24hours. The experiment was carried out intriplicates.v. Comparison <strong>of</strong> b<strong>and</strong>s <strong>and</strong> Rf values:Aluminum-backed silica gel 60 F 254TLC foils(10X20cm) <strong>of</strong> 0.25mm thickness (Merck,Dramstadt, Germany) were run with methanolas the mobile phase <strong>and</strong> then dried in an oven at120°C for 15-20 mins before sample loading.HPTLC <strong>of</strong> FCF from 7 th to 21 st days was carriedout using Linomat 5 applicator (CAMAG, Muttenz,Germany) using a 100ìl Hamilton syringe. TheTLC foils were developed in a solvent system <strong>of</strong>chlor<strong>of</strong>orm:ethanol (93:7). After running the foilswere dried in an oven at 120°C for 5 mins <strong>and</strong>the developed plates were observed under a UVsource at 254 <strong>and</strong> 366nm. The Rf value <strong>of</strong> b<strong>and</strong>sobserved was compared with the Rf value <strong>of</strong> theobserved b<strong>and</strong> <strong>of</strong> crystalline alternaric acid.vi. In vivo bioassay: Tomato seeds weresoaked in sterile water. The seeds were allowedto germinate in autoclaved Petri plates lined withWhatman No. 1 paper. After the seedlingsreached two leaf stages, they were transferredin pots. Tomato plants were grown for about 4weeks <strong>and</strong> were used in in vivo bioassay. 0.5 ml<strong>of</strong> crystal solution along with water was taken inan injection syringe <strong>and</strong> this was injected in thepetiole region <strong>of</strong> the plant. Sterile water was usedin the control plants. The surface <strong>of</strong> the leaf wasslightly pricked with sterilized syringe in order toenhance the rate <strong>of</strong> infection. The plants weremonitored regularly for the development <strong>of</strong> visiblesymptoms.ResultsThe crystals <strong>of</strong> alternaric acid wereobserved under 10X<strong>and</strong> 40X magnifications(Fig.1).The yield <strong>of</strong> alternaric acid obtained bythis method is 0.05 g per100 ml. This isolateproduced maximum toxin from 18 th day onwards<strong>and</strong> the Rf value was 0.5. (Fig. 2a). The toxinsappear as blue fluorescing spots when placedunder a short wavelength UV lamp (366nm)(Fig. 2b). The Rf value <strong>of</strong> crystalline alternaricacid <strong>and</strong> that <strong>of</strong> alternaric acid separated fromcrude FCF by HPTLC was 0.5. TLC foil <strong>of</strong>10X10 dimensions was scanned under Scanner 3at 254 <strong>and</strong> 366nm. A distinct peak <strong>of</strong> separatedcrystalline alternaric acid was observed at 366nm(Fig. 3). Symptoms appeared on the treated leavesafter 24 hours from inoculation (Fig. 4). Morethan one peak was observed in the undilutedsamples (Fig. 5a). By UV- spectrophotometricscanning the absorption maxima <strong>of</strong> the alternaricacid was 254 nm (Fig. 5b). Necrosis on the leafwas clearly visible on the Tomato plant treatedwith crystals whereas on the control plant therewere no symptoms observed (Fig. 6).DiscussionEarly blight, caused by the deuteromycetefungus A. solani is a destructive disease <strong>of</strong> tomatoworld-wide. Alternaric acid, a phytotoxicmetabolite <strong>of</strong> A. solani, (13) is a highly crystalline,colorless <strong>and</strong> an optically inactive unsaturateddibasic acid. It contains no methoxyl groups <strong>and</strong>Isolation <strong>of</strong> Alternaric acid