full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and PharmacyVol. 5 (2) 1098-1103 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1100undiluted and diluted samples was done. Dilutionswere carried out using ethanol. The sample wasdiluted with ethanol. Thus, 50ìl of sample and2ml of ethanol was taken in a quartz cuvette. 3ml of 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 andplaced in sterile Petri plates containing WhatmanNo. 1 paper. 100ìl of crystals along with waterwas applied gently on leaf surface using amicropipette and 100ìl of water was applied oncontrol leaves. These plates were allowed toincubate at 25°C±1°C for 1 to 2 days and themoisture in dishes was maintained throughout theexperiment. The results were recorded after 24hours. The experiment was carried out intriplicates.v. Comparison of bands and Rf values:Aluminum-backed silica gel 60 F 254TLC foils(10X20cm) of 0.25mm thickness (Merck,Dramstadt, Germany) were run with methanolas the mobile phase and then dried in an oven at120°C for 15-20 mins before sample loading.HPTLC of 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 ofchloroform:ethanol (93:7). After running the foilswere dried in an oven at 120°C for 5 mins andthe developed plates were observed under a UVsource at 254 and 366nm. The Rf value of bandsobserved was compared with the Rf value of theobserved band of 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 and were used in in vivo bioassay. 0.5 mlof crystal solution along with water was taken inan injection syringe and this was injected in thepetiole region of the plant. Sterile water was usedin the control plants. The surface of the leaf wasslightly pricked with sterilized syringe in order toenhance the rate of infection. The plants weremonitored regularly for the development of visiblesymptoms.ResultsThe crystals of alternaric acid wereobserved under 10Xand 40X magnifications(Fig.1).The yield of alternaric acid obtained bythis method is 0.05 g per100 ml. This isolateproduced maximum toxin from 18 th day onwardsand 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 of crystalline alternaricacid and that of alternaric acid separated fromcrude FCF by HPTLC was 0.5. TLC foil of10X10 dimensions was scanned under Scanner 3at 254 and 366nm. A distinct peak of 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 of 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 of tomatoworld-wide. Alternaric acid, a phytotoxicmetabolite of A. solani, (13) is a highly crystalline,colorless and an optically inactive unsaturateddibasic acid. It contains no methoxyl groups andIsolation of Alternaric acid