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Current Trends in Biotechnology and PharmacyVol. 5 (2) 1098-1103 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1099commercially. Isolation of alternaric acid byfollowing the method proposed in this report offersbetter yield than the previous methods. Moreover,the time required for the process of isolation isless and there is a significant increase in the yield.By following this method a good quantity ofpurified form of alternaric acid can be obtained.The aim of this study is to develop a simple andrapid method which facilitates isolation ofalternaric acid for use in various aspects of plantpathology.Materials and methodsFungal culture and its maintenance:Alternaria solani isolate was grown on potatodextroseagar (PDA) plates and these plates wereincubated at 25º C ± 1º C. After sporulation theseplates were maintained at 4ºC until use.Fungal Culture Filtrate (FCF) production andits analysis: An 8 mm mycelial mat was cut witha sterile cork borer from 1 month old A. solani,grown on a PDA plate and transferred into a 250mL Erlenmeyer flask containing 100 mL ofautoclaved potato-dextrose broth (PDB). Flaskswere incubated from 21 days at 25º C ±1ºC instatic submerged condition. Thereafter, the brothwas filtered using Whatman filter paper No. 1.Fungal culture filtrate from 7 th to 21 st days wascollected and subjected for HPTLC analysis.Isolation of Alternaric acid : For developmentof crystals an 8mm mycelial mat was cut with asterile cork borer from 1 month old A. solani,grown on a PDA plate and transferred into a250mL Erlenmeyer flask containing 100mL ofPDB. Flasks were incubated for 18 days at25ºC±1ºC in static submerged condition. Mycelialmat from 18 days old culture grown on PDB wasremoved and the broth was filtered usingWhatman filter paper No. 1. The pH of the filtratewas adjusted between 3.0-3.5 by 1N HCl andextracted with equal volume of ethanol. Thecolorless solution obtained was dissolved byadding drop by drop boiling carbon tetrachloride(2mL) using a glass dropper. Residual ethanol wasremoved by evaporation and crystalline alternaricacid appeared on cooling.Characterization of alternaric acid: Theisolated compound is alternaric acid wasconfirmed by the following analytical techniquesand assay.i. Microscopic analysis: A small drop ofcrystalline alternaric acid was taken on a cleanconcavity glass slide. Cover slip was gently putonto the drop and the slide was observed undermicroscope (Axioplan, Image Analyzer) at 10Xand 40X magnifications.ii. HPTLC (High Performance Thin LayerChromatography): Purification of alternaric acidwas carried out by HPTLC technique. Aluminumbackedsilica gel 60 F 254TLC foils (10X10cm) of0.25mm thickness (Merck, Dramstadt, Germany)were run with methanol as the mobile phase andthen dried in an oven at 120°C for 15-20 minutesbefore sample loading. Sample loading onto TLCfoils were performed with a Linomat 5 applicator(CAMAG, Muttenz, Germany) using a 100ìlHamilton syringe. The TLC foils were developedin a solvent system of isopropanol:ammonia:water(5:1:5). After running the foils were dried in anoven at 120°C for 5mins and the developed plateswere observed under a UV source at 254 and366nm. Rf values were calculated for theobserved metabolite. The presence of alternaricacid was further confirmed by scanning thedeveloped TLC foils. The scanning of foils inwhich crystalline alternaric acid was separatedwere scanned in Scanner 3 at 254 and 366nmand observed for the presence of peak.iii. Spectrophotometric analysis: The observedband was scraped and subjected to spectralscanning (Unicam Alpha) from 190 to 400nm tofind out its absorbance maxima. The scanning ofPatel et al
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
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(December 7-9, 2011)Venue: Karunya
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