PLANTA DANINHA. BIOEFFICACY OF A COMMON WEED Datura metel AGAINST Colletotrichum gloeosporioides

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1 PLANTA DANINHA KARIM, M. et l. Bioefficcy of common weed Dtur metel ginst Colletotrichum gloeosporioides PD-2016 (9 págins) PROVA GRÁFICA < SOCIEDADE BRASILEIRA DA CIÊNCIA DAS PLANTAS DANINHAS ISSN (print) (online) Article KARIM, M. 1 JABEEN, K. 1 * IQBAL, S. 1 JAVAID, A. 2 BIOEFFICACY OF A COMMON WEED Dtur metel AGAINST Colletotrichum gloeosporioides Bioeficáci d Plnt Dtur metel contr Colletotrichum gloeosporioides ABSTRACT - Mngo (Mngifer indic) is one of the most importnt trees grown minly in the tropicl nd sutropicl countries. Anthrcnose disese cused y Colletotrichum gloeosporioides, is the most dmging disese cusing reduction of flower set nd yield losses in mngo. The present study ws designed to evlute ntifungl ctivity of Dtur metel extrcts ginst C. gloeosporioides. Extrcts (1% to 3%) of different prts of D. metel viz. leves, seeds nd roots were ssessed for their ntifungl potentil. Methnolic seed extrct of D. metel depicted the highest ntifungl potentil ginst the test fungus. All the pplied concentrtions effectively reduced growth of the test fungus. However, 1.5% conc. of methnolic seeds extrct showed mximum reduction (80%) in comprison with control. This extrct ws further sujected to frctiontion using vrious orgnic solvents viz. n-hexne, chloroform, ethyl cette nd n-utnol. Chloroform frction ws proved to e the est for the inhiition of the trget fungus tht ws nlyzed y GC-MS nd vrious ioctive constituents like n-hexdecnoic cid, phytol, octdecnoic cid, oleic cid, o-xylene nd cyclohexnol were identified. Keywords: ntifungl, GC-MS, methnolic extrcts, phytochemicl nlysis. * Corresponding uthor: <khjist_1@hotmil.com> Received: My 31, 2016 Approved: July 25, 2016 RESUMO - A mngueir (Mngifer indic) é um ds árvores mis importntes do mundo, cultivds principlmente nos píses tropicis e sutropicis. A ntrcnose, cusd pelo fungo Colletotrichum gloeosporioides, é doenç mis prejudicil, que cus redução do número de flores e perds de rendimento d mng. O presente estudo foi desenvolvido pr vlir tividde ntifúngic de extrtos de Dtur metel contr C. gloeosporioides. Extrtos (1% 3%) de diferentes prtes de D. metel viz. folhs, sementes e rízes form vlidos qunto o potencil ntifúngico. O extrto metnólico de sementes de D. metel presentou o mior potencil ntifúngico contr o fungo de teste. Tods s concentrções plicds reduzirm com eficáci o crescimento do fungo de teste. No entnto, o extrto metnólico de sementes n concentrção de 1,5% presentou redução máxim (80%) em relção o controle. Este extrto foi ind sumetido frccionmento utilizndo vários solventes orgânicos viz. n-hexno, clorofórmio, cetto de etil e n-utnol. A frção de clorofórmio mostrou ser melhor pr iniição do fungo-lvo, pós nálise por meio de GC-MS, e form identificdos vários constituintes iotivos, por exemplo, ácido n-hexdecnóico, fitol, ácido octdecnóico, ácido oleico, o-xileno e ciclo hexnol. Plvrs-chve: ntifúngico, GC-MS, extrtos metnólicos, nálise fitoquímic. 1 Deprtment of Botny, Lhore College for Women University, Jil Rod, Lhore, Pkistn; 2 Institute of Agriculturl Sciences, University of the Punj, Lhore, Pkistn. Doi: /S

2 KARIM, M. et l. Bioefficcy of common weed Dtur metel ginst Colletotrichum gloeosporioides 2 INTRODUCTION Mngo is one of the most delicious fruits in the world nd it hs ttined specil position in the line of delightful fruits; it hs significnt nutritive nd therpeutic elements (Thir et l., 2012). Pkistn rnks fourth in terms of mngo production worldwide (Ghfoor et l., 2010). Reserch studies on mngo shows the presence of mny nutritionl contents viz. crude proteins, crohydrtes nd vitmins (Srkiyyi et l., 2013). Mngo crops re dmged y mny pre- nd post-hrvest diseses, including nthrcnose, which is cused y Colletotrichum gloeosporioides (Pndey et l., 2012). Under wrm nd humid conditions, nthrcnose is one of the most dmging nd persistent diseses, nd it cuses mss losses in mngo fields (Sju et l., 2013). This fungus contins vrious pectinolytic enzymes (polyglcturonse trns eliminse, pectin trns eliminse, polyglcturonse, pectin methyl esterse) nd cellulolytic enzymes (cellulse) which re responsile for its pthogenicity (Jt et l., 2013). To control this disese, synthetic fungicides re very importnt. Hot enomyl dips nd orte degrde spore germintion nd reduce hrmful effects of C. goleosporioides (Kim et l., 2007). However, severe hrmful effects nd environmentl prolems rise s result of fungicides. These prolems give new pproch towrds the use of environmentl friendly plnt products tht cn retrd fungl pthogens (Jvid nd Akhtr, 2015). Becuse of fewer hrmful effects of nturl products on the environment, they re ssumed to e good sustitutes to synthetic fungicides (Pwr, 2011; Jvid et l., 2015). Dtur metel (fmily Solncee) is n importnt medicinl plnt. It is well known for its insecticidl, hericidl, ntifungl, nticteril, nticncer, ntiinflmmtory nd ntirheumtoid ctivity (Monir nd Munn, 2012; Hossin et l., 2013). D. metel contins flvonoids, phenols, tnnins, sponins nd sterols s mjor phytoconstituents (Dontus nd Ephrim, 2009). Therefore, the present study ws designed to evlute the ntifungl potentil of different prts of D. metel ginst C. gloeosporioides. MATERIALS AND METHODS Leves, roots nd seeds of D. metel were collected from Rilwy Rod Sheikhupur, Punj, Pkistn. Different plnt prts were thoroughly wshed nd completely dried t 35 o C nd stored in polythene gs. The culture of C. gloeosporioides ws isolted from the disesed mngo plnt on 2% mlt extrct gr (MEA) medium. Antifungl iossy One hundred grms of dried leves, seeds nd roots of D. metel were soked in 250 ml methnol for week t room temperture; fterwrds, the soked mterils were filtered through utoclved muslin cloth. The extrct ws then llowed to evporte until volume ws reduced to 5 g. This plnt extrct ws further diluted y dding 55 ml of distilled wter to mke 20% stock solution. Mlt extrct gr (2 g destilledwter) ws prepred in 250 ml flsk y dding the required mount of MEA in 51, 52.5, 54, 55.5, 57 nd 60 ml of distilled wter. This medium ws utoclved t 121 o C for 30 minutes. Five concentrtions viz. 3%, 2.5%, 2%, 1.5% nd 1% of leves, roots nd seeds were prepred. For preprtion of these concentrtions 9, 7.5, 6, 4.5 nd 3 ml stock solutions were dded to 51, 52.5, 54, 55.5, 57 ml of MEA, respectively, to mke finl volume up to 60 ml. Chloromycetin (250 mg L -1 ) ws dded in ech concentrtion to void cteril contmintion nd 20 ml of ech concentrtion ws poured in 9 x 9 cm sterilized Petri pltes. All Petri pltes were inoculted y C. gloeosporioides with sterilized cork orer from the tips of two-week old fungl culture nd tightly pcked. All the concentrtions were replicted three times. After 7 dys, fungl growth dimeter ws mesured in centimeters y tking verge of three dimeters t right ngle of ech colony. Percentge growth inhiition of the fungl colonies ws determined y using the formul: Growth inhiition (%) = Growth in control - Growth in tretment 100 Growth in control

3 KARIM, M. et l. Bioefficcy of common weed Dtur metel ginst Colletotrichum gloeosporioides 3 Phytochemicl nlysis Biochemicl constituents of Dtur metel seeds were evluted y phytochemicl nlysis of the methnolic extrct. Stndrd techniques nd methodologies of Edeog et l. (2005), nd Prekh nd Chnd (2007) were followed for these tests. In vitro iossy with isolted frctions The most effectul plnt prt (seed) ws selected for iossy-guided frctiontion to check its ntifungl potentil ginst C. gloeosporioides. One hundred nd fifty grms powdered seeds of D. metel were extrcted with 400 ml methnol. This extrct ws evported on rotry evportor nd further portioned with vrious orgnic solvents in incresing order of polrity including n-hexne, chloroform, ethyl cette nd n-utnol (Jeen et l., 2013). This prtitioning resulted in the gummy mss of n-hexne (1.56 g), chloroform (2.66 g), ethyl cette (2.23 g) nd n-utnol (1.86 g). All the four frctions were serilly diluted to check their ioctivity ginst the trget fungus y following the protocol of Sherzi et l. (2016). GC-MS Anlysis The chloroform frction of the methnolic seed extrct of D. metel ws found the most effective in previous ssy hence it ws selected for GC-MS nlysis. This frction ws seprted through HP-5MS cpillry column (30 m x 0.25 mm x 1.0 μl) y pplying the following temperture progrm; 40 o C for 5 min, o C t 2 o C min -1, 70 o C for 2 min, o C t 3 o C min -1, o C t 5 o C min -1, o C t 10 o C min -1 nd then 220 o C for 2 min. 280 o C ws set s trnsfer line temperture. Mss detector conditions were: electronic impct (EI) mode t 70 ev; scnning rte 2.88 scn S-1; temperture source: 200 o C: mss scnning rnge: m/z ; helium gs t 1.0 ml min -1 (Wheed et l., 2016). The constituents of voltile components were identified fter comprison with the computer dt system lirry (NIST 98) ttched to the GC-MS instrument nd document. Sttisticl nlysis All the dt were sttisticlly nlyzed y using nlysis of vrince (ANOVA) followed y Duncn s Multiple Rnge Test t 5% level of significnce with the id of the computer softwre COSTAT. RESULTS AND DISCUSSION In the present study, the ntifungl potentil of D. metel ws ssessed ginst C. gloeosporioides. All the pplied concentrtions of D. metel cused significnt reduction in rdil growth of the test fungus (Figure 1A nd B). The methnolic seed extrct of D. metel ws found to e highly effectul in in vitro iossy s ll the pplied concentrtions significntly retrded the rdil growth of C. gloeosporoides. Both the higher concentrtions viz. 3% nd 2.5% cused mximum reduction in the growth of the test fungus y 76% nd 80%, respectively. The methnolic extrct of roots of D. metel ws lso found effective s there ws 35% inhiition in test fungus growth in the 1.5% concentrtion. The methnolic lef extrct of D. metel ws found to e less effective in the in vitro iossy. Shhnz et l. (2010) investigted tht D. metel extrcts successfully reduced the growth of Mcrophomin phseolin nd Rhizoctoni solni. In similr study, Rinez et l. (2013) suggested tht D. metel extrcts cn e used s fungicide to control Fusrium oxysporum. The results of the phytochemicl nlysis showed tht phlotnins, flvonoids, terpenoids, lkloids nd coumrins were present in the methnolic extrct of D. metel seeds (Tle 1). Previously, Monir nd Munn (2012) reported tht lkloids, flvonoids nd tnnins re present in D. metel. Srkiyyi et l. (2013) suggested tht the ntifungl ctivity D. metel might e due to the presence of lkloids, terpenoid, steroids, flvonoid, triterpenes, phenolic nd tnnin compounds.

4 KARIM, M. et l. Bioefficcy of common weed Dtur metel ginst Colletotrichum gloeosporioides 4 Control 1% 1.5% 2% 2.5% 3% A Fungl dimeter (cm) c 0.00 Lef Extrct Root Extrct Seed Extrct Lef Extrct Root Extrct Seed Extrct B -2 Decrese in fungl dimeter over control (%) Vlues with different letters t the top show significnt difference (P 0.05) s determined y the DMR Test. Figure 1 - Effect of methnolic lef, root nd seed extrcts of D. metel on growth of C. gleosporioides. Tle 1 - Phytochemicl nlysis of the methnolic extrct of D. metel seeds Phytochemicl Indiction Results Glycosides No rown color ppers -ve Sponins No frothing ppers -ve Phlotnins Formtion of Red ppt. occur +ve Terpenoids Ring formtion occur +ve Flvonoids Yellow color ppers +ve Alkloids Ornge color ppers +ve Coumrins Color ppers under UV +ve Tnnins -ve The in vitro iossy with isolted orgnic frctions showed tht the chloroform frction of the methnolic seed extrct of D. metel proved etter thn other frctions, nmely n hexne, n-utnol nd ethyl cette, t retrding the growth of the mycelil mss of C. gloeosporioides s it cused 53% reduction in fungl iomss (Figure 2). This effective frction ws sujected to GC-MS nlysis for seprtion nd identifiction of ioctive constituents (Figure 3). Six compounds were identified in the GC-MS nlysis from the chloroform frction of the methnolic seed extrct (Tle 2). As seeds re rich in ftty cid, the mjor proportion of this frction consists of ftty cid. Sturted ftty cids such s octdecdienoic cid, n-hexdecnoic cid, hydrocrons e.g. o-xylene nd phytol were identified in the chloroform frction of D. metel.

5 KARIM, M. et l. Bioefficcy of common weed Dtur metel ginst Colletotrichum gloeosporioides 5 0% 0.52% 1.04% Fungl dimeter (cm) c c c n-hexne Chloroform Ethyl cette n-utnol Vlues with different letters show significnt difference s determined y the DMR test. Figure 2 - Effect of different concentrtions of isolted frctions from the D. metel methnolic seed extrct on the in vitro growth of C. gloeosporioides. Figure 3 - GC-MS Chromtogrm of chloroform extrcts from D. metel seeds. Tle 2 - GC-MS nlysis of the chloroform extrct of D. metel seeds Sr. # Compound nme Chemicl formul Moleculr weight Retention time (min) 1 Cyclohexnol C 10 H 18 O o-xylene C 8 H Phytol C 20 H 40 O Octdecnoic cid C 18 H 36 O Oleic cid C 18 H 34 O n-hexdecnoic cid C 16 H 32 O n-hexdecnoic cid is highly ioctive compound nd Aucker nd Deeplkshmi (2013) stted tht the methyl ester of hexdecnoic cid isolted from leves of Annon murict proved to hve extreme ntifungl potentil ginst Alternri solni, Aspergillus fumigtus nd Penicillium chrysogenum. A flvonoid, Cyclohexnol ws lso identified in the GC-MS nlysis of D. metel

6 KARIM, M. et l. Bioefficcy of common weed Dtur metel ginst Colletotrichum gloeosporioides 6 seed extrct tht is known to hve ctive ntimicroil potentil (Hussin et l., 2014). Phytol, diterpene monosturted lcohol (drug), ws lso found in the seeds of D. metel. The ntimicroil potentil of this importnt drug hs een studied y mny reserchers s Rj et l. (1998) studied the ntimycocteril potentil of phytol. Likewise, Inoue et l. (2005) lso ssessed the ntimicroil ctivity of phytol ginst Stphylococcus ureus. One of the mjor ioctive compound oleic cid ws lso found in the present investigtion. Aucker nd Devi (2014) reported tht oleic cid hs ntifungl potentil ginst vrious phytopthogenic fungi such s Colletotrichum fulctum, Rhizoctoni solni nd Fusrium oxysporum. The present study concludes tht the methnolic seed extrct of D. metel is highly effective ginst C. gloeosporioides, especilly the chloroform su-frction of this extrct, which contins potent ntifungl gents. Ftty cids nd other compounds present in this su-frction might e responsile for ntifungl ctivity. REFERENCES Aucker M.N., Deeplkshmi T. In vitro Antifungl potentils of ioctive compound methyl ester of hexdecnoic cid isolted from Annon murict Linn. leves. Biosci Biotechnol Res Asi. 2013;10: Aucker M.N., Devi P.K. In vitro ntifungl potentils of ioctive compound oleic cid, 3-(octdecyloxy) propyl ester isolted from Lepidgthis cristt Willd. (Acnthcee) inflorescence. Asin Pc J Trop Dis. 2014;7: Edeog H.O. et l. Phytochemicl constituents of some Nigerin medicinl Plnts. Afr J Biotechnol. 2005;4: Dontus EO., Ephrim CI. Isoltion, chrcteriztion nd nticteril ctivity of lkloid from Dtur metel Linn leves. Afr J Phrm Phrmcol. 2009; 3(5): Ghfoor A. et l. Determinnts of mngo export from Pkistn. J Agric Res. 2010;48:105. Hossin M.A. et l. Gs chromtogrphy mss spectrometry nlysis nd totl phenolic contents of vrious crude extrcts from the fruits of Dtur metel L. J Tih Univ Sci. 2013;7: Hussin H.T. et l. Stereostructure, ntimicroil nd cytotoxic ctivity of cyclo-hexene, cyclohexnol nd pyridine derivtives synthesized from chlcones. Inter J Phrm Sci Res. 2014;5: Inoue Y. et l. Biphsic effects of gernylgerniol, teprenone, nd phytol on the growth of Stphylococcus ureus. Antimicro Agents Chemother. 2005;49: Jeen K. et l. Antifungl ctivity of Azdircht indic ginst Alternri solni. J Life Sci Technol. 2013;1: Jt B.L., Shrm P., Gour H.N. Production of enzymes nd culture filtrtes y Colletotrichum gloeosporioides Penz. cusing nn fruit rot. Proc Nt Acd Sci Indi Section B: Biol Sci. 2013;83: Jvid A., Akhtr R. Antifungl ctivity of methnolic root extrct of Withni sommnifer ginst Fusrium oxysporum f. sp. cepe. Africn J Trdit. 2015;12:22-7. Jvid A. et l. Mngement of Mcrophomin phseolin y extrcts of n llelopthic grss Impert cylindric. Pkistn J Agric Sci. 2015;15: Kim Y. et l. Antioxidnt phytochemicl nd fruit qulity chnges in mngo (Mngifer indic L.) following hot wter immersion nd controlled tmosphere storge. Food Chem. 2007;105: Monir M.K., Munn S.M. Review on Dtur metel: A potentil medicinl plnt. Glo J Res Med Plnts Indig Med. 2012;1: Pndey A. et l. In Vitro Studies for resistnce to nthrcnose disese (Colletotrichum gloeosporioides Penz.) in different mngo hyrid seedlings. Inter J Fruit Sci. 2012;12: Prekh J., Chnd S.V. In vitro nticteril ctivity nd phytochemicl nlysis of some Indin medicinl plnts. Turk J Biol. 2007;3:53-8.

7 KARIM, M. et l. Bioefficcy of common weed Dtur metel ginst Colletotrichum gloeosporioides 7 Pwr B.T. Antifungl ctivity of some stem extrcts ginst seed-orne pthogenic fungi. J Phytol. 2011;12: Rj M.S. et l. Antimycocteril ctivity of (E)-phytol nd derivtives: preliminry structure-ctivity study. Plnt Med. 1998;64:2-4. Rinez A. et l. Antifungl ctivity of Dtur metel L. orgnic nd queous extrcts on some pthogenic nd ntgonistic fungi. Afr J Microiol Res. 2013;16: Sju K.A. et l. Yield loss of lrge crdmom (Amomum suultum Rox.) due to Colletotrichum light in Sikkim. J Spices Arom Crop. 2013;22: Srkiyyi S. et l. Comprtive nlysis of nutritionl nd nti-nutritionl contents of some vrieties of mngo (Mngifer indic) in Kdun Metropolis-Nigeri. Res J Appl Sci Eng Technol. 2013;5: Shhnz D. et l. Comprtive effect of plnt extrcts of Dtur l Ness nd Cynodon dctylon (L.) Pers., lone or in comintion with microil ntgonists for the control of root dis ese of cowpe nd okr. Pkistn J Bot. 2010;42: Sherzi A.Z. et l. Mngement of Ascochyt riei (Pss). L y Chenopodium lum L. extrcts. Plnt Dninh. 2016;34: Thir W.A. et l. An nlysis of the effectiveness of extension work conducted y pulic sector with specil reference to mngo in the southern Punj, Pkistn. Pkistn J Agric Sci. 2012;49: Wheed N. et l. Biopesticidl ctivity of Clotropis procer L. ginst Mcrophomin phseolin. Afr J Trdit Complement Altern Med. 2016;13: