Antifungal activity of volatile components extracted from leaves, stems and flowers of four plants growing in Tunisia

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1 Phytopthol. Mediterr. (2005) 44, Antifungl ctivity of voltile components extrcted from leves, stems nd flowers of four plnts growing in Tunisi NAIMA BOUGHALLEB 1, NOURI DÉBBABI 2, HICHEM BEN JANNET 2, ZINE MIGHRI 2 nd MOHAMED EL MAHJOUB 1 1 Lbortoire de Phytopthologie, Ecole Supérieure d Horticulture et d Elevge, Chott Mriem 4042, Sousse, Tunisie 2 Lbortoire de Chimie des Substnces Nturelles et de Synthèse Orgnique (UR/99/12-26), Fculté des Sciences de Monstir, 5000 Monstir, Tunisie Summry. Voltile components extrcted from the leves, stems nd flowers of Lntn cmr, Mlvviscus rboreus, Hibiscus ros-sinensis cv. red flowers nd white flowers were tested ginst the fungi Alternri solni, Botrytis cinere, Fusrium solni f. sp. cucurbite, F. oxysporum f. sp. niveum, Pythium ultimum, Rhizoctoni solni nd Verticillium dhlie. The strongest inhibitory effect of the extrcts ws found with voltile components extrcted from the stems nd the flowers. Complete inhibition ws chieved ginst V. dhlie. The wekest effect ws ginst P. ultimum. Voltile components extrcted from the leves were not effective. Key words: Mlvcee, Verbencee, ntimicrobil components. Introduction Wild plnts re widespred in Tunisi, where they represent very rich nd chrcteristic flor (Pottier-Alpetite, 1979, 1981). Mny of these plnts re used s nturl medicines. In order to discover new biologiclly ctive compounds tht cn be used to control disese, mny wild plnts hve been in the pst, nd still re tody, the subject of chemicl investigtions. For exmple, number of species tht re used in folk medicine, such s Euphorbi mcrocld, E. bougheii, E. stritell, E. serrt, E. virgt, E. fortissimo nd E. cooperi, hve lso been found Corresponding uthor: N. Boughlleb Fx: E-mil: bougtn@yhoo.fr to cuse mrked suppression of root infecting fungi including Fusrium oxysporum, Rhizoctoni solni, Alternri solni nd Verticillium dhlie (Gundidz et l., 1992; Gundidz nd Kuf, 1993; Shudt nd Siddiqui, 2002; Al-Mughrbi, 2003). Chrysnthemum coronrium hs been found to be ctive ginst Aspergillus spp., Pythium ultimum, Fusrium moniliforme, Sclerotini sclerotiorum nd Botrytis cinere. Other species, such s Lntn cmr, hve been reported to hve ntibcteril ctivity (Khn et l. 1988; Vijy et l. 1995; Junior nd Zni, 2000; Sutthiviykit et l. 2000). Lntn cmr, Hibiscus ros-sinensis (cv. red nd white flowers) nd Mlvviscus rboreus re widely distributed in Tunisi nd re commonly used s ornmentl plnts ll over the country. These species hve not hitherto been the subject of biologicl 307

2 N. Boughlleb et l. nd chemicl investigtion in Tunisi. However these plnts when collected from other countries were the source of severl metbolites, including some tht were biologiclly ctive. Previous studies on L. cmr reported on the chemicl composition of the essentil oil of this plnt. This oil contins 25 components, the most importnt of which re sbinene ( %), β-cryophyllene ( %), 1,8-cineole ( %), bicyclogermcrene ( %) nd α-humulene ( %). These voltiles hve been found to possess ntimicrobil nd ntimutgenic properties s well s being biologiclly ctive ginst tics (Amblyom vriegtum) nd some fungi (Deen nd Thoppil, 2000; Alitonou et l., 2004). The effect of hydrolcoholic extrct of L. cmr leves on fertility, generl reproductive performnce nd tertology in rts hve lso been studied (Mollo et l., 2005). This plnt ws further reported to be source of some bioctive pentcyclic triterpenoids: lntdenes A-D, of which lntden C ws found to be heptotoxicnt (Shrm et l., 1992; Shrm et l., 2000). For the genus Hibiscus, the essentil oil of H. cnnbinus ws rich in (E)-phytol (28.16%), (Z)- phytol (8.02%), n-nonnl (5.7%), benzene cetldehyde (4.39%), (E)-2-hexnl (3.10%) nd 5-methylfurfurl (3%) nd hd ntifungl ctivity ginst Colletotrichum frgri, C. gloeosporioides nd C. cuttum (Kobisy et l., 2001). To further reserch on L. cmr, H. rossinensis nd M. rboreus nd if possible to encourge cultivtion of these species in Tunisi, in the present work the ntifungl ctivity of voltile extrcts from vrious ir orgns of these plnts ws studied. As trget orgnisms 7 fungl species were used tht re frequently isolted from crops, fruits or soil in Tunisi. Mteril nd methods Plnt mteril Four plnts were tested in the study: Lntn cmr, Mlvviscus rboreus, Hibiscus rossinensis cv. red flower nd Hibiscus ros-sinensis cv. white flower. The plnts were collected nd identified ccording to their morphologicl chrcteristics. Identifiction ws confirmed by compring collected voucher species with species of known identity. Extrction of voltile components Ech plnt ws divided into three prts: leves, stems nd flowers. An pproprite mount (Tble 1) of ech plnt smple ws subjected to stemdistilltion for 3 5 h. Five hundred ml of ech queous distillte ws subjected to two successive extrctions with chloroform (CHCl 3 ) (2 100ml). After decnttion nd seprtion, the recuperted orgnic lyer contining voltiles ws dried over nhydrous sodium sulfte, then filtered nd desiccted under reduced pressure. Fungl isoltes nd testing of extrcts The fungi used in the biologicl ssys were collected from vrious loctions nd hosts in Tunisi (Tble 2). All fungl isoltes were identified nd smples of ech fungus were deposited in the collection bnk t the Plnt Pthology Lbortory (Ecole Supérieure d Horticulture et d Elevge d Horticulture, Sousse, Tunisi). Fungl isoltes were mintined on potto dextrose gr (PDA) (Difco Lbortories, Inc., Detroit, MI, USA), stored t room temperture nd subcultured once month (Dens nd Svobod, 1990) when needed. The isoltes were grown for 7 10 dys on PDA t 25 C. Extrcts were tested s in Al-Mughrbi et l. (2001). Twelve voltile components extrcted from L. cmr, M. rboreus, H. ros-sinensis cv. red flower nd white flower (Tble 1) were diluted with sterile distilled wter (SDW) to give finl concentrtion of 1000 mg l -1 for ech component (Crter, 1968). Two milliliters of ech solution of ech extrct ws evenly dispersed on PDA in the pproprite Petri dishes. Control dishes received 2 ml of SDW ech. Pltes were left overnight for the solutions to be bsorbed through the medium. A 6-mm-dimeter plug of inoculum tken from the ctively growing mrgin of colony of ech isolte ws plced in the centre of ech Petri dish with the mycelium fce down. Ech isolte for ech voltile component ws inoculted on five dishes nd incubted t 22 C for 8 dys. Five control dishes were run long with ech fungl isolte nd voltile frction nd tested in the sme wy. Rdil growth ws mrked every dy, strting two dys fter incubtion, for 7 9 dys or until the dishes were overgrown. The percent growth inhibition cused by ech voltile component ws clculted s follows: [% inhibition = growth in con- 308 Phytopthologi Mediterrne

3 Antifungl ctivity of compounds from four plnts growing in Tunisi trol - growth in smple/growth in control] 100, where growth ws expressed s colony dimeter in mm (Douk et l., 1995). The percent inhibition vlues were the mens of five replictions. Pooled verge percent inhibition vlues nd stndrd errors were lso clculted. Results nd discussion Voltile components extrcted from the leves, stems nd flowers of the four plnts tested in this work hd ntifungl effects on the mjority of fungi under study. In generl, extrcts from flowers exhibited stronger ntifungl ctivity (pooled verge from 38 to 41.2%) thn extrcts from the stems (pooled verge between 23.1 nd 29.8%) or the leves (15.7 to 27.1%). Voltile components extrcted from the flowers of L. cmr hd the strongest ntifungl effect (38%), followed by extrcts from the leves (27.1%) nd stems (26.6%) (Tble 3). This ntifungl ctivity could be due to the presence of some terpenic components such s sbinene, β- cryophyllene, 1,8-cineole, bicyclogemcrene nd α-humilene, which re the min constituents of the oils (Sefidkon, 2002). Tble 1. Plnt prts, dry weight, weights nd yield of the extrcts of the four plnts used in this study. Plnt prt nd species Dry weight (g) Weight of voltile frction (mg) Yield (%) Lntn cmr Leves Stems Flowers Hibiscus ros-sinensis cv. red flower Leves Stems Flowers Hibiscus ros-sinensis cv. white flower Leves Stems Flowers Mlvviscus rboreus Leves Stems Flowers Tble 2. Fungl isoltes used to test the ntifungl ctivity of extrct plnts. Fungus Source Plnt-prt smpled Loction Collection dte Alternri solni Tomto Leves Chott mriem 12/11/2002 Botrytis cinere Vine Fruit Bou Argoub 11/05/2000 Fusrium oxysporum f. sp. niveum Wtermelon Roots Skhir 23/05/2001 F. solni f. sp. cucurbite Wtermelon Roots Skhir 23/05/2001 Pythium ultimum Wtermelon Roots nd stem Regueb 23/05/2001 Rhizoctoni solni Wtermelon Roots nd stem Jebenien 05/07/2001 Verticillium dhlie Potto Roots Boufich 03/11/1999 Vol. 44, No. 3, December,

4 N. Boughlleb et l. Hibiscus ros-sinensis cv. red flower nd white flower showed respectively the highest pooled inhibition verge for voltile component extrcts from flowers (38.8 nd 41.2%), with lower vlues for extrcts from stems (29.8 nd 25.4%) nd leves (18.4 nd 19.9%) (Tbles 4 nd 5). The present study is the first to exmine the chemicl composition of the voltile components of the different orgns of this plnt. However, essentil oil of the leves of H. cnnbinus L. hs been investigted: it hd significnt fungitoxic ctivity, probbly due to its mjor constituents such s (Z) nd (E)-phytol, n-nonnl, benzene cetldehyde, 2-hexenl nd 5-methylfurfurl (Kobisy et l. 2001). Tble 3. Inhibition (%) of fungl growth with different prts of Lntn cmr. Alternri solni 28.0± ± ± Botrytis cinere 31.3± ± ± Fusrium oxysporum f.sp. niveum 27.5± ± ± F. solni f.sp. cucurbite 16.0± ± ± Pythium ultimum Rhizoctoni solni 23.0± ± ± Verticillium dhlie 64.0± Pooled verge Inhibition percent is the men ± stndrd error of five determintions per fungus nd per voltile component. Tble 4. Inhibition (%) of fungl growth with different prts of Hibiscus ros-sinensis cv. red flower. Alternri solni 37.0± ± ± Botrytis cinere 28.3± ± ± Fusrium oxysporum f. sp. niveum 24.6± ± ± F. solni f. sp. cucurbite 15.4± ± ± Pythium ultimum Rhizoctoni solni ± ± Verticillium dhlie 23.4± ± Pooled verge See Tble 3. Tble 5. Inhibition (%) of fungl growth with different prts of Hibiscus ros-sinensis cv. white flower. Alternri solni 28.0± ± ± Botrytis cinere 35.0± ± ± Fusrium oxysporum f. sp. niveum 30.8± ± ± F. solni f. sp. cucurbite 25.0± ± ± Pythium ultimum ± Rhizoctoni solni 23.5± ± ± Verticillium dhlie ± Pooled verge See Tble Phytopthologi Mediterrne

5 Antifungl ctivity of compounds from four plnts growing in Tunisi Tble 6. Inhibition (%) of fungl growth with different prts of Mlvviscus rboreus. Alternri solni 35.2± ± ± Botrytis cinere 25.0± ± Fusrium oxysporum f. sp. niveum 31.0± ± ± F. solni f. sp. cucurbite 11.0± ± ± Pythium ultimum Rhizoctoni solni 23.0± ± Verticillium dhlie 15.8± Pooled verge See Tble 3. As regrds Mlvviscus rboreus, the pooled verge of voltile components extrcted from the stems of this plnt ws more strongly ntifungl thn the extrct from the leves. The extrct from the flowers ws lest effective (pooled verge 7.9%) (Tble 6). The metbolites responsible for such ctivity re not known t present becuse the chemicl composition of the voltile extrcts of the different orgns of this plnt ws not studied before. However, certin ftty cids hve been identified by GC- MS nd these cids could be ingredients of the voltile frctions mentioned bove (Crblleir nd Cruz, 1997). Complete inhibition of V. dhlie ws chieved with stem nd flower components extrcted from L. cmr nd with flower components from H. ros-sinensis cv. red flower nd white flower. A high level of inhibition (75.3%) ginst V. dhlie ws lso obtined with the voltile component from the stems of H. ros-sinensis cv. red flower, nd with leves of Lntn cmr (64.0%). The voltile component extrcted from M. rboreus leves exhibited low inhibition percentge (15.8%), while the components from the stems nd flowers were ineffective ginst V. dhlie. The lowest percent inhibition overll ws chieved ginst Pythium ultimum (Tbles 3, 4, 5 nd 6). This fungus ws not inhibited by the leves, stems or flower extrcts of ny of the four plnts tested, with the exception of flower extrct from H. ros-sinensis cv. white flower, which hd low inhibitory effect (11.4%). Fusrium solni f. sp. cucurbite ws strongly inhibited by voltile components from the flowers of L. cmr (26.3%) nd Mlvviscus rboreus (30.2%), by the stem components of H. ros-sinensis cv. white flower (28%) nd of H. ros-sinensis cv. red flower (26.5%) (Tbles 3, 4, 5 nd 6). The strongest inhibition of F. oxysporum f. sp. niveum ws obtined with flower components extrcted from L. cmr (32.4%) nd Mlvviscus rboreus (31%), nd with stem components of H. ros-sinensis cv. white flower (56.6%) nd H. rossinensis cv. red flower (32.4%) (Tbles 3, 4, 5 nd 6). Botrytis cinere ws strongly inhibited by ll extrcts (44%) except flower extrct from H. rossinensis cv. white flower. Alternri solni nd R. solni were modertely inhibited by ll extrcts, with inhibition vrying from 26 to 39.33% (Tbles 3, 4, 5 nd 6). The results of the study show tht L. cmr, H. ros-sinensis nd M. rboreus my be promising sources of ntimicrobil compounds to be used s lterntive pesticides in the control of plnt diseses. Literture cited Alitonou G., F. Alvessi, I. Bocoss, E. Ahoussi, J. Dngou nd D.C.K. Sohounhloué, Composition chimique et ctivités biologiques de l huile essentielles de Lntn cmr Linn. Comptes Rendus Chimie 7, Al-Mughrbi K.I., Antimicrobil ctivity of extrcts from leves, stems nd flowers of Euphorbi mcrocld ginst plnt pthogenic fungi. Phytopthologi Mediterrne 42, Al-Mughrbi K.I., T.A. Abuji, G.H. Anfok nd W. Shhrour, Antifungl ctivity of olivecke extrcts. Phytopthogi Mediterrne 40, Crblleir N.M. nd C. Cruz, ,9-Nondecdienoic cids in Mlvviscus rboreus nd Allmnd cthrtic. Phytochemistry 49(5), Vol. 44, No. 3, December,

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