www.elsevier.de/micresAntifungalactivityofTagetespatulaextractsonsomephytopathogenicfungi:ultrastructuralevidenceonPythiumultimumD.Maresa,*,B.Tosia,F.Polib,E.Andreottia,C.RomagnolicaDepartmentofNaturalandCulturalResources,UniversityofFerrara,C.soPortaMare2,I-44100Ferrara,ItalybDepartmentofEvolutionaryandExperimentalBiology,UniversityofBologna,viaIrnerio42,I-40126Bologna,ItalycDepartmentoftheMuseumofPaleobiologyandoftheBotanicalGarden,UniversityofModenaandReggioEmilia,V.leCadutiinGuerra127,I-41100Modena,ItalyReceived10June2004AbstractMethanol extract, obtainedfromTagetes patulaplant, was assayedagainst threephytopathogenic fungi: Botrytis cinerea, Fusarium moniliforme and Pythiumultimum.Theantifungalactivitywastestedbothinthedarkandinthelight,usingtwodifferentlightingsystems.Thedatashowedthattheextractprovedtohaveadose-dependent activity on all the fungi with a marked difference betweentreatments inthelight thaninthedark. Goodgrowthinhibitionwas observedinfungi only when these were treated with the highest dose of the extract andirradiated, whereas the same dose gave only a modest inhibition when the experimentwasconductedinthedark. At5and10 mg/ml inthedark, growthincreased. Theresults indicatedthat thepresenceof aluminous sourceenhances theantifungalactivity, with small differences between UV-A and solar spectrum light. SEM and TEMobservationsonPythiumultimumrevealedthattheTagetespatulaextractinducedalterations oncell fungal membranes with a photoactivationmechanismpossiblyinvolvingtheproductionof freeradicals andleadingtoaprematureagingof themycelium.& 2004ElsevierGmbH.Allrightsreserved.IntroductionDevelopment of newandnovel antifungal agents(natural or synthetic) can be useful in the control ofinfections causedbyphytopathogenicfungi. Withthis aim, screening is oriented more and moretowardsmoleculesthatpossessaselectiveactionagainstthesefungi, withouteitherbeingtoxictoARTICLEINPRESSKEYWORDSThiophenes;Tagetespatula;Antifungalactivity;Phytopathogenicfungi*Correspondingauthor.E-mailaddress:mrd@dns.unife.it(D.Mares).0944-5013/$ - seefrontmatter& 2004ElsevierGmbH. Allrightsreserved.doi:10.1016/j.micres.2004.06.001MicrobiologicalResearch159(2004)295304manortoecosystem.Thediscoveryofphotosensi-tizers(moleculeswhichexpressfulltoxicityinthepresenceofirradiationonly)andtheirpresenceinnumerous plant families, presents the possibility toexamine the biological and ecological roles of theseplants, with the further possibility of isolating newactive molecules. This type of phenomenon hasoccurred, for example, withsomefuranocoumar-ins, currentlybeingusedinthephototherapeutictreatment of psoriasis (Simonet al., 2000), or inthe agriculture eld, where natural pesticidescouldassumeanimportant rolecombatingpestsanddiseases, maintaining, atthesametime, theecological balance (de Matos and Pinto Ricardo,2003).Theobjectiveofthispaperistoverify,invitro,theantifungal activity of theextract of Tagetespatula, a dicotyledon belonging to the family of theAsteraceae, on some phytopathogenic fungi. T.patulaaccumulatesinitstissueshighamountsofthiophenes,wellknownfortheirphototoxicactiv-ity towards bacteria, nematodes (Hudson andTowers, 1991) and also fungi (Romagnoli et al.,1994;Maresetal.,2002).Inthis paper, theactivity of theextract of T.patula was tested both in the dark and in the light,inorder toevaluatetherolecarriedout bytheirradiation in the process of the photodynamicactivationoftheextract.Inadditionweusedtwodifferent lighting systems: UV-A and Solar lightspectrumlamps.Thesameexperimentswerealsorepeatedusingtwopurestandardsnamelyalpha-terthienyl (a-T) and5-(4-hydroxy-1-butinyl)-2,20-bithienyl (BBTOH), also present in the extract. Thephytopatogenicfungi usedas tests wereBotrytiscinerea, FusariummoniliformeandPythiumulti-mum.Finally, scanningandtransmissionelectronmi-croscopywas performedinorder toevaluatetheeffect of extracts of T. patula on the ultrastructuralmorphology of the fungusPythium ultimum, whichactually proved to be the most sensitive. Thus, withthe following series of experiments, we attemptedto nd out if a possible commercial use of thisproductinagriculturecouldexistsinsteadofusingsyntheticpesticides.MaterialsandmethodsPlantmaterialandextractionTheextractusedintheexperimentswasobtainedfromTagetespatulaL.growinginanexperimentaleld owned by Mazzoni vivai in Tresigallo(Ferrara, Italy). The methanol extraction wasperformedasreportedbyTosietal.(1991).TestorganismsThethreephytopathogenicfungi usedduringthegrowth experiments were as follows: Botrytiscinerea Person, strain no. 48339, Pythium ultimumTrow, strainno. 58812andFusariummoniliformeSheldon, strainno. 36541, suppliedbytheAmer-ican Type Culture Collection (ATCC, USA). Thesamples of myceliumnecessary for the in vitroexperiments, weretakenfromcultures growninslantsand kept at 26721C on PotatoDextrose Agar(PDA,Difco).MediapreparationwithextractsofT: patulaThe culture media for the treatments wereprepared by adding different volumes of a solutionoftheextractofT.patulaindimethylsulphoxyde(DMSO) to each Petri plate (each still containing thePDA uid), with the aimof obtaining the nalconcentrationsof5,10,50 mg/ml.Purestandardsa-T andBBTOHweredissolvedinDMSOandafter-wardsaddedtothePetri platescontainingPDAinorder toobtainthenal concentrationsof 5, 10,50 mg/ml. The DMSO concentration in the nalsolutionwas0.1%. Equivalentquantities(0.1%) ofthesolvent(DMSO)wereaddedtothecontrols.For each of the three fungi, two differentexperiments werepreparedfor thevarious typesof luminous sources used during the phase ofirradiation. Threereplicates wereusedfor eachconcentrationandall experimentswererepeatedtwice.SolarspectrumlightForeachfungustwoseriesofplatestreatedwithvariousdoseswereprepared. Therstserieswaspreparedforirradiationwithsolarspectrumlight,the second was designed for incubation in darkconditions.Intheplates designedfor light treatment, theinoculumwasplaceddirectlyonthemedium.Thesampleswerethenexposedtodailyirradiationfor16 h by means of the solar spectrum light, using twolampsBiolux(OsramL36W/72,temperatureheat6500 K, owtotal 1600 lumen/m2to 1 mof dis-tance),keptataconstantdistanceof50 cmfromthe Petri plates. Some samples treated with 50 mg/ml of extract were exposed to irradiation with solarspectrum light for 90 min, for TEM observation. TheARTICLEINPRESS296 D.Maresetal.second series was placed in dark conditions at26721C.Every 24 h the diameter of fungal colony ontreated and control plates was measured. The totaldurationoftheexperimentwas5days.UV-AlightTwoseriesofplatespreparedinexactlythesameway as those for the rst experiment werepreparedforeachfungus.Thefungifromtherstseries were kept in contact with the treatedmediumfor 24 hinthedark, at 26721C. Subse-quentlythesampleswereirradiatedwithUV-Afor90 min, usingauorescentlamp(blacklightblueuorescentlamp,Sylvania,F20,T12-BLB)withalightintensityof0.5 mW/cm2,keptataconstantdistanceof10 cmfromthesamples.Whenthephaseof irradiationwas completed,theplateswereplacedagaininthedark.Inthiscasealso,thediameteroffungal colonywas measuredevery24 hfor 5days. Thesecondseries on the other hand, after the inoculum of thefungus, wasplacedindarkconditionsat26721C,for5days.ExperimentwiththestandardsThethreefungi weretreatedwitha-T andBBTOHstandard following the same methods used with theextracts.Allexperimentswerepreparedindoublequantity,andtwicerepeated.ElectronmicroscopyFourexperimentalserieswereprepared:eachonewas composed of one control sample and onesampletreatedwith50 mg/ml of extract. For therstandthesecondseriesasampletreatedwith10 mg/ml wasalsoprepared,andeveryserieswasexposed to a different kind of treatment, asfollows:Firstseries:control,treatedwith10and50 mg/ml:keptat26721Cinthedarkfor16 h.Second series: control, treated with 10 and50 mg/ml: irradiation with solar spectrum light(Biolux)for16 h.Third series: control, treated with 50 mg/ml:irradiationwithUV-Afor90 min.Fourth series: control, treated with 50 mg/ml:irradiation with solar spectrumlight (Biolux) for90 min and successive transfer to the dark at26721C.For the irradiation of the colonies, the samemodalities and the same materials used in thegrowthexperimentswerealsoused.Attheendofthisphasetheprocedureofcollectionandsamplepreparation for observation by the TEM and the SEMwasstarted.Transmissionelectronmicroscopy(TEM)andscanningelectronmicroscopy(SEM)The youngest P. ultimum hyphae were chosen fromthe margin of the mycelia; the samples wereroutinely xed with 6% glutaraldehyde (GA) in0.1 Msodiumcacodylatebuffer,pH7.2,for3 hat41C.Afterhavingbeenrinsedinthesamebuffer,thefungi whichweretoundergoTEMwerepost-xedfor 20 h at 41C in 1% OsO4 in the same buffer.Theywerethendehydratedinagradedseries ofethanol solutions andembeddedinEpon-Aralditeresin.SectionswerecutwithanLKBUltratomeIII,stainedwithuranyl acetateandleadcitrate,andobservedwithaHitachiH-800,at100 kV.For SEM the fungi were xed in 6% GA in a sodiumcacodylate buffer, briey post-xed for 1 h at 41C in1% OsO4 in the same buffer and then dehydrated inacetone,critical pointdriedandgoldcoatedwithanS150Sputter coater (Edwards). SEMobserva-tions were performed with a Cambridge Stereoscan360scanningelectronmicroscopeatanaccelerat-ing voltage of 20 kV. Both microscopes are owned bytheElectronMicroscopyCenterofFerraraUniver-sity.ResultsBotrytis cinereaAll theinhibitiondatarelatedtothis fungus areshown in Table 1. The treatment of B. cinerea withtheextractofT.patulaandirradiationwithsolarlightshowedadose-dependentinhibitionofmyce-lial growth, that reached the highest value (39.3%)in colonies treated with the maximum dose (50 mg/ml). At the same dose, irradiation with UV-Aenhancedtheactionof theextract (57.4%) com-pared to irradiation with solar light. By comparison,the experimental series kept in the dark andtreatedwith50 mg/ml showedalower inhibition(24.8%).Attheinferior doses,thesamplestreatedwith the extract and irradiated with UV-A or kept inthedark,weregrownevenmorethanthecontrol.Also, the responses of the two standards were verydifferent if the fungi were kept in the dark orirradiated with UV-A. In fact, while a-Tshowed verylow inhibition values at the three concentrations inthese conditions, BBTOH showed higher values,ARTICLEINPRESSAntifungalactivityofTagetespatulaextracts 297thatreached the maximum of inhibition(85.3%)atthehighestdose,afterirradiationwithUV-A.FusariummoniliformeThe inhibition data on this phytopathogen arereportedinTable2. Therst experiment, madeusingtheextractofT.patulaincombinationwithsolar light, showed a marked inhibitory dose-dependent effect. The range of values startedfrom 21.4% inhibition, for that treated with 5mg/mlreaching 50.9% for that treated with 50 mg/ml. Theexposure to the UV-A lead to similar results, despitethe fact that in the complex, the results wereactuallylowerthanthoseobservedintheexperi-mentdoneusingthesolarspectrumlight.On the other hand, in dark conditions, thosetreated with 5 and 10 mg/ml showed a growthincrease, a phenomenon already observed on B.cinerea, whereas at the highest dose (50 mg/ml) theinhibitionwas33.8%.Among the standards used, the most active on F.moniliforme,aftertheirradiationwithsolarlight,was BBTOH, whichinducedthehighest inhibitionvalueonthosetreatedwith50 mg/ml(75.4%).Thisvalue was very similar to that obtained afterirradiation with UV-A (73.2%). The samples treatedwith the lowest concentrations and kept in the darkorexposedtosolarlightshowedsimilarvaluesofinhibition, while those irradiated with UV-A showedhighervalues,asshowninTable2.Evenifa-T, onthewhole,waslessactivethanBBTOH, it showed the highest inhibition values onlyafterirradiation.PythiumultimumThe experiments on P. ultimum with the extract ofT.patulabothwiththeexposuretothesolarlightARTICLEINPRESSTable1. Percentageinhibitionrateafter5daysoftreatmentonB.cinereaExtract a-T BBTOHDark Controls 0 0 05 mg/ml 16.770.5 7.171.4 15.77210 mg/ml 15.670.9 8.171.4 36.071.550 mg/ml 24.871.2 12.471.6 67.871.7Solarspectrumlight Controls 0 0 05 mg/ml 11.871.8 3.071.3 5.371.510 mg/ml 18.672.8 16.671.6 13.371.750 mg/ml 39.371.7 73.471.8 78.671.1UV-Alight Controls 0 0 05 mg/ml 1.572.6 9.471.3 56.672.110 mg/ml 4.072.2 10.471.8 67.071.450 mg/ml 57.472.0 14.470.9 85.370.5Alldatashownareaveragesandstandarderrorsfromthreedeterminationsoftwoindependentexperiments.Table2. Percentageinhibitionrateafter5daysoftreatmentonF.moniliformeExtract a-T BBTOHDark Controls 0 0 05 mg/ml 23.271.1 3.770.7 27.771.710 mg/ml 2.070.2 4.271.0 33.970.950 mg/ml 33.871.3 5.471.7 64.171.2Solarspectrumlight Controls 0 0 05 mg/ml 21.471.6 14.871.3 18.171.210 mg/ml 37.071.2 16.171.3 34.071.350 mg/ml 50.971.7 43.771.7 75.470.6UV-Alight Controls 0 0 05 mg/ml 6.771.3 29.871.1 44.771.510 mg/ml 13.570.8 32.071.6 51.870.750 mg/ml 47.371.0 36.271.4 73.271.1Alldatashownareaveragesandstandarderrorsfromthreedeterminationsoftwoindependentexperiments.298 D.Maresetal.and the UV-A exposure (Table 3) showed apercentageinhibitionhigherthanthatfoundonB.cinereaandF.moniliforme.Alsothesamplestreatedwiththehighestdose(50 mg/ml) and kept in the dark showed highpercentages of inhibition (51.4%), while thosetreated with the lower doses (510 mg/ml) grewmore than the controls, as already observed with B.cinereaandF.moniliforme.ThetreatmentofP. ultimumwiththe extractofT. patulaUV-A demonstrated a dose-dependentinhibition,withsmallervalueshowever,comparedto those observed after exposure to the solar light.Following treatment with both the standards thedatashowedaninhibitoryactivityofBBTOHinalltheexperimentalconditions(dark,solarlight,UV-A) higher thanthat of a-T. Thehighest inhibitionvalues were obtained however, after irradiationwithUV-A.P. ultimumresulted the most sensitive of thethreetestedfungi andforthisreasonthisfungustreatedwiththeextractswaschosenforscanningand transmission electron microscopy observations.PythiumultimumSEMobservationsThe control (Fig. 1) showed the characteristicmorphology of P. ultimum, with lengthened hy-phae,ofconstantdiameter,sub-parallel andwithrounded or lightly tapering apex and smoothexternal surface. In the samples treated with10 mg/mloftheextract,andkeptinthedark(Fig.2), themorphologyreects that of thecontrols,withtheexceptionofthepresenceofundulationsalong the hyphal border. The irradiation with Biolux(Fig. 3) provokedrather, littleswellinglocalizedalong the hyphae and at their extremities, and theformationofanomalousapexbifurcations.In those treated with 50 mg/ml macroscopicmorphologic alterations areevident, bothinthesamples irradiated and in those kept in the dark. Inthe latter, the apexes showed an irregular growth indimension, with multiple ramications in subapicalexpandedareaswithirregularshape(Fig.4).Alsointhetreatedsamplesexposedtothesolarlight,severalswellingsofovoidalorspherical shapeareevident, localized in the subterminal position, nearthe apexes, or in the middle position along thehyphae(Fig.5).PythiumultimumTEMobservationsBoththecontrols kept inthedark (Fig. 6), andthoseirradiated,maintainedanormalmorphologywithmitochondria, nuclei, ribosomes andnumer-ous granules of glycogen. The plasmalemma,adherent with the cellular wall, and the innersystemof endomembrane, showedanormal mor-phology, indicating that they were not damaged bytheirradiation. Inhyphae treated withthehighestdoseoftheextract(50 mg/ml)aftergrowthinthedark, normal cells are present (Fig. 7) besidedeeply altered cells, in an advanced necroticcondition(Fig. 8). Intherst case(Fig. 7) therearenotanyevidentalterations:theorganulesareintegral and the endomembrane systemis welldeveloped. In the second case inner membranes aredecaying andthe organules are hardly recogniz-able,whilethecellwallseemstobeaffected.Theextendedirradiation(16 h)withsolarspec-trum light caused deep alterations in fungi treatedwiththesamedoseofextract(Fig.9).An80%ofthecellsappeartobeinastateofpartialortotalnecrosis,oftenwithplasmolysedcytoplasm.After shorter irradiation with solar spectrum light(90 min) the samples treated with 50 mg/ml ofARTICLEINPRESSTable3. Percentageinhibitionrateafter5daysoftreatmentonP.ultimumExtract a-T BBTOHDark Controls 0 0 05 mg/ml 21.370.8 17.871.0 20.471.310 mg/ml 28.171.3 22.071.5 32.771.150 mg/ml 51.471.1 36.871.3 58.771.5Solarspectrumlight Controls 0 0 05 mg/ml 22.771.6 22.971.2 28.070.710 mg/ml 40.271.3 44.171.0 46.670.550 mg/ml 72.671.2 66.571.0 68.370.5UV-Alight Controls 0 0 05 mg/ml 18.671.2 37.071.1 45.171.310 mg/ml 24.371.0 38.072.4 46.771.750 mg/ml 62.771.2 41.471.7 85.970.6Alldatashownareaveragesandstandarderrorsfromthreedeterminationsoftwoindependentexperiments.AntifungalactivityofTagetespatulaextracts 299ARTICLEINPRESSFigures 15.1. Control of P. ultimum, observedat SEM, showinglengthenedandsubparallel hyphaewithsmoothsurface and rounded apex. SEM; bar 5 mm. 2. Hyphae of P. ultimum, after treatment with 10 mg/ml of T. patula extractinthedark,maintainanalmostnormalmorphology.Theonlydifferenceisthatthemarginlooseslinearity,showingalternatediametralvariations.SEM;bar5 mm.3.HyphaofP.ultimumaftertreatmentwith10 mg/mlofextractandirradiatedwithBioluxfor16 h. Someanomaliessuchaslittleswellingsalongthehyphaandapexbifurcations arevisible.SEM;bar5 mm.4.InP.ultimum,aftertreatmentwiththeextractatthehighestdose(50 mg/ml)inthedark,apical swellings and multiple anomalous ramications are evident. SEM; bar 5 mm. 5. Effects of irradiation with Bioluxaftertreatmentwithextractat50 mg/ml. Thepresenceofseveral stressedswellingsandaroughsurfaceisworthnoting.SEM;bar5 mm.300 D.Maresetal.extract showed similar, but softer aspects (Fig. 10).The presence of alterations at cell wall level can benoticed,consistingofitsthickeningandmoderateprotrusionsinsidethecell cytoplasm.Theirradia-tion with the same light for 16 h induced damage tothe cells even in those treated with 10 mg/ml.Therewas damagetothemembranes, as canbeobservedinFig. 11, wherethenuclear envelopeappearstobepartiallydestroyed.The irradiation with UV-A for 90 min of thesamples treated with 50 mg/ml, determined thedeath of the cells (Fig. 12). In those treated,moreover, complete cellular disorganization isaccompaniedbydeepparietalalterations,formedby marked protrusions containing material incorpo-ratedinthewall.DiscussionThe extract of T. patula exercises a phototoxicdose-dependentactiononall threetestedphyto-patogens. Thecomparisonof theinhibitionaftertreatment with the extract, both in the presence ofirradiation and in the dark, shows a correlationbetween the toxicity and the process of photo-activation of the thiophenes present in the extract.Inthedark,theextractinhibitsthegrowthofthethree fungi only at the highest dose, equal to 50 mg/ml,whileatthelowerdosesof5and10 mg/mlanincrement of the mycelial growth comparedto thecontrolsisobserved.Thisbehaviourisnotunusualwhenfungi weretreatedwithcompoundsatsub-inhibitoryconcentrations.ItwasalreadyobservedwiththethiopheneBBTOHonthedermatophyteNannizia cajetani (Romagnoli et al., 1998), and alsowith the extracts of ten different cultivars ofTagetes onthephytopathogens B. cinereaandF.moniliforme(Maresetal.,2002).By comparing the results of UV-A and solar light itis evident that prolonged irradiation with solar lightover a period of 5 days (even at lowintensity)generallyprovokesgreaterdamageonthetreatedfungi thanthosecausedbyastronger irradiationwith UV-A for a short time. This higher activity afterARTICLEINPRESSFigures 68.6. Control hypha of P. ultimum observed at TEM. The regular disposition of organules and the integrity ofthe endomembrane system is evident. The nucleus, the plasmalemma, the mitochondria, glycogen granules and lipidicdrops are clearly visible. TEM; bar 1 mm. 78. P. ultimum treated with 50 mg/ml of extract and kept in the dark. Normalcellswithelectron-opaquevacuoles(7),anddeeplyalteredcells,withhardlyrecognizableorganules(8)arevisible.TEM;bar1 mm.AntifungalactivityofTagetespatulaextracts 301irradiation with the solar spectrumlight can beattributed also to the long light exposure thatfavoursthedryingofthemediumfurtherslowingdownthemycelialgrowth.P. ultimumhas beenshowntobe, among thethreephytopathogens, themost sensitivetothephotodynamicactionoftheextract,especiallyatthelower doses, as demonstratedbytheresultsobtained after irradiation with both the lightsources.Infactthegrowthobserved,inboththelightingconditions andat thesameextract con-centration, is generally lower than that of B.cinereaandF.moniliforme.The observation at SEMon the most sensitivefungusP.ultimum,conrmsthetendencynoticedinthegrowthexperiments, i.e. theentityof themorphological alterations correlates withextractconcentrationanddependsontheirradiation.Thefact that deepest alterationsareobservedintheirradiatedsamplessuggestsaninvolvementintheextract actionof thiophenes, that possess photo-toxicproperties (HudsonandTowers, 1991). Theobserved light-independent fungitoxic activity ofthe extract and the standards is probably due to thepresence of BBTOHwhich, in the dark, resultedmore active than a-T. The strong antifungal activityof BBTOH at high concentrations, also in theabsence of photoactivation, was also shown onother fungi, for example on dermatophytes (Ro-magnolietal.,1998).The ultrastructural examination of P. ultimumwith TEM allows the formulation of a hypothesis onARTICLEINPRESSFigures912.9. HyphaeofP.ultimumaftertreatmentwith50 mg/mlofextractandirradiatedwithBioluxfor16 h.Plasmolysed cells with irregular cell wall outline are clearly visible. TEM; bar 1 mm. 10. P. ultimum treated with 50 mg/ml of extract and irradiated with Biolux for 90 min. The cytoplasm is almost completely disorganized: only membranousfragment are recognizable.A marked parietal protrusionis also worth noting. TEM; bar 1 mm. 11. The treatmentwithextractat10 mg/mlandwithsolarspectrumlightfor16 h,causesonlypartialdamagetothemembranes.Anucleus,with a little damaged envelope, is hardly recognizable. TEM; bar 1 mm. 12. Treated sample with 50 mg/ml of extract andirradiated with UV-A for 90 min. After UV-A irradiation, the extract determined the complete cellular degeneration. Thecellwallshowsnumerousanddeepprotrusions.TEM;bar1 mm.302 D.Maresetal.the mechanism of the phototoxicity of the extract,ahypothesissupportedbytheanalogywithresultsobtainedinprevious studies (Mares et al., 1990)thatindicatethemaintargetofthiophenesintheendomembrane system, and the cause of thestructural alterations of the cells in the photactiva-tionmechanism. Infact, insamples treatedwith50 mg/ml and kept in the dark, necrotic cells can beobserved alongside normal cells with electron-opaquebodies withgranular aspect, encircledbyone single membrane, indicating that they arevacuoles.Thepresenceofanalogousstructures,isalready documented for the dermatophyte N.cajetani, after treatmentinthedarkwith10 mg/ml of BBTOH (Romagnoli et al., 1998) and forMicrosporumcookei,treatedwitha-T andkeptinthedark, (Mares et al., 1990). Inthelatter, theexistenceofa-Tinsidevacuoleshasbeendemon-strated with the optical microscope in uorescenceas these vacuoles, irradiated with the correct UV-Alight (350 nm), emitted a characteristic blueuorescence (Zechmeister and Sease, 1947). Itappearsthereforereasonabletoassertthat,aftertreatment with the extract of T. patula, thethiophenes present are segregated inside thevacuoles, where they remain inactive in darkconditions.On the contrary, the same treated samples, afterirradiationwithUV-Aor withsolar light, demon-strated the presence of plasmolysed or at leaststronglyalteredcells, inwhichtheentireproto-plasm appears to be in an advanced necrotic state,allowing only the recognition of fragments ofmembrane, belonging to the decaying organulesinside.The appearance of such alterations only afterirradiation, indicates that the toxicity of theextract of T. patulais determinedbythephoto-induced activation of the thiophenes in its content.Evenifthemechanismwithwhichthethiophenesact on the cellular structures has not beencompletelyclaried, itispossibletorefertothehypotheses formulatedbyseveral authors onthemechanismof action of the a-T (Towers, 1984;Hudson et al., 1986; Mares et al., 1990, 1994;Romagnolietal.,1994)andoftheBBTOH(Romag-noli et al., 1998). Inthese studies, the damageobserved on membranes, indicates that the a-Tacts, in the presence of UV-A, with a photodynamicmechanismthat involves theformationof singletoxygenor of freeradicals (Hudsonet al., 1986).This highly reactive chemical species, could cause,rstly the breaking of the tonoplast of vacuoleswhere the thiophenes are contained with theconsequence, secondly, of the diffusion of lyticenzymes which cause nally the damage of theentire endomembrane systemof the fungal cell(Maresetal.,1990).The specic interaction with membrane proteinshas been suggested by other authors (Hudson et al.,1986), that attribute the fast degradation ofnuclear and mitochondrial membranes to theoxidationof aminoacids, such ashistidine,trypto-phan and methionine, and to the oxidation ofunsaturated compounds such as fatty acids presentinthelipidsofmembranes.Theanomalous swellingsintheapical or inter-calary position, observed at the SEM on P. ultimumtreatedwiththeextract, arenoteasilyinterpre-table: they can be considered as simply cellswellings or oogonia formations or beginning ofsporangia. These latter survival structures alsoappear in normal conditions without treatment,whenP.ultimumndsitselfinadverseconditions,such as lack of food (Stanghellini and Hancock,1971).It can therefore be concluded that the extract ofT. patula possesses a fungitoxic activity dose-dependent in presence of irradiation, indepen-dentlyfromthekindofluminoussource,succeed-ingtoinhibitthegrowthofthethreetestedfungialreadyatverylowdoses.Thatcanbecorrelatedtothepresenceof thiophenes intheextract, inparticulara-T andBBTOHthatseemtoactonthefunguswithamechanismofphotoactivationinvol-ving, most likely, theformationof freeradicals,causing deep damage at the level of the endomem-brane system and production of sporangia assurvivalstructures.AcknowledgementsInvestigations supportedbygrantsfromMazzoniVivai Tresigallo, FE (Italy) and the MinisteroIstruzioneUniversit"aeRicerca(MIUR).Special thanks are due to the staff of theElectronMicroscopy Center of Ferrara Universityfor their skillful work and to Orna Crinion for adviceonlanguagetranslation.ReferencesDeMatos,O.,PintoRicardo,C.P.,2003.Plantscreeningfor light-activated antifungal activity. In: Rai, M.,Mares, D. (Eds.), Plant-Derived Antimycotics. FoodProducts Press, an Imprint of The Haworth Press, Inc.,NewYork,London,Oxford,pp.229255.Hudson,J.B.,Towers,G.H.N.,1991.Therapeuticpoten-tialofplantphotosensitizers.Pharmacol.Therapeut.49,181222.ARTICLEINPRESSAntifungalactivityofTagetespatulaextracts 303Hudson, J.B., Graham, E.A., Chan, G.C., Finlayson, A.J.,Towers, G.H.N., 1986. Comparison of the antiviraleffects of naturally occurring thiophenes andpoly-acetylenes.PlantaMed.6,453458.Mares, D., Fasulo, M.P., Bruni, A., 1990. Ultraviolet-mediated antimycotic activity of a-terthienyl onMicrosporum cookei. J. Med. Vet. Mycol. 28, 469477.Mares, D., Romagnoli, C., Rossi, R., Carpita, A., Ciofalo, M.,Bruni,A.,1994.Antifungal activityofsome2,20:50,500-tertiophene derivatives. Mycoses 37, 377383.Mares, D., Tosi, B., Romagnoli, C., Poli, F., 2002.Antifungal activity of Tagetes patula extracts. Pharm.Biol.40,400404.Romagnoli,C.,Mares,D.,Fasulo,M.P.,Bruni,A.,1994.Antifungal effects of a-terthienyl from Tagetes patulaonvedermatophytes.Phytother.Res.8,332336.Romagnoli, C., Mares, D., Sacchetti, G., Bruni, A., 1998.Thephotodynamiceffect of 5-(4-hydroxy-1-butinyl)-2,20-bithienyl on dermatophytes. Mycol. Res. 102,15191524.Simon, J.C., Peger, D., Schopf, E., 2000. Recentadvances in phototherapy. Eur. J. Dermatol. 10,642645.Stanghellini,M.E.,Hancock, J.G.,1971.The sporangiumof Pythiumultimumas asurvival structureinsoil.Phytopathology61,157164.Tosi, B., Bonora, A., DallOlio, G., Bruni, A., 1991.Screeningfortoxicthiophenecompoundsfromcrudedrugs of the family Compositae used in Northern Italy.Phytother.Res.5,5962.Towers, G.H.N., 1984. Interactionof light withphyto-chemicalsinsomenatural andnovel system.Can.J.Bot.62,29002911.Zechmeister,L.Z.,Sease,J.W.,1947.Ablue-uorescingcompound, a-terthienyl, isolated from Marigolds.J.Am.Chem.Soc.69,273275.ARTICLEINPRESS304 D.Maresetal.