In Vitro and in Planta Activity of Some Essential Oils against Venturia inaequalis (Cooke) G. Winter

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1 Ngy ville G. et l. / online: Not Bot Horti grobo, 2014, 42(1): Print ISSN X; Electronic Not Bot Horti grobo, 2014, 42(1): In Vitro nd in Plnt ctivity of Some Essentil Oils ginst Venturi inequlis (Cooke) G. Winter Géz NGY 1 *, Tmás HOCHBUM 1, Szilvi SÁROSI 2, Márt LDÁNYI 3 1 Corvinus University of Budpest, Fculty of Horticulturl Sciences, Deprtment of Plnt Pthology, 44. Ménesi Str., H-1118 Budpest, Hungry; gez.ngy@uni-corvinus.hu (*corresponding uthor); HochbumT@nebih.gov.hu 2 Corvinus University of Budpest, Fculty of Horticulturl Sciences, Deprtment of Medicinl nd romtic Plnts, Villányi Str., H-1118 Budpest, Hungry; szilvi.srosi@uni-corvinus.hu 3 Corvinus University of Budpest, Fculty of Horticulturl Sciences, Deprtment of Biometrics nd griculturl Informtics, Villányi Str., H-1118 Budpest, Hungry; mrt.ldnyi@uni-corvinus.hu bstrct The effect of the essentil oils of thyme (Thymus vulgris), cinnmon (Cinnmomum verum) nd sweet ornge (Citrus sinensis) on conidil germintion of Venturi inequlis ws investigted in vitro. Moreover, the effect of cinnmon oil ws further studied by nlyzing its preventive nd curtive ctivity s well s its rinfstness in n pple seedling-venturi pthosystem under growth chmber nd greenhouse conditions. The essentil oils suppressed the germintion of conidi t concentrtions of 0.01% nd 0.1% effectively. The highest inhibition level ws chieved with thyme oil. Cinnmon oil (0.2%) with Silwet Str djuvnt (0.02%) ws protective nd curtive ginst Venturi inequlis when pplied 72 h, 24 h, 1 h before or 24 h, 72 h fter inocultion of plnts. Effective control ws chieved when essentil oil ws pplied by 24 h, 1 h or 24 h pre- or post-inocultion, respectively. The 72 h ppliction ws less effective, however, it still showed decent control level. lthough the simulted rinfll of light (1 mm), moderte (3 mm) nd hevy (5 mm) rin decresed the control efficcy of cinnmon oil, the wter mounts did not influence control level. The oil ppliction resulted in moderte nd long lsting control (92% efficcy without rinfll, 65% efficcy with rinfll). The results of this study show tht the tested essentil oils re pproprite cndidtes for the development of biofungicides ginst pple sc. Keywords: pple sc, conidi, essentil oils, preventive nd curtive ctivity, rinfstness Introduction pple sc (Venturi inequlis (Cooke) G. Winter) is widespred nd common disese round the world wherever pples re grown (Villncourt nd Hrtmn, 2000). The pthogen cuses significnt loss in the Hungrin pple production. In epidemic yers, the number of fungicide pplictions ginst the disese often exceeds twenty spryings in vegettion period. In recent yers, the use of fungicides ginst pple sc is strictly regulted by EU legisltion (eg. Directive 2009/128/EC, Commission regultions No 834/2013, No 1004/2013, No 1138/2013, No 61/2014). In orgnic production the use of copper fungicides is limited due to soil ccumultion s well (Commission Regultion No. 473/2002, Council Regultion (EEC) No. 2092/91). Therefore, the reserch of lterntive solutions to synthetic or copper bsed fungicides is prticulrly needed due to the potentil risks of the environment pollution nd of fungicide resistnce. Essentil oils of medicinl nd romtic plnts re suitle cndidtes for this purpose. Their ntimicrobil nd ntifungl ctivity hs been known for centuries nd it is still n intensively studied field. Since essentil oils contin severl chemicl compounds, the inhibition is bsed on different biologicl modes of ction. The primry trget of essentil oils is the fungl plsm membrne. ctive compounds cuse lesions nd reduce ergosterol biosynthesis resulting in severe morphologicl chnges of the hyphl wll (Pinto et l., 2006; Soylu et l., 2010; Tin et l., 2012). lthough there re numerous dt ville on the reserch of the effect of plnt extrcts nd essentil oils on plnt pthogens, fewer results cn be found out the effect on Venturi species. The plnt extrcts from Citrus sp., Gleg officinlis, Inul viscos, Quillj sponri, Polypodium vulgre nd Sponri officinlis inhibited conidil germintion of Venturi inequlis (Cooke) G. Winter effectively nd gve good control of pple sc on seedlings in greenhouse studies, however, simulted rinfll decresed the efficcy significntly (Pfeiffer, 2002; Pfeiffer et l., 2004). Plnt extrcts contining high contents of ctive ingredients of rtemisinin (rtemisi nnu), chelidonine (Chelidonium mjus), menthol (Menth piperit), populin (Populus nigr), linlool + linlyl cette (Slvi sclre) nd thymol (Thymus vulgris) were tested ginst the scospore nd conidi germinton of Venturi inequlis (Cooke) G. Winter. lmost ll extrcts showed high inhibition of scospore germintion. rtemisinin, thymol nd populin

2 Ngy G. et l. / Not Bot Horti grobo, 2014, 42(1): extrcts effectively inhibited conidil germintion s well. The 1% Populus extrct gve similr control efficcy ginst pple sc on pple trees s synthetic fungicides in 2 yers field study (Bálint et l., 2014; Theisz et l., 2007). The preventive ppliction of six plnt oils (cnol, corn, grpeseed, olive, soyben nd sunflower) reduced sc infection on pple leves significntly under controlled condition, however, orchrd ppliction of cnol nd soyben oil with emulsifier did not give better control level thn tht of the ppliction of emulsifier lone (Northover nd Schneider, 1993). The essentil oils of Phellodendron chinense, Russowi sogdin nd Znthoxylum bungenum inhibited in vitro the mycelil growth of Venturi pirin nd other fungi effectively. The wide ntifungl ctivity could be ttributed to the high contents of monoterpene compounds (Gong et l., 2009, 2011; Tn et l., 2007). Berngtsson et l. (2006, 2009) compred the effect of plnt extrcts nd the resistnce inducer cibenzolr-s-methyl on conidil germintion with their effect on sc infection on pple seedlings. Severl extrcts inhibited effectively conidil germintion, however, showed no or moderte in vivo efficcy. They pointed out tht besides the direct fungisttic or fungicide effect of the plnt extrcts, induction of plnt resistnce might lso ply n importnt role in their mode of ction. lthough the effect of plnt extrcts on Venturi inequlis (Cooke) G. Winter hs been ctively investigted, the effect of essentil oils is little-known. This is prticulrly true to their in vivo mode of ction, durtion of efficcy nd rinfstness, which ltter highly influence the control efficcy in outdoor conditions. The effect of the essentil oils of thyme (Thymus vulgris) nd cinnmon (Cinnmomum verum) in different plnt pthogen reltionships hs been investigted in the Deprtment of Plnt Pthology, Corvinus University of Budpest since 2005 (eg. Hochbum nd Ngy, 2013; Horváth et l., 2013; Petróczy et l., 2006). The objectives of this study were to (i) investigte the effect of thyme, cinnmon nd sweet ornge oil on the germintion of the conidi of Venturi inequlis (Cooke) G. Winter, (ii) revel in vivo mode of ction of cinnmon oil ginst sc on pple seedlings, (iii) ssess the length of efficcy of cinnmon oil nd (iv) evlute the effect of rin mount on the efficcy of essentil oil tretment. Mterils nd methods Essentil oils nd djuvnt The essentil oils of cinnmon (Cinnmomum verum) (romx Inc.), thyme (Thymus vulgris) (romx Inc.) nd sweet ornge (Citrus sinensis) (Nturol Ltd.) were selected for in vitro nd in vivo ssys. Chemicl composition ws determined by GC 6890N Gs Chromtogrph equipped with 5975 Inert mss selective detector (T. 1). ccording to previous studies (Hochbum nd Ngy, 2013), Silwet Str djuvnt ws dded in 0.02% concentrtion to queous essentil oil dilutions in order to fcilitte the dispersion of the oils. The pplied concentrtion did not influence conidium germintion. In the wsh-off test the djuvnt ws not pplied. T. 1. Composition (%) of the selected essentil oils Component RT LRI Cinnmon (%) Thyme (%) 110 Ornge (%) α-pinene cmphene sinene β-myrcene α-terpinene ρ-cymene limonene ,8-cineol γ-terpinene linlool borneol terpinen-4-ol α-terpineol phenylethyl lcohol thymol crvcrol cinnmyl ldehyde eugenol gernyl cette β-cryophylene α-humulene methyl trnscinnmte ethyl cinnmte cinnmylcette cryophylene oxide benzyl benzote Totl Note: RT - Retention time, LRI - Liner retention index reltive to C8-C23 n- lknes on HP-5 column Test plnts nd fungl inoculum pple seedlings with 4-6 fully expnded leves were used for in vivo ssys. The seedlings were produced from seeds of pple cultivrs Gl nd Jongold. Plnts were grown in light-room t C with 12 h photoperiod in vegetle trys nd were lter trnsferred into individul pots. Disesed pple leves were collected from non-treted orchrds nd home grdens from different cultivrs. Leves were stored in pper bgs in refrigertor (4 C) until inocultion. Conidi were hrvested from the leves by smll brush into sterile distilled wter to obtin conidil suspension of mixed popultion. Inoculum concentrtion ws mesured by hemocytometer.

3 111 In vitro ssy of the inhibition of conidil germintion Two-fold dilutions of essentil oils dissolved either in wter or in 0.1% pple lef ( Jongold ) brew were prepred. Oil dispersion ws fcilitted by Silwet Str (0.02%) µl of conidium suspensions ( conidi/ml) ws mesured into the wells of single concve microscope slides µl essentil oil suspensions were dded to the conidium suspensions. Finl concentrtions of essentil oils were 0.001%, 0.01% nd 0.1%. The wells were covered by cover slips to prevent evportion of the oils. Conidi suspensions were incubted on 20 C in the drk. Germintion ws ssessed microscopiclly fter 96 h by counting 3 30 spores per tretment nd compred to the germintion in wter, in pple lef brew nd in 0.02% Silwet Str solution. Conidi were considered germinted when the germ tube ws t lest twice s long s the one of the conidi. The tril ws performed in triplictes. Efficcy of essentil oils ws clculted by bbott s formul (bbott, 1925). In vivo plnt ssys The essentil oil of cinnmon ws selected for plnt ssys. ccording to our erlier in vitro studies (Cseh et l., 2014), the essentil oil of thyme could be hrmful to plnts under forced conditions. Ornge oil does not show significnt in vivo inhibition ginst Venturi inequlis (Cooke) G. Winter in ongoing orchrd trils. ssessment of preventive nd curtive ctivity of cinnmon oil The experiment ws crried out in greenhouse during the winter of Potted Gl seedlings were plced into plstic boxes. Plnts were kept between 10 C nd 27 C under nturl photoperiod supplemented with 12 h rtificil light. Reltive humidity rnged between 7 nd 97%. rtificil inocultion ws performed by sprying conidium suspension ( conidi/ml) onto the upper nd lower side of the leves. Seedlings were spryed with 0.2% cinnmon oil diluted in 0.02% Silwet Str 72 h, 24 h nd 1 h prior to inocultion s protective tretments nd 24 h or 72 h fter inocultion s curtive tretments. Conidium nd essentil oil suspensions were spryed onto the plnts by hnd spryer until run-off. fter inocultion, reltive humidity ws mintined ove 9 for three dys by covering boxes by plstic lid in order to fcilitte conidil germintion. Evlution ws crried out on the 14 th nd 21 st dys fter inocultion by the ssessment of 30 leves per tretment in six blocks ccording to their infected lef re. Inhibition of essentil oil tretments ws compred to inoculted control plnts. Wsh-off study of cinnmon oil The experiment ws crried out in growth room (14-16 C, 12 h photoperiod, 80-95% reltive humidity). Potted Jongold seedlings were spryed until run-off by singlenozzled hnd spryer with 0.2% cinnmon oil diluted in distilled wter without djuvnt. In order to ensure dilution homogeneity spryer ws being shken continuously. Treted plnts were left to dry for 4 hours (15 C, 8 RH) before rinfll simultion. To ssess the effect of nturl Ngy G. et l. / Not Bot Horti grobo, 2014, 42(1): rinfll on the rinfstness of the essentil oil different quntity of distilled wter ws spryed onto the seedlings for simulting light (1 mm), moderte (3 mm) nd hevy (5 mm) rinfll. Treted plnts were left to dry for 4 hours (15 C, 8 RH) before rtificil inocultion. Untreted, nowter-exposed (0 mm) nd wter-exposed seedlings were inoculted with conidium suspension ( conidi/ml) in the sme mnner s described erlier. Evlution of rinfstness ws crried out on the 14 th nd 21 st dys by the ssessment of 30 leves per tretment in six blocks ccording to their infected lef re. Sttisticl nlysis In order to normlize the dt nd homogenize the vrinces, in the cse of the rte of germinted conidi, rcsin trnsformtion; in the cses of infected lef re dt, ln(x ) trnsformtion were used. Normlity ws proved by skewness nd kurtosis (Tchnick nd Fidell, 2006). The effect of essentil oil tretment on conidil germintion ws evluted by univrite NOV model hving fixed fctors s tretments (essentil oil types cinnmon, thyme nd ornge in concentrtion of 0.001%, 0.01% nd 0.1%, Silwet Str nd Untreted control) s well s germintion medi (wter, pple lef brew). The preventive nd curtive effect of cinnmon oil ws evluted by univrite NOV with rndom block design hving fixed fctors s tretments (Control, 1 h, 24 h, 72 h or Control, 24 h, 72 h, respectively) s well s the length of time period fter inocultion (dys 14 nd 21) nd blocks s different plnts. The effect of cinnmon oil under simulted rinfll ws evluted by univrite NOV with rndom block design hving fixed fctors s tretments (Control, 0 mm rin, 1 mm rin, 3 mm rin nd 5 mm rin) s well s time period fter inocultion (dys 14 nd 21) nd blocks s different plnts. Homogeneity of vrinces nd the normlity of the residuls of the NOV models run with the trnsformed dt ws yet slightly violted, however, considering the homogeneity of smple sizes nd the robustness of NOV in cse the rtio of mximl nd miniml vrinces is under 2, we ccepted the results s relile. In cses of significnt fctor effects Gmes-Howell s post hoc test ws run. Results nd discussion Effect on conidil germintion Germintion ws more intensive in wter thn in pple lef brew, however, similr tendency ws observed in the inhibition of the germintion by the essentil oils in both types of pplied germintion medi. In the lowest pplied concentrtion (0.001%), none of the essentil oils suppressed germintion significntly in comprison with Silwet Str control. Inhibition level ws more remrkle in 0.01% concentrtion. Thyme nd sweet ornge suppressed germintion in pple brew significntly t 94.3% nd 82.9% efficcy, respectively. In wter, only the inhibition of thyme (96. of efficcy) proved to be significnt. In 0.1% concentrtions, ll essentil oils suppressed germintion in pple brew effectively (p<0.05) t efficcy,

4 however, cinnmon oil did not show significnt inhibition in wter. Both the effects of essentil oil tretment nd germintion medi were significnt on conidil germintion (F tr(10;44)=46.35 with p<0.001; F med(1;44)=12.84 with p<0.001, resp.). The interction ws lso significnt (F tr*med(10;44)=2.71 with p<0.05). The tretment effect size ws high, menwhile the medi effect size ws low (prtil η 2 =0.91 nd 0.27, resp.). The observed power ws ove 0.9 in ll cses (Fig. 1). ll tested essentil oils suppressed conidil germintion effectively. The highest inhibition level ws chieved with thyme oil. Bálint et l. (2014) nd Theisz et l. (2007) found thyme extrcts effective s well. The high contents of phenolic monoterpens, especilly thymol (40.6%) nd its precursore p-cymene (24.6%), might be responsible for the ntifungl effect of thyme oil (Gong et l., 2009), however, the synergistic effect of minor oil components might lso contribute to the ntifungl ctivity (Zmbonelli et l., 2004). ccording to Tn et l. (2007), the high content of limonene (97.6%) might hve cused the good inhibitory effect of ornge oil. In pple lef brew the germintion rtes of conidi were lower thn in wter in most tretment, which could be explined by the ccumultion of ntifungl substnces in old pple leves (grios, 2005). Preventive nd curtive ctivity of cinnmon oil The preventive nd curtive pplictions of 0.02% cinnmon oil decresed the disese severity on inoculted plnts significntly compred to inoculted control plnts. Oil ppliction 1 h or 24 h before inocultion showed lmost complete inhibition (100 to 99% efficcy) by the first while n outstnding inhibition (91 to 92% efficcy) by the second evlution. Cinnmon oil pplied 72 h before inocultion decresed infection level t rte of 65% (first evlution) nd 51% (second evlution) efficcy. verge conidil germintion B BC Ngy G. et l. / Not Bot Horti grobo, 2014, 42(1): pple brew c bc BC * * bc b B BC * c C * c B Wter C 112 Curtive ppliction of cinnmon showed good disese control even when pplied 72 h fter inocultion. t the first evlution, efficcy rnged from 97% (24 h curtive ppliction) to 83% (72 h curtive ppliction). By the second evlution, efficcy of cinnmon lmost remined the sme (96.8%) when pplied 24 h fter inocultion nd decresed by 2 when pplied 72 h fter inocultion. Both the effects of preventive nd curtive ppliction were significnt (preventive: Ftr(3;165)=37.03 with p<0.001; curtive: Ftr(2;123)=65.13 with p<0.001) while both the effect of time length fter inocultion nd the interction were insignificnt in both cses (preventive: Ftime(1;165)=3.73 with p=0.06; Ftr*time(3;165)=0.04 with p=0.99, resp.; curtive: Ftime(1;123)=0.093 with p=0.76; Ftr*time(2;123)=0.28 with p=0.76, resp.). The tretment effect size ws moderte (prtil η 2 =0.51 nd 0.40 for preventive nd curtive tretments, respectively) with observed power ove 0.99 (Fig. 2). Folir ppliction of cinnmon oil inhibited sc infection on pple seedlings effectively under controlled conditions. Interestingly, curtive pplictions gve similr control efficcy s tht of the preventive pplictions. Since curtive ctivity of ctive substnces is bsed on the inhibition of mycelil growth rther thn spore germintion, it cn be concluded, tht the min compounds of the essentil oil (cinnmyl ldehyde (67.9%), cinnmylcette (6.6%) nd eugenol (6.8%)) effect both spore germintion nd mycelil growth (Soylu et l., 2010). Infected lef re Infected lef re 5 45% 4 35% 3 25% 2 15% 1 5% 5 45% 4 35% 3 25% 2 15% 1 5% c B 1st evlution 2nd evlution Control 1 h 24 h 72 h Time prior to inocultion c C Control 24 h 72 h Time fter inocultion b 1st evlution 2nd evlution b B Fig. 1. Men nd stndrd devition of conidium germintion (%) of Venturi inequlis (Cooke) G. Winter in 0.05% pple brew nd in wter under different tretments. Different letters re for significntly different groups (pple lef brew: lower cse; wter: upper cse; p<0.05; Gmes Howell s test). sterisk is for significntly different types of medi per tretment level. Fig. 2. Men nd stndrd devition of infected lef re (%) of pple leves ffected by preventive (ove) nd curtive (below) tretments of 0.2% cinnmon oil, compred to inoculted untreted control. Different letters re for significntly different groups (1 st evlution: lower cse; 2 nd evlution: upper cse; p<0.05; Gmes Howell s test).

5 113 ccording to Velluti et l. (2004) cinnmyl ldehyde nd eugenol re responsible for the ntifungl ctivity. strong curtive effect of cinnmon oil ws observed ginst Fusrium hed blight of winter whet in previous field experiments s well (Horváth et l., 2013; Ngy et l., 2013). However, Berngtsson et l. (2006) found curtive ppliction of plnt extrcts less effective. The oil components eugenol, cinnmyl ldehyde, cinnmyl-cette belong to phenylpropnoid molecules, which ply n importnt role in plnt defense mechnisms (Bhttchry et l., 2010; Dixon et l., 2002). Therefore, ccording to Berngtsson et l. (2006, 2009), beside direct ntifungl ctivity cinnmon oil my influence plnt defense s well. The ppliction of cinnmon oil within 24 h pre- or post-inocultion resulted in very effective nd long lsting disese control. Decent control level ws chieved with its ppliction within 72 h, especilly by the curtive timing (Fig. 3). This observtion emphsizes the importnce of disese forecsting for outdoor ppliction of essentil oils. Further reserch is needed for the determintion of the trnsloction of cinnmon oil within the plnt. Rinfstness of cinnmon oil Without simulted rinfll cinnmon oil inhibited sc infection on pple seedlings effectively. Control efficcy decresed s result of rin. However, significnt difference in disese severity ws not observed mong the different rin mounts. The effect of tretment ws significnt (Ftretm(4;149)=5.96 with p<0.001) tht is to sy, untreted control reveled significntly different results tht those of treted ones. Both the effect of time length fter inocultion nd the interction were insignificnt (Ftime(1;149)=1.32 with p=0.25; Ftretm*time(4;149)=0.48 with p=0.75, resp.). The tretment effect size ws low (prtil η 2 =0.14) with Ngy G. et l. / Not Bot Horti grobo, 2014, 42(1): Infected lef re b observed power ove 0.98 (Fig. 4). lthough control efficcy decresed by simulted rinfll, the wter mounts did not influence control level. It cn be concluded tht prt of the ctive compounds of cinnmon oil remins on the lef surfce due to their lipophilic properties. However, Pfeiffer et l. (2004) found tht lrger mount of rin decresed the efficcy of plnt extrcts on pple seedlings more considerly. In spite of simulted rinfll, oil ppliction resulted in moderte (pproximtely 65% efficcy) nd long-lsting disese control in lortory conditions. Conclusions B Control 0 mm 1 mm 3 mm 5 mm pplied rin 1st evlution 2nd evlution The tested essentil oils, especilly Thymus vulgris suppress the conidil germintion of Venturi inequlis effectively. Cinnmon oil pplied with djuvnt hs preventive nd strong curtive effect ginst pple sc on plnts which could be observed within 3 dys prior- or postinocultion. lthough the efficcy of cinnmon oil ppliction decresed by simulted rinfll, moderte control effect ws still exhibited even fter the plnts were exposed to reltively high mount of rin. The results of this study show tht the tested essentil oils my represent bses for the development of biofungicides ginst pple sc. However, further field experiments re needed for the investigtion of control efficcy in outdoor growing conditions. Moreover, the trnsloction of essentil oils in plnt should lso be tested in lortory. B B Fig. 4. Men nd stndrd devition of infected lef re (%) of pple leves ffected by cinnmon oil tretment followed by different mount of simulted rinfll, compred to inoculted untreted control. Different letters re for significntly different groups (1 st evlution: lower cse; 2 nd evlution: upper cse; p<0.05; Gmes Howell s test). cknowledgments Fig. 3. Sc symptoms on the leves of inoculted control plnt (ove) nd tht of curtively (72 h) treted plnt by cinnmon oil (below) on the 21 th dy This reserch ws supported by the Europen Union nd the Stte of Hungry, co-finnced by the Europen Socil Fund in the frmework of TÁMOP /

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