Some plants can inhibit seed germination and growth

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1 Journl of Horticulturl Science & Biotechnology (2013) 88 (4) Effects of Persin wlnut lef extrcts on seed germintion, seedling growth, nd some physiologicl chrcteristics of the sil (Ocimum silicum L.) cultivr Genovese By MONAD DADI*, DAVOOD BAKHSHI, GHOLAMALI PEYVAST nd ZAHRA BALOUCHI Deprtment of Horticulturl Science, Fculty of Agriculture, University of Guiln, Rsht 41376, Rsht-Qzvin Rod, Irn (e-mil: (Accepted 21 Mrch 2013) SUMMARY This study ws conducted to elucidte the llelopthic effects of series of dilutions of Persin wlnut (Juglns regi) lef extrcts [i.e., undiluted, 1:2 (v/v), 1:4 (v/v), or 1:8 (v/v) dilutions] nd distilled wter (control) on the chrcteristics of seed germintion, seedling growth, nd susequent physiologicl chnges in the sil (Ocimum silicum L.) cultivr, Genovese. The results indicted tht ll queous lef extrcts of wlnut hd sttisticlly significnt inhiitory effects on the germintion of sil seeds nd on seedling growth chrcteristics. Seed germintion percentges, the lengths nd fresh weights (FWs) of the plumule nd the rdicle, the FWs nd dry weights (DWs) of the shoots nd roots, nd lef res decresed significntly with incresing concentrtions of queous wlnut lef extrct. The DWs of the plumules nd rdicles were incresed y the undiluted wlnut lef extrct. Moreover, it ws found tht the yields of essentil oils nd totl phenolics, nd the nti-oxidnt ctivities of sil seedlings incresed with incresing concentrtions of queous wlnut lef extrct, wheres the reltive wter contents of sil leves, lef wter potentils, s well s the totl chlorophyll nd crotenoid contents of sil leves decresed significntly. The highest protein contents nd peroxidse (POD) ctivities were chieved using undiluted or 1:2 (v/v) diluted queous wlnut lef extrcts, respectively. The results of this study suggest tht cultivtion of the sil cultivr, Genovese my e pproprite for wlnut tree-sed intercropping systems, sed on the increses in nti-oxidnt cpcity nd the ccumultion of secondry metolites. Some plnts cn inhiit seed germintion nd growth in other plnts y producing toxic llelochemicls (or llelopthins). The chemicl interctions tht occur etween living orgnisms including plnts, insects, nd microorgnisms re referred to s llelopthy (Rizvi nd Rizvi, 1992), The orgnic compounds involved in llelopthy re collectively clled llelochemicls (Einhellig, 1986). Such compounds re relesed y voltilistion, y leching from leves, stems, uds, flowers, fruit or seed, y exudtion from roots, nd y the degrdtion of ded plnt tissues. All plnt prts hve een shown to contin llelochemicls, ut leves nd roots re the most undnt sources (Rizvi nd Rizvi, 1992). One prominent exmple of llelopthy occurs in wlnut (Juglns regi). The min chemicl responsile for llelopthy in wlnut is juglone (5-hydroxy-1,4 nphthoquinone). Juglone hs een isolted from mny species in the wlnut fmily (the Juglndcee) including J. nigr nd J. regi. A colourless, non-toxic, reduced form clled hydrojuglone is undnt (e.g., t 750 1,000 µg ml 1 ), especilly in the leves, fruit hulls, nd roots of wlnut trees. When exposed to oxidising gents (ir), hydrojuglone is oxidised to its toxic form, juglone. Rin then wshes the juglone from the leves to the soil. Thus, neighouring plnts cn e negtively ffected y *Author for correspondence. soring juglone through their roots (Segur-Aguilr et l., 1992). The llelopthic effects of juglone on plnts re generlly toxic, ut my e eneficil in some species. In previous studies, seedling growth in tomto, cucumer, grden cress, nd lflf ws strongly inhiited y juglone or wlnut lef extrcts (Terzi nd Kocçlişkn, 2010); however, muskmelon seedling growth incresed following juglone tretment (Kocçliflkn nd Terzi, 2001). Bsil (Ocimum silicum L.) is n economiclly importnt crop tht is grown in severl Mediterrnen countries. It is populr culinry, medicinl, nd ornmentl her. Bsil is mrketed fresh or dry, nd is lso cultivted on n gro-industril scle for extrction of its essentil oils. Bsil oil is rich in phenolic compounds (Simon et l., 1990) nd is used in the phrmceuticl nd perfume industries throughout the World. Other memers of the fmily Lmicee re lso vlule due to their phrmceuticl properties. For exmple, the voltile oils produced in their leves re used s nti-oxidnts (Hrsh et l., 2002). Recent interest in Mediterrnen cuisine hs led to significnt increse in the consumption of sil, world-wide. The concentrtions of secondry products in some medicinl nd romtic plnts such s sil depend on the growing conditions. Aiotic stresses hve strong positive impct on secondry metolic pthwys,

2 434 Effects of Persin wlnut lef extrcts on sil seed including those responsile for the ccumultion of nturl products such s essentil oils (Selmr, 2008). Intercropping with wlnut is commonly-used cultivtion system in severl countries. Its high vlue, esthetic qulities, cpcity for nut production, rpid growth potentil, nd dptility to mngement, mke wlnut suitle to e intercropped (Thevthsn et l., 1999). However, the gronomic yields of some sensitive species cn e reduced significntly y the deleterious effects of juglone (Terzi nd Kocçlişkn, 2010). There is little or no relile informtion on the physiologicl effects of wlnut lef extrcts or juglone during seed germintion nd seedling growth in medicinl or romtic plnts. Therefore, the im of this work ws to evlute whether sil ws susceptile or resistnt species for intercropping with wlnut, nd its physiologicl responses to juglone. MATERIALS AND METHODS Two studies were undertken. In the first, severl vriles of sil seed germintion were investigted in response to different dilutions of Persin wlnut lef extrct [i.e., undiluted, 1:2 (v/v), 1:4 (v/v), or 1:8 (v/v)]. In the second study, queous extrcts of wlnut leves were pplied to the growing medi used for sil seedlings, to evlute the physiologicl effects of wlnut lef extrcts. Preprtion of queous wlnut lef extrcts Studies were crried out using Persin wlnut leves (J. regi) grown in Irn. Fresh leves (20 kg in totl) were collected from 20 trees t the eginning of Septemer, towrds the end of the vegettive growth period (Cosmulescu et l., 2011) from n orchrd in Shirz, in the South of Irn. Trees were plnted t spcing of 7 m 7 m (204 trees h 1 ) nd ll trees were 20 yers old. Wlnut trees younger thn 7 yers do not contin sufficient juglone to cuse toxic llelopthic effects (Ercisli et l., 2005). Leves (pprox. 1 kg per tree) were collected from the middle-third of those rnches exposed to sunlight. Wlnut lef extrcts were prepred from leves y drying them t 70 C in n oven for 48 h. The dried leves were then powdered nd 10 g ws homogenised in 100 ml distilled wter in Wring lender nd filtered through Whtmn No.1 filter pper. The filtrte ws centrifuged t 850 g nd the superntnt ws decnted. Different dilutions were prepred y diluting this extrct 1:8 (v/v), 1:4 (v/v), or 1:2 (v/v). Distilled wter ws used s control tretment. Effect of queous wlnut lef extrcts on sil seed germintion chrcteristics Seeds were surfce-sterilised with 1% (v/v) sodium hypochlorite for 15 min t 24 C, followed y sterile wter rinses. At lest 20 seeds were plced in ech 9-cm Petri dish on two sheets of filter pper moistened with the different dilutions of queous wlnut lef extrct, undiluted extrct, or wter (control). The Petri dishes were then plced in growth chmer t 22 ± 1 C nd 70 ± 5% reltive humidity (RH). Ech tretment consisted of three Petri dishes. After 7 d, germintion percentges, the lengths of the rdicles nd plumules, nd the fresh weights (FWs) nd dry weights (DWs) of the rdicles nd plumules were mesured. Effect of queous wlnut lef extrcts on the morphologicl fetures of sil seedlings The experiment ws conducted in greenhouse t the Agriculturl Fculty, University of Guiln, Rsht, Irn (37 16 N) in Seeds of sil (O. silicum L.) Genovese were sown in plstic pots (25 cm in dimeter) filled with 5:2:2:1(v/v/v/v) mix of pet moss:composted rk:pomix:cly with ph of 6.5 tht hd een stem sterilised t 100 C for 30 min. Pomix is highly porous, pet-sed growing medium tht is idel for wtersensitive crops, rooting cuttings, or low-light growing conditions. After germintion, the seedlings were thinned to three plnts per pot. Conditions within the greenhouse were s follows: dy/night tempertures of 27 C/20 C (± 2 C) nd 80%/75% (± 5%) RH with 16 h photoperiod t light intensity of pprox. 150 µmol photons m 2 s 1 provided y cool-white fluorescent lights, controlled utomticlly. Prior to ech tretment, the field cpcity (FC) of the medium in the pot ws determined. Three pots were sturted with wter, covered with plstic to void evportion nd llowed to drin for 24 h. Soil smples from ech pot were then tken to determine their moisture content. An verge moisture content ws clculted ccording to Krmer nd Boyer (1995). When the plnts reched the three-to-four true-lef stge, 150 ml of wlnut lef extrct (estimted ccording to the FC nd soil wter content) ws pplied directly y hnd onto the sustrte, twice month, using grduted glss eker to void direct contct with the folige. This ws continued until the full-flowering stge. Plnts were hrvested, nd selected morphologicl nd physiologicl chrcteristics of ech seedling were determined. Effect of queous wlnut lef extrcts on sil lef wter reltions Lef tissue is most commonly used to determine reltive wter contents (RWC). RWC ws nlysed t full-flowering in three lef smples from the third node from the top of the shoot (three replictions per tretment). Lef discs (n = 3; dimeter = 12.7 mm) were cut from ech lef. After determining their FW, the discs were immersed in distilled wter for 6 h to estimte their turgid weight (TW), then the discs were dried t 70 C for 24 h to mesure their DW. RWC ws clculted using the following eqution (Reigos nd Oonzález, 2001): RWC (%) = [(FW - DW)/(TW - DW)] 100 The wter potentil of plnts ws determined using leves t the second nd third nodes from the top of the shoots. The mesurements were crried out t the fullflowering stge using pressure chmer (Model 600; PMS Instruments, Corvllis, OR, USA) with three replictions per tretment. CO 2 ws used s the gs for the chmer (Turner, 1988). Determintion of juglone concentrtions in wlnut lef extrcts y HPLC Juglone ws mesured using high-performnce liquid chromtogrphy (HPLC), s descried y Girzu et l. (1998) with three replictions of ech extrct. Ech lef extrct ws filtered through 0.45-µm syringe filter, then 50 µl ws injected into the HPLC system (Wters,

3 M. DADI, D. BAKHSHI, G. PEYVAST nd Z. BALOUCHI 435 Milford, CT, USA) equipped with UV-visile detector (Wters Dul-Asornce 2487), column (Wters Symmetry C 18 ; 5 µm pore size; 4.6 mm 150 mm; Wters, Dulin, Irelnd). The moile phse ws mixture of solvent A [95 : 5 (v/v) wter : cetic cid] nd solvent B [90 : 10 (v/v) cetonitrile : wter]. Smples nd the column were held t 25 C, the eluent flow ws t 1.0 ml min 1, nd the run time ws 40 min.a liner grdient ws progrmmed s follows: t 0 10 min : 80% A plus 20% B; t min: 60% A plus 40% B; t min : 20% A plus 80% B (isocrtic). Juglone ws identified y compring its retention time with tht of corresponding stndrd nd y spiking smples with the stndrd. Its concentrtion ws clculted from the sornce t 420 nm using its molr extinction coefficient ( = 3,811 M 1 cm 1 ). Juglone concentrtions were expressed in mg 100 g 1 FW of lef. The juglone stndrd ws purchsed from Sigm-Aldrich (Okville, ON, Cnd). Evlution of the physiologicl chrcteristics of sil plnts To mesure essentil oil concentrtions, fresh sil plnts were collected t the full-flowering stge. The yield of essentil oil otined ws expressed s percentge of the solute DW of herge (Khlid, 2006). The concentrtions of chlorophyll, chlorophyll, totl chlorophyll, nd totl crotenoids were determined y UV-visile spectrophotometry (PG Instruments Ltd., Leicester, UK), s descried y Wellurn (1994). Peroxidse (POD) ctivity ws ssyed y mesuring the formtion of guicol, spectro-photometriclly, ccording to In et l. (2007).The increse in sornce t 470 nm ws recorded for 2 min following the ddition of H 2 O 2. POD ctivity ws expressed in µmol g 1 FW min 1. The totl protein contents of plnts were estimted ccording to Brdford (1976). Totl phenolics contents were mesured using the Folin-Cioclteu method, s descried y Singleton et l. (1999) nd were expressed in mg gllic cid equivlents (mg GAE) 100 g 1 FW of herge. Anti-oxidnt cpcities were determined y mesuring the scvenging of 2, 2-diphenyl-2- picrylhydrzyl hydrte (DPPH) rdicls, ccording to Brnd-Willims et l. (1995). Sttisticl nlysis Both experiments were crried out ccording to completely rndomised design. For the seed germintion experiment, ech tretment contined four replictions of ech wlnut lef extrct preprtion. For physiologicl nlysis, there were three replictions of ech tretment. Before nlysis of vrince (ANOVA), dt were tested for normlity nd homoscedsticity using the Kolmogorov-Smirnov nd Cochrn tests, respectively. To determine sttisticl differences etween tretments, vrince nlysis, nd lest significnt difference (LSD) tests were performed (P 0.01). RESULTS AND DISCUSSION Juglone concentrtions Juglone is well-known component of wlnut nd occurs t high concentrtions in ll green nd growing prts of wlnut trees. The juglone concentrtion in fresh leves ws quntified y HPLC. Bsed on its spectrl chrcteristics, the verge juglone concentrtion ws 8.63 mg 100 g 1 FW. This ws in greement with Cosmulescu et l. (2011), who reported mg juglone 100 g 1 FW in leves from different wlnut cultivrs. Bsil seed germintion nd the lengths, FWs nd DWs of rdicles nd plumules Bsil seed germintion ws significntly inhiited y ll wlnut lef extrcts (Tle I). Juglone significntly decresed the germintion percentges, from 95% in the wter control to 67.5% in the undiluted lef extrct. The lengths nd FWs of plumules nd rdicles were ffected y ll wlnut lef extrcts. The mximum plumule nd rdicle lengths were otined in the wter controls, while the minimum lengths were oserved using the undiluted wlnut lef extrct. All queous extrcts decresed the FWs of plumules nd rdicles. However, plumule DWs were enhnced y the undiluted lef extrct. A significnt difference ws lso found etween the undiluted lef extrct nd ll other tretments. Rdicle DWs incresed significntly from 16 mg per seedling in the controls to 20 mg with the undiluted lef extrct. These results gree with those of Kocçlişkn nd Terzi (2001), who reported tht the germintion of cucumer seed ws inhiited y juglone or wlnut lef extrcts, wheres Terzi et l. (2004) found tht juglone hd no significnt effect on seed germintion in muskmelon. In previous study, Segur-Aguilr et l. (1992) oserved tht juglone cused oxidtive stress during seed germintion. During seed germintion, the most importnt event for emryo growth is protein synthesis. Juglone locks protein synthesis t the trnscription stge y inhiiting RNA polymerses (Cho et l., 2001). Chnges in the morphologicl chrcteristics of sil seedlings The FWs nd DWs of sil shoots nd roots decresed following tretment with ll wlnut lef extrcts. Also, the lef res of seedlings decresed mrkedly following ll wlnut lef extrct tretments (Tle II). Kocçlişkn nd Terzi (2001) demonstrted tht oth juglone nd wlnut lef extrcts inhiited seed germintion nd seedling growth in severl plnt species TABLE I Influence of dilutions of Persin wlnut lef extrct on the men vlues of selected seed germintion chrcteristics in sil (Ocimum silicum L. Genovese ) Proportion of Percentge Plumule Rdicle Plumule fresh Rdicle Plumule dry Rdicle dry lef extrct (v/v) germintion length (mm) length (mm) weight (g) fresh weight (g) weight (g) weight (g) : : c c c : c c d c c Undiluted 67.5 c 7.70 d 3.83 e c c Men vlues (n = 4) in ech column followed y the sme lower-cse letters re not significntly different t P 0.01 ccording to the LSD test.

4 436 Effects of Persin wlnut lef extrcts on sil seed TABLE II Influence of dilutions of Persin wlnut lef extrct on the men vlues of selected seedling growth chrcteristics in sil (Ocimum silicum L. Genovese ) Proportion of Shoot fresh Shoot dry Root fresh Root dry Lef re Lef Lef wter lef extrct (v/v) weight (g) weight (g) weight (g) weight (g) (cm 2 ) RWC (%) potentil (MP) : : c 1.43 c 0.13 c 9.22 c c : c c 0.13 c 7.04 dc cd 0.70 c Undiluted c 0.76 c 0.08 c 6.59 d d 0.93 d Men vlues (n = 3) in ech column followed y the sme lower-cse letters re not significntly different t P 0.01 ccording to the LSD test. such s wtermelon, tomto, grden cress, nd lflf. In lter studies, similr results were reported with wlnut lef extrcts on strwerry (Ercisli et l., 2005), lthough seedling growth in muskmelon incresed significntly following tretment with wlnut lef extrcts (Kocçlişkn nd Terzi, 2001). Juglone inhiited plnt growth y reducing the rtes of photosynthesis nd respirtion (Jose nd Gillespie, 1998) nd incresing oxidtive stress (Segur-Aguilr et l., 1992). In ddition, in cucumer, it ws shown tht juglone decresed seedling growth y stimulting the synthesis of scisic cid (ABA), growth-inhiiting hormone, or y preventing the synthesis of growth-promoting hormone such s gierellic cid (GA; Terzi, 2008). Effects of queous wlnut lef extrcts on lef wter reltions in sil Tle III shows the influence of wlnut lef extrcts on sil lef wter reltions. Lef RWC vlues nd wter potentils decresed significntly with incresing concentrtions of wlnut lef extrct. The minimum vlues were found using undiluted wlnut lef extrct. Juglone-medited reductions in growth rose from the decresed ility of roots to trnslocte wter, y the inhiition of plsm memrne H + -ATPse ctivity, nd y cusing drought stress (Hejl nd Koster, 2004; El- Hdrmi et l., 2005). A numer of studies hve reported llelochemiclinduced disruptions of plnt wter lnce. Some hve suggested tht this is due to interference with norml memrne function nd the disruption of ctive trnsport (Glss nd Dunlp, 1974; Brkosky et l., 1999; or 2000). Brkosky et l. (1999) reported tht disruptions in wter reltions ppered to e the primry mode of ction tht led to n overll reduction in growth in lefy spurge (Euphori esul L.) in the presence of the phytotoxin, hydroquinone. Chnges in the physiologicl chrcteristics of sil plnts Essentil oil yields: As shown in Figure 1, essentil oil yields incresed significntly (P 0.01) with n increse in wlnut lef extrct concentrtion. As mentioned ove, Persin wlnut lef extrcts reduced lef wter potentils nd cused wter stress in plnts. Drought stress incresed essentil oils yields in other medicinl nd romtic plnts s these compounds cn prevent oxidtion in plnt cells (Alidi et l., 2008). In ddition, the stimultion of essentil oil production under wter stress could e due to the fct tht plnts produce high concentrtions of terpenes under environmentl stress conditions ecuse of the reduced lloction of cron to growth, suggesting trde-off etween plnt growth nd defence (Turtol et l., 2003). Pigment contents: Totl chlorophyll nd chlorophyll concentrtions decresed significntly following undiluted wlnut lef extrct tretment; while the mximum level of chlorophyll ws otined in the 1:2 (v/v) lef extrct tretment (Tle III). Totl crotenoid contents decresed grdully with incresing concentrtions of wlnut lef extrct. The mximum nd minimum crotenoid contents were found in the wter control nd undiluted lef extrcts, respectively (Tle III). These results gree with Hejl et l. (1993), who reported tht juglone inhiited plnt growth y reducing the rte of photosynthesis nd chlorophyll contents in Lemm minor. Biossys indicted tht the inhiitory effect of wlnut lef extrcts ws proportionl to its concentrtion. The mximum concentrtion hd the strongest inhiitory effect, wheres in some cses lower concentrtions showed no, or only slight effects. Einhellig (1986) showed tht juglone cted directly on the photosynthetic pthwy nd not y indirect effects such s stomtl interference or ffecting wter reltions. POD ctivity: To verify whether the llelochemicl stress cused y wlnut lef extrcts induced oxidtive stress in sil leves, we mesured the ctivity of the key ntioxidnt enzyme, POD, in sil. The results showed tht POD ctivity ws significntly incresed (P 0.01) in the 1:2 (v/v) extrct tretment (Tle III). Incresed POD ctivity my contriute to the protection of sil from oxidtive stress. Similr increses in POD ctivity hve een reported in soyen roots treted with juglone. Bohm et l. (2006) showed tht, t low juglone concentrtions ( 1.0 µm), POD ctivity incresed, while TABLE III Influence of dilutions of Persin wlnut lef extrct on the men vlues of some physiologicl chrcteristics of sil (Ocimum silicum L. Genovese ) Proportion of Chlorophyll (mg g 1 FW) POD ctivity Totl protein Totl crotenoids lef extrct (v/v) Totl (µmol g 1 FW min 1 ) (mg g 1 FW) (mg 100 g 1 FW) c : c : c : c Undiluted 0.65 c c Men vlues (n = 3) in ech column followed y the sme lower-cse letters re not significntly different t P 0.01 ccording to the LSD test.

5 M. DADI, D. BAKHSHI, G. PEYVAST nd Z. BALOUCHI c 0.06 d d :8 1:4 1:2 Undiluted Proportion of wlnut lef extrct (v/v) FIG.1 Chnges in the essentil oil yields of sil (Ocimum silicum L. Genovese ) plnts treted with different concentrtions of Persin wlnut lef extrct. Columns with the sme lower-cse letter were not significntly different t P 0.01 ccording to the LSD test. Verticl rs indicte ± stndrd errors (n = 3). Essentil oil yields [% (v/w)] t high concentrtions ( 10 µm) it ws inhiited. POD ctivity tended to e higher in juglone-tolernt plnt species, enling the plnts to protect themselves ginst oxidtive stress, wheres such ctivity ws not oserved in juglone-sensitive plnts (Sclet et l., 1995). Peroxidses re widely distriuted in ll higher plnts. One of their min functions is s prt of the defence complex in cells, ensuring detoxifiction of rective oxygen species (ROS). This function is importnt during plnt metolic responses to different stress fctors. Peroxidses protect cells ginst the hrmful effects of peroxides nd hydroperoxides (Cstillo, 1992). Since POD ctivity ws enhnced following tretment with the 1:2 (v/v) wlnut lef extrct, nd decresed t the mximum extrct concentrtion, it is possile tht this ROS-scvenging system ws impired y the undiluted extrct. However, the cpcity of the cells to scvenge ROS my e exceeded y the undiluted extrct nd they susequently decrese POD ctivity (Bohm et l., 2006). Proteins: As shown in Tle III, totl lef protein contents were significntly incresed y the undiluted wlnut lef extrct. An increse in protein contents y juglone tretment ws indicted in previous study (Terzi et l., 2004). Increses in protein contents my include increses in some enzyme proteins. In previous study, phenol oxidses incresed significntly following juglone tretment (Kocçlişkn et l., 2008). These ctivities my e incresed y the oxidtive defence mechnisms induced y llelochemicl (juglone) stress (Segur-Aguilr et l., 1992). Totl phenolics: Chnges in totl phenolics contents re shown in Figure 2. Totl phenolics concentrtions incresed slightly with incresing wlnut lef extrct concentrtion, nd the mximum content of phenolic compounds ws oserved following the undiluted wlnut lef extrct tretment. Phenolic compounds re importnt for the prevention of stress-induced oxidtive dmge s they ply importnt roles in removl of toxic sustnces (Di Crlo et l., 1999). Coder (2011) reported tht phenolic compounds cn ct s nti-oxidnts in wlnut. The production of ROS such s H 2 O 2,O 2., nd OH. due to non-host specific toxins such s juglone is responsile for tissue dmge in mny plnt species (Du nd Ehrenshft, 2000). The oxidtive urst is n erly event in Anti-oxidnt ctivity Anti-oxidnt ctivity r = 0.78 ** Totl phenolic compounds :8 1:4 1:2 Undiluted Proportion of wlnut lef extrct (v/v) FIG.2 Chnges in the concentrtion of totl phenolic compounds nd in ntioxidnt ctivity (DPPH rdicl-scvenging ctivity) in sil (Ocimum silicum L. Genovese ) plnts treted with different concentrtions of Persin wlnut lef extrct. r = correltion coefficient etween totl phenolic compounds concentrtion nd nti-oxidnt ctivity. Columns or dtum points with the sme lower-cse letters re not significntly different t P 0.01 ccording to the LSD test. Verticl rs indicte ± stndrd errors (n = 3). the plnt defence response to different stresses nd cts s secondry messenger to signl susequent defence rections in plnts (Low nd Merid, 1996). Anti-oxidnt ctivity: Anti-oxidnt ctivity incresed significntly (P 0.01) in the 1:4 (v/v), 1:2 (v/v), nd undiluted wlnut lef extrct tretments compred with the controls nd the 1:8 (v/v) extrct (Figure 2). This result my e due to the oxidtive defence mechnisms induced y juglone stress (Segur-Aguilr et l., 1992). This result grees with El-Hdrmi et l. (2005) who reported tht, under juglone stress, the ctivities of mny nti-oxidnt enzymes such s ctlse (CAT), superoxide dismutse (SOD), glutthione peroxidse (GPO), nd scorte peroxidse (APO) were stimulted. This condition could distur the lnce etween ROSgenerting fctors nd ROS-scvenging systems. As shown in Figure 2, there ws significnt (P 0.01) positive reltionship etween totl phenolics concentrtion nd nti-oxidnt ctivity (r = 0.78). The mechnisms y which phenolic compounds scvenge free rdicls is yet to e fully estlished. Polyphenols in plnts contriute to the nti-oxidnt nd phrmcologicl ctivities of plnts. Anti-oxidnts such s phenolic cids, polyphenols, nd flvonoids hve eneficil role in scvenging free rdicls nd thus reduce oxidtive stress (Shetgiri et l., 2010). In conclusion, Persin wlnut lef extrcts induced oxidtive nd wter stress, resulting in incresed enzymtic nd non-enzymtic ROS-scvenging cpcity. Concentrtions of secondry metolites such s essentil oils nd phenolic compounds incresed in sil leves exposed to ll wlnut lef extrct tretments. Therefore, the ppliction of wlnut lef extrcts could e wy to increse the concentrtions of secondry metolites. The cultivtion of sil s medicinl plnt in wlnut-sed intercropping system might e profitle re of reserch, given recent trends towrds the greter commercilistion of intercropping. We thnk the University of Guiln, Irn for finncil support Totl phenolic compounds (mg GAE 100 g 1 FW)

6 438 Effects of Persin wlnut lef extrcts on sil seed REFERENCES ALIABADI, F., LEBASCHI, H., HUSSEIN, M., SHIRANIRAD, A. H., VAL- ADABADI, A. R. nd DANESHIAN, J. (2008). Effects of rusculr mycorrhizl fungi, different levels of phosphorus nd drought stress on wter use efficiency, reltive wter content, nd proline ccumultion rte of corinder (Corindrum stivum L.). Journl of Medicinl Plnts Reserch, 2, BARKOSKY, R. R., BUTLER, J. L. nd EINHELLIG, F. A. (1999). Mechnisms of hydroquinone-induced growth reduction in lefy spurge. Journl of Chemicl Ecology, 25, BARKOSKY, R. R., EINHELLIG, F. A. nd BUTLER, J. L. (2000). Cffeic cid-induced chnges in plnt wter reltionships nd photosynthesis in lefy spurge. Journl of Chemicl Ecology, 26, BOHM, P.A.F.,ZANARDO, F. M. L., FERRARESE, M. L. L. nd FER- RARESE-FILHO, O. (2006). Peroxidse ctivity nd lignifiction in soyen root growth inhiition y juglone. Biologi Plntrum, 50, BRADFORD, M. M. (1976). A rpid nd sensitive method for the quntittion of microgrm quntities of protein utilizing the principle of protein dye inding. Anlyticl Biochemistry, 72, BRAND-WILLIAMS, W., CUVELIER, M. E. nd BERSET, C. (1995). Use of free rdicl method to evlute nti-oxidnt ctivity. Food Science nd Technology, 28, CASTILLO, F. J. (1992). Peroxidse nd stress. In: Plnt Peroxidses ( ). Topics nd Detiled Literture on Moleculr, Biochemicl nd Physiologicl Aspects. (Penel, C., Gsper, T. nd Greppin, H., Eds.). University of Genev Press, Genev, Switzerlnd CHAO, S. H., GREENLEAF, A. L. nd PRICE, D. H. (2001). Juglone, n inhiitor of the peptidyl-prolyl isomerse Pin l, lso directly locks trnscription. Nucleic Acids Reserch, 29, CODER, K. D. (2011). Blck Wlnut Allelopthy: Tree Chemicl Wrfre (Allelopthy Series). Wrnell School of Forestry & Nturl Resources, University of Georgi, Athens, GA, USA. Outrech Puliction WSFNR pp. COSMULESCU, S., TRANDAFIR, I., ACHIM, G. nd BACIU, A. (2011). Juglone contents in lef nd green husks of five wlnut (Juglns regi L.) cultivrs. Notule Botnice Horti Agrootnici Cluj - Npoc, 39, DAUB, M. E. nd EHRENSHAFT, M. (2000). The photo-ctivted Cercospor toxin cercosporin: contriution to plnt disese nd fundmentl iology. Annul Review of Phytopthology, 38, DI CARLO, G., MASCOLO, N., IZZO, A. A. nd CAPASSO, F. (1999). Flvonoid: old nd new spects of clss of nturl therpeutic drugs. Life Science, 65, EINHELLIG, F. A. (1986). Mechnisms nd modes of ction of llelochemicls. In: The Science of Allelopthy. (Putmn,A.R. nd Tng, C.S., Eds.). Willey-Interscience, New York, NY, USA EL-HADRAMI, A., KOME, D. nd LEPOIVRE, P. (2005). Effect of juglone on ctive oxygen species nd nti-oxidnt enzymes in susceptile nd prtilly resistnt nn cultivrs to Blck Lef Strek Disese. Europen Journl of Plnt Pthology, 113, ERCISLI, S., ESITKEN, A., TURKKAL, C. nd ORHAN, E. (2005). The llelopthic effects of juglone nd wlnut lef extrcts on yield, growth, chemicl nd PNE compositions of strwerry cv. Fern. Plnt, Soil nd Environment, 51, GIRZU, M., FRAISSE, D., CARNAT, A. P., CARNAT, A. nd LAMAISON, J. L. (1998). High-performnce liquid chromtogrphic method for the determintion of juglone in fresh wlnut leves. Journl of Chromtogrphy A, 805, GLASS, A. D. M. nd DUNLAP, J. (1974). Influence of phenolic cids on ion uptke. IV. Depolriztion of memrne potentils. Plnt Physiology, 54, HARSH, P. B.,WALKER, T. S., SCHWEIZER, H. P. nd VIVANCO, J. M. (2002). Root specific elicittion nd ntimicroil ctivity of rosmrinic cid in hiry root culture of Ocimum silicum. Plnt Physiology nd Biochemistry, 40, HEJL, A. M. nd KOSTER, K. L. (2004). Juglone disrupts root plsm memrne H + -ATPse ctivity nd impirs wter uptke, root respirtion, nd growth in soyen (Glycine mx) nd corn (Ze mys). Journl of Chemicl Ecology, 30, HEJL, A. M., EINHELLING, F. A. nd RASMUSSEN, J. A. (1993). Effects of juglone on growth, photosynthesis nd respirtion. Journl of Chemicl Ecology, 9, IN, B. C., MOTOMURA, S., INAMOTO, K., DOI, M. nd MORI, G. (2007). Multivrite nlysis of reltion etween prehrvest environmentl fctor, posthrvest morphologicl nd physiologicl fctors, nd vse-life of cut Asomi Red roses. Journl of the Jpnese Society for Horticulturl Science, 76, JOSE, S. nd GILLESPIE, A. R. (1998). Allelopthy in lck wlnut (Juglns nigr L.) lley cropping. II. Effects of juglone on hydroponiclly grown corn (Ze mys L.) nd soyen (Glycine mx L. Merr.) growth nd physiology. Plnt nd Soil, 203, KHALID, K. H. A. (2006). Influence of wter stress on growth, essentil oil, nd chemicl composition of hers (Ocimum sp.). Interntionl Agrophysics, 20, KOCAÇALIŞKAN, I. nd TERZI, I. (2001). Allelopthic effects of wlnut lef extrcts nd juglone on seed germintion nd seedling growth. Journl of Horticulturl Science & Biotechnology, 76, KOCAÇALIŞKAN, I., CEYLAN, M. nd TERZI, I. (2008). Effects of juglone on seedling growth in intct nd cotless seeds of cucumer (Cucumis stivus cv. Beith Alph). Scientific Reserch nd Essys, 4, KRAMER, P. nd BOYER, J. S. (1995). Wter Reltions of Plnts nd Soils. Acdemic Press, Sn Diego, CA, USA. 495 pp. LOW, P. S. nd MERIDA, J. R. (1996). The oxidtive urst in plnt defense: function nd signl trnsduction. Physiologi Plntrum, 96, REIGOSA, M. J. nd GONZÁLEZ, L. (2001). Plnt wter sttus. In: Hndook of Plnt Ecophysiology Techniques. Kluwer Acdemic Pulishers, Dordrecht, The Netherlnds RIZVI, S. J. H. nd RIZVI, V. (1992). Allelopthy: Bsic nd Applied Aspects. Chpmn nd Hll, London, UK. 480 pp. SCALET, M., FEDERICE, R., GUIDO, M. C. nd MANES, F. (1995). Peroxidse ctivity nd polymine chnges in response to ozone nd simulted cid rin in Aleppo pine needles. Environmentl nd Experimentl Botny, 35, SEGURA-AGUILAR, J., HAKMAN, I. nd RYDSTROM, J. (1992). The effect of 5OH-1,4-nphthoquinone on Norwy spruce seeds during germintion. Plnt Physiology, 100, SELMAR, D. (2008). Potentil of slt nd drought stress to increse phrmceuticl significnt secondry compounds in plnts. Agriculture nd Forestry Reserch, 58, SHETGIRI, P. P., DARJI, K. K. nd D MELLO, P. M. (2010). Evlution of nti-oxidnt nd nti-hyperlipidemic ctivity of extrcts rich in polyphenols. Interntionl Journl of Phytomedicine, 2, SIMON, J. E., QUINN, J. nd MURRAY, R.G. (1990). Bsil: source of essentil oils. In: Advnces in New Crops. (Jnick, J. nd Simon, J. E., Eds.). Timer Press, Portlnd, OR, USA SINGLETON, V. L., ORTHOFER, R. nd LAMUELA-RAVENTOS, R. S. (1999). Anlysis of totl phenols nd other oxidtion sustrtes nd nti-oxidnts y mens of Folin-Cioclteu regent. Methods in Enzymology, 299, TERZI, I. (2008). Allelopthic effects of juglone nd decomposed wlnut lef juice on muskmelon nd cucumer seed germintion nd seedling growth. Africn Journl of Biotechnology, 7, TERZI, I. nd KOCAÇLIŞKAN, I. (2010). Effects of gierellic cid nd kinetin in overcoming the effects of juglone stress on seed germintion nd seedling growth. Turkish Journl of Botny, 34, TERZI, I., KOCAÇLIŞKAN, I., BENLIOGLU, O. nd SOLAK, K. (2004). Effects of juglone on growth of muskmelon seedlings with respect to physiologicl nd ntomicl prmeters. Biologi Plntrum, 47, THEVATHASAN, N. V., GORDON, A. M. nd VORONEY, R.P. (1999). Juglone (5-hydroxy-1,4-npthhoquinone) nd soil nitrogen trnsformtion interction under wlnut plnttion in southern Ontrio, Cnd. Agroforestry Systems, 44, TURNER, N. C. (1988). Mesurement of plnt wter sttus y the pressure chmer technique. Irrigtion Science, 9, TURTOLA, S., MANNINEN, A. M., RIKALA, R. nd KAINULAINEN, P. (2003). Drought stress lters the concentrtion of wood terpenoids in Scots pine nd Norwy spruce seedlings. Journl of Chemicl Ecology, 29, WELLBURN, A. R. (1994). The spectrl determintion of chlorophylls nd, s well s totl crotenoids, using vrious solvents with spectrophotometers of different resolution. Journl of Plnt Physiology, 144,