Corresponding author: * ABSTRACT

Transcription

1 Meiinl n Aromti Plnt Siene n Biotehnology 213 Glol Siene Books Effet of Folir Applition of Dimmomium Phosphte on Morphologil Chrteristis n Constituents of Essentil Oil of Mexin Mrigol (Tgetes minut L.) Mrzieh Neghn 1 Kmel Ms 2* Enytollh Tfzoli 3 Aolrsool Zkerin 4 1 Islmi Az University, Unit Jhrom, Jhrom, Irn n Young Reserhers Clu of Jhrom University, Irn 2 Lortory of Biotive Sustnes, Biotehnology Center in Borj-Ceri Tehnopol, BP. 91, 25 Hmmm-Life, Tunisi 3 Deprtment of Hortiulturl Sienes, College of Agriulture, Shirz University, Shirz, Irn 4 Deprtment of Hortiulturl Sienes, Islmi Az University, Unit Jhrom, Jhrom, Irn Corresponing uthor: * ms_kmel@hotmil.om ABSTRACT Pot trils were rrie out in greenhouse in Shirz, Irn to etermine the effet of folir pplition of immonium phosphte (DAP) on morphologil hrteristis n qulity of essentil oil (EO) of Mexin mrigol (Tgetes minut L.). DAP ws pplie t, 2.4, 4.8, 7.2, 9.6 n 12% (w/v). Growth prmeters inrese s DAP levels inrese. The ry weight of shoots inrese t 2.4% DAP. At 7.2% DAP, the height, lef re, length of xillry shoots n fresh weight of eril prts peke. Moreover, the numer of xillry shoots, numer of flowers per plnt n the EO yiel rehe mximum t 9.6% DAP. Regring the EO onstituents, the ontent of ihyro tgetone n Z-tgetone inrese s DAP level inrese while Z- -oimene n Z-oimenone ontent erese s the DAP level inrese. The role of phosphorus s entrl n pivotl metoli n regultory nutrient element is isusse. Keywors: Asteree, essentil oil omposition, growth prmeters Arevitions: DAP, immomium phosphte; EO, essentil oil INTRODUCTION Asteree is the lrgest fmily of vsulr plnts with more thn 23, speies (Jeffrey 27), rih in seonry metolites n essentil oils (EOs) (Teixeir Silv 23, 24; Teixeir Silv et l. 25). The genus Tgetes, with the ommon nme of mrigol, onsists of 3-4 speies tht re enemi from Arizon to Argentin (Sefikon et l. 24). Mexin mrigol (Tgetes minut L.) is n nnul plnt elongs to the Asteree fmily. It is ntive of grsslns n mountinous regions of South Ameri (Dole 1999), inluing Argentin, Chile, Bolivi, Peru, n in the Cho region of Prguy (Reihe 193; Perkins 1912; MVugh 1943; Soule 1993). T. minut grows wil from spring to erly winter when it ompletes its life yle (Bu n Kul 27). The plnts reh 1 to 2 m in height. Leves re slightly glossy green, n re pinntely issete into 4 to 6 pirs of pinne. The unersurfe of leves er numer of smll, puntute, multiellulr glns, orngish in olor, whih exue liorie-like rom when rupture. Glns my lso e foun on the stems n involure rts (Dole 1999). Frtions of T. minut EO hve the potentil for phi ontrol n the EO lso hs the potentil s nturl heriie for mnging rie wees (Singh et l. 23; Tomov et l. 25; Btten et l. 26; Pritekel et l. 26; Btish et l. 27). Due to its ompetitive nture, T. minut is resistnt to nturl rought n survives esily on poor soils s wee, lthough it is now lso ultivte s rop for grohemil n phrmeutil purposes (Hulin 28). T. minut plnt orgns ontin llelohemils tht inhiit the germintion n root growth of Ai sk sees (Arif n Alhmmi 28). New Worl people were si to use T. minut s flvorful everge, meiinl te, n oniment sine pre-ontt times (Rees 1817). T. minut is ommerilly grown n hrveste for its EOs whih re use in the flvor n perfume inustry s Tgetes oil. The EO is use in perfumes, n s flvor omponent in most mjor foo prouts, inluing ol everges, loholi everges, frozen iry esserts, ny, ke goos, geltins, puings, oniments, n relishes (Crveiro et l. 1988). Tgetes EO is use to tret hest infetions, oughs n trrh, ilting ronhi, filitting the flow of muus n isloging ongestion n n e use in ses of skin infetions (Chmorro et l. 28; Meshktlst et l. 21). It hs heling effet on wouns, uts, lluses n unions (Singh et l. 22). T. minut is rih in mny seonry metolites, inluing yli, monoyli n iyli monoterpenes, sesquiterpenes, flvonois, thiophenes, n romtis (Roriguez n Mry 1977). Vrious stuies on T. minut reporte tht there re vritions in the EO omposition oring to the hrvesting lotion, the growth stge n the ifferent prts of the plnt (Ester et l. 28). EO ompositions of T. minut L. hve iologil tivities ginst ertin pthogens (Kéit et l. 2) n re well known for their ioil properties (Gillij et l. 28). Also, T. minut hs een reporte to hve ntimiroil tivity ginst Stphyloous ureus n Stphyloous epiermiis (Kterere et l. 212). It is use in the tretment of ols, irrhoe n suspete liver ilments (Rios n Reio 25). A. spinosus ws use in the tretment of menorrhgi, gonorrhoe, ezem n oli (Azhr et l. 24) n is lso ite in the tretment of ietes (Kterere n Eloff 25). Previous stuies showe tht the seonry ompouns in Tgetes re effetive eterrents of numerous orgnisms, inluing fungi, fungi pthenogeni on humns, teri, roun worms in generl, tremtoes (Wng et l. 23), nemtoes (Hmwi et l. 24), n numerous inset pests through severl ifferent mehnisms (Srivnti et l. 26; Krueger et l. 21; Meshktlst et l. 21). The nti- Reeive: 11 June, 212. Aepte: 3 Otoer, 212. Originl Reserh Pper

2 Meiinl n Aromti Plnt Siene n Biotehnology 7 (1), Glol Siene Books miroil tivity of five seonry ompouns in T. minut ( -oimene, ihyrotgetone, tgetone, Z-oimenone, n E-oimenone), when teste on 4 strins of teri n fungi, showe tht the EO of T. minut h % inhiitory effet on Grm-positive teri, 95% inhiitory effet on Grm-negtive teri, n % inhiitory effet on fungi (Hethelyi et l. 1986; Cespees et l. 26). Huson (199) teste mny ifferent seonry ompouns for ntivirl tivity, n etermine tht thiophenes emonstrte the gretest ntivirl tion t the lowest oses, n with the lest toxiity overll. Of the thiophenes, moleules with two or more thiophene units showe the highest tivity. Huson (199) teste 32 thiophenes, evlute their effiy n etermine the 1 most effetive ones. Atkinson et l. (1964) were the first uthors to report thiophenes in T. minut. A omprison of Atkinson s results to those of Huson shows tht 7 of 1 most effetive ntivirl thiophenes re foun in T. minut. Frtions of T. minut EO hve the potentil for phi ontrol n lso the potentil s nturl heriie for mnging rie wees (Shhzi et l. 21). Despite vritions in their reltive onentrtions, the min onstituents of T. minut EOs re limonene n is-oimene (monoterpenes), ihyrotgetone, trns- n is-tgetone, trns- n istgetenone (oxygente monoterpenois). Other oxygente monoterpenois (terpinolene, rvrol n rvone) n sesquiterpenes ( -ryophyllen, germrene-d, -elemene hve lso een reporte t lower onentrtions (Vázquez et l. 211). Dihyrotgetone ws the min ompoun of T. minut oil extrte from leves (Chlht et l. 1995). Anlysis of T. minut EO y GC/MS showe tht the min ompouns were -oimene, Z-tgetenone, E-tgetenone, E-tgetone n Z-tgetone (Lwrene 1996). Vrious isoltion n instrumentl tehniques hve een use to stuy the oil omponents of Tgetes, ut stem istilltion n hromtogrphy hve een shown to e the most equte (Vázquez et l. 211). However, hyroistilltion is still the most ommon extrtion tehnique employe to otin EOs from romti plnts (Kákoniová et l. 26; Mgw et l. 26; Sroglou et l. 26; Beerr et l. 21; Moreno et l. 21; Urzu et l. 21; Buitrgo et l. 211). It is lorious n time-onsuming proess tht requires lrge mounts of smple. Moreover, when investigtors extrt EOs from plnt mtrix for nlysis, little ttention is pi to the possiility tht the extrtion methos my yiel ifferent EO profiles, or even worse, smple egrtion, espite it eing well known tht hemil retions n our uring the istilltion proess (Bu n Kul 27). For this reson, the finl omposition of the prout my not e representtive of the originl mteril, n the oserve vritions in oil omposition my strongly epen on the type of istilltion metho use (Bu n Kul 25). Thus, it is importnt tht reserhers explore the vrious vntges n isvntges of given extrtion or instrumentl tehnique efore rrying out n nlysis. Sine Mexin mrigol is n importnt meiinl plnt, ny stuy s relte to the ultivtion spets of this plnt is of prime interest. In ommeril meiinl plnt proution, the min ojetive is to proue high iomss yiels per hetre with high levels of seonry metolites. Nutritionl requirements hve mjor effet on the yiel n growth of ll hortiulturl n gronomi rops (Defult et l. 23). On the other hn, the level of seonry metolites in meiinl plnts my e positively or negtively ffete y the kin n mount of nutrient elements. Phosphte plys entrl, pivotl metoli n regultory role on the nexus of severl physiologil n iohemil proesses in plnts, inluing photosynthesis, energy onservtion, inter n intrellulr oorintion of rohyrte metolism (Ael 22) n in energy trnsfer (Hrley 1971). These effets re exerte y phosphte oth s n essentil nutrient n s signl for metoli moultion. Phosphorus from the rhizosphere is the lest essile nutrient require y plnts n its vilility is rrely equte for optiml growth euse of poor ontent n its immoile form in the soil (Ael et l. 22). Aoringly, it is speulte tht folirly pplie inorgni phosphte my help plnts to hve ner norml moe of metolism. This my e one of the working moels for future experimenttion. Of the vrious growth n yiel hrteristis of the rop, the importne in iomss n iogenesis of EO in the leves ws the most importnt for oil proution (Sngwn et l. 21). In plnts, phosphorus is require in reltively lrge mounts for the iosynthesis of primry n seonry metolites, sine phosphorus hs essentil funtions s onstituent of nulei is n phospholipis (iomemrnes) n plys key role in the energy metolism of ells (Mrshner 2). Moreover, phosphorus is known to hve multifrious ellulr funtions in plnts, inluing signlling n trnsmemrne metolite flux. Seonry metolism is highly moulte y these mehnisms (Nell et l. 29). Of the vrious growth n yiel hrteristis of the rop, the improvement in iomss iogenesis of essentil oil in the leves is the most importnt for oil proution (Sngwn et l. 21). Some hemils when pplie through folige, re known to elerte the sorption n ssimiltion mehnism of nutrients n improve the yiels in severl rops. It hs een shown tht soil pplition of immonium phosphte (DAP) inreses the grin yiel in whet, n lso, it hs enhne the proution of essentil oil in rose, sge n lvener plnts (Rm et l. 23). The present stuy ws unertken to investigte the effets of folir pplition of immonium phosphte on morphologil hrteristis n qulity of EO of Mexin mrigol. MATERIALS AND METHODS Plnt mteril The sees of Mexin mrigol were ultivte in Sr greenhouse (Shirz, Irn) in Ferury 27. Plnts were grown in sny lom soil. Some of the physiologil hrteristis of the soil re shown in Tle 1. The experiment ws rrnge s ompletely rnomize esign (CRD) with four replitions. The tretment onsiste of 6 DAP levels (, 2.4, 4.8, 7.2, 9.6 n 12%). The seelings were hn trnsplnte from the nursery e to min pots when they h 7-8 leves on April 14 th. All plnts were irrigte immeitely fter trnsferring the seelings eqully mong pots. Irrigtion ws rrie out every y for one week to estlish the seelings in pots. There were 12 pots in totl. DAP ws sprye t 3 stges se on Rm et l. (23). First, the soil ws overe y plsti in orer to prevent DAP from eing sore into the soil. Then, plnts were sprye t the stge of stem formtion t 5 pm in the fternoon. The next y, the plsti ws remove. The seon folir sprying ws one 2 weeks lter, n the thir folir sprying ws one when flower-us forme, 35 ys lter the seon folir sprying, n plnts were t this stge for 2 weeks. Plnt height, the mount of hlorophyll, the numer n the length of xillry shoots n lef re were mesure t the full flowering stge. Lrge folir stlks were hrveste with pruning-shers, leving out 5 m ove the groun surfe. The shoots n roots fresh weight n flower numer were mesure. Also, hlorophyll ontent ws mesure y hlorophyll meter SPAD-52 (Koni-Minolt, Osk, Jpn). All smples were she-rie (uring 15 ys). EO ws extrte y sujeting flowers n leves together (5 g) to hyroistilltion for 2 h using n ll glss Clevenger-type pprtus (Golis, Tehrn, Irn), oring to the metho outline y the Europen phrmopoei (Anonymous 1996). EO yiel ws expresse s perentge w/w on ry mtter sis. The oils were rie over nhyrous N 2 SO 4 n store in sele vils t low temperture (4 C) efore nlysis. Then the oils were nlyze y GC n GC-MS. Dt were sujete to vrine nlysis n mens were ompre y using Dunn s New Multiple Rnge Test (DNMRT). 12

3 Folir pplition of immomium phosphte on Mexin mrigol. Neghn et l. Tle 1 Some physil n hemil hrteristis of the experimentl soil. EC s m -1 ph OC (%) TN (%) P (mg kg -1 ) K(mg kg -1 ) Silt (%) Sn (%) Cly (%) Orgni mtter (OC), Totl Nitrogen (TN) Plnt height (m) Lef re (mm 2 ) Numer of xillry shoots Dimmonium phosphte (%) 3 4 e Lenght of xillry shoots (m) Dimmonium phosphte (%) Flower numer Dimmonium phosphte (%) Dimmonium phosphte (%) Chlorophyll ontent Dimmonium phosphte (%) Dimmonium phosphte (%) Effet of folir pplition of DAP on plnt height (Fig. 1), lef re (Fig. 2), numer of xillry shoots (Fig. 3), length of xillry shoots (Fig. 4), flower numer (Fig. 5) n hlorophyll ontent (mg/g fresh weight of lef smple) (Fig. 6) of Tgetes minut L. Mens followe y ifferent letters re signifintly ifferent (P.5), s inite y DNMRT. Chemils DAP ws purhse from LSn (Duline, Ireln), n Anhyrous N 2 SO 4 ws purhse from Fluk (Buhs, Switzerln). The 1-hexnol use s internl stnr ws purhse from Sigm- Alrih (Buhs, Switzerln). Hyroistilltion After reoring their fresh iomss, 5 g of eh T. minut L. orgn (leves, stems, flowers n sees) were ir rie uring 15 ys. In orer to extrt their EO, they were sujete to onventionl hyroistilltion for 2 h. Then, extrtions of ll replitions elonging to eh tretment were mixe, n finl extrtions were nlyze. The hyroistilltion ws performe y simple lortory Quikfit pprtus whih onsiste of 2 ml istilltion flsk, onenser n reeiving vessel. The otine istillte ws extrte twie with n-pentne n rie over nhyrous N 2 SO 4 n store in tightly lose rk vils t 4 C until nlysis. Choie of the solvent ws se on its ility to extrt the mjor onstituents of the EO without loss of the high voltile omponents uring the onentrtion step (Hosni et l. 211). For the etermintion of the yiel, the EO ws weighe on n nlytil sle. After weighing, the whole smple ws reilute in 1 ml of the extrtion solvent n 1 μl ws susequently nlyze. Gs hromtogrphy Anlytil gs hromtogrphy ws performe using Shimzu GC-9A gs hromtogrph equippe with DB-1 fuse sili olumn (6 m.25 mm,.25 μm film thikness). Oven temperture ws hel t 4 C for 5 min n then progrmme to 28 C t rte of 4 C /min; injetor n etetor Flme Ioniztion Detetor (FID) temperture ws 29 C; rrier gs, helium with liner veloity of 32 m/s. Perentges of omponents were lulte y eletroni integrtion of FID pek res without the use of response ftor orretion. 13

4 Meiinl n Aromti Plnt Siene n Biotehnology 7 (1), Glol Siene Books Root fresh weight (g) Shoot fresh weight (g) Dimmonium phosphte (%) Dimmonium phosphte (%) Shoot ry weight (g) Root ry weight (g) Dimmonium phosphte (%) Dimmonium phosphte (%) 5 Essentil oil (w/w) Dimmonium phosphte (%) Effet of folir pplition of DAP on shoot fresh weight (Fig. 7), shoot ry weight (Fig. 8), root fresh weight (Fig. 9), root ry weight (Fig. 1) n essentil oil ontent (Fig. 11) of Tgetes minut L. Mens followe y ifferent letters re signifintly ifferent (P.5), s inite y DNMRT. Gs hromtogrphy-mss spetrometry nlyses The Gs hromtogrphy-mss spetrometry nlyses were rrie out on Vrin 34 GC-MS system equippe with DB-1 fuse sili olumn (6 m.25 mm i..). Oven temperture ws 4-25 C t rte of 4 C; trnsfer line temperture ws 26 C; rrier gs ws helium with liner veloity of 31.5 m/s; split rtio ws 1/6; ioniztion energy ws 7 ev; sn time ws 1s; mss rnge ws 4-3 mu. Ientifition of omponents The omponents of the oils were ientifie y omprison of their mss spetr with those of omputer lirry or with uthenti ompouns ville in our lortory n onfirme y omprison of their retention inies either with those of uthenti ompouns or with t pulishe in the literture (Shimoto 1987; Dvies 199; Ams 21; Sefikon et l. 24; Ms et l. 27). Quntitive t (%) were otine from the eletroni integrtion of the FID pek res without the use of the orretion ftors. Dt (EO yiels n other prmeters) were nlyze y MSTATC using ANOVA with the lest signifint ifferene (LSD) t the.5 proility level. RESULTS The t of this investigtion showe tht ll growth prmeters were positively ffete y folir pplition of DAP. The plnt height, the numer n length of xillry shoots, the numer of flowers, the fresh n ry weight of shoots, lef re n the EO yiel inrese signifintly y folir pplition of DAP s ompre to ontrol (P.1). In ition, sprying DAP ffete the EO omposition (Figs. 1-11). The totl ry mtter is n importnt riterion for rop proution. At 7.2% DAP, the height notiely inrese, n the length of plnts rehe 2.9 m, ut s DAP levels inrese, the height of plnts erese to roun m (t 12% DAP) (Fig. 1). Surprisingly, folir sprying DAP inrese lef re of T. minut when ompre to ontrol plnts (869.8 mm²). However, it inrese rmtilly to 2184 mm² (t 7.2% DAP) efore sinking to low of mm² (t 12% DAP) (Fig. 2). Although the numer of xillry shoots inrese from in ontrol plnts to 166 t 9.6% DAP, it showe ip t 12% DAP to 154 (Fig. 3). From the results of this reserh, it ppere tht DAP oul hve the positive influene on the inresing of T. minut shoot numer. It n e lerly seen tht the length of xillry shoots signifintly inrese to m t 7.2% DAP in omprison to ontrol plnts (78 m), ut t 2.4% (124.2 m), 4.8% (131 m), n 9.6% DAP (

5 Folir pplition of immomium phosphte on Mexin mrigol. Neghn et l. Tle 2 Effet of folir pplition of DAP on essentil oil omposition of Tgetes minut L. No. Constituent RI DAP (%) Sinene Z- -Oimene E- -Oimene ihyro tgetone pinene oxie is-thujone llo oimene pinene oxie E-tgetone Z-tgetone methyl sliylte Verenone Z-oimenone E-oimenone isoeugenyl phenylette trns-ryophyllene humulene Biylogermrene Cryophyllene oxie neophytiene Limonene Terpinolene is-epoxy-oimene Linlyl propionte Piperitone oxie Heptene Otene Nonene Heneiosne Doosne Triosne Groupe ompouns Monoterpene hyrorons Oxygente monoterpenes Sesquiterpene hyrorons Oxygente sesquiterpenes Others Retention Inex (RI), -: not etete m) length of xillry shoots i not show signifint ifferenes (Fig. 4). As regrs to the numer of flowers, it showe n upwr tren. Control plnts h the lest numer of flowers (212). However, it peke t 2771 t 2.4% DAP. The numer of flowers rehe mximum t 9.6% DAP (3287), ut there were not signifint ifferene etween the numer of flowers t 9.6% n other levels of DAP (Fig. 5). On the other hn, hlorophyll ontent flutute signifintly with inresing levels of DAP. The mount of hlorophyll in ontrol plnts ws though it erese to t 4.8% DAP n rehe 32 t 7.2% DAP (Fig. 6). Aoring to the fresh weight of shoots, ontrol plnts h the lest mount of fresh weight of shoots (239.5 g), n sprying 2.4% DAP inrese the fresh weight to g. Though t 4.8 n 7.2% DAP, the fresh weight of shoots inrese to out 31 g (Fig. 7). Regring the ry weight of shoots, it ws low-nerly 8.5 g in ontrol plnts, while it inrese t higher onentrtions n rehe g t 2.4% DAP, whih n e enefiil for the proution of EO. Afterwrs, 12% DAP erese the ry weight of shoots to g, ut there were no signifint ifferene etween ry weight of shoots in ontrol plnts with those of t 7.2, 9.6 n 12% DAP (Fig. 8). In terms of the fresh weight of root, it showe some flututions. The ontrol plnts h the mximum fresh weight of root (79 g). However, it erese t 2.4 n 4.8% DAP to n g, respetively n then inrese to g t 12% DAP (Fig. 9). Aoring to the ry weight of root, Fig. 1 shows rop in the ry weight of root whih erese from to 22 g in ontrol plnts n 4.8% DAP, respetively, ut it rehe 33 g t 2.4% DAP n erese to 17.5 g t 9.6% DAP. On the other hn, the most importnt hrteristi of meiinl plnts is the EO yiel. Fortuntely, EO of T. minut in this experiment inrese signifintly. As mtter of ft, there ws grul inrese in the EO yiel with the inrese of DAP level (Fig. 11). The EO yiel ws.85% in ontrol plnts. Afterwrs, it peke 1.35% t 12% DAP. As result, there ws signifint ifferene etween the EO yiel in ifferent DAP levels, n the highest mount ws otine t 9.6% DAP. Generlly, folir sprying of 9.6% DAP inrese the iosynthesis of the EO in the leves to the extent of.5% over the ontrol whih ws of signifint importne. EO omposition The list of etete ompouns with their retention inies n reltive perentges re given in Tle 2. The results of this stuy in sitution of onstituents of the EO showe tht there were 2 ompouns in the EO of T. minut (Tle 2). Min ompouns of EO of Mexin mrigol (ompouns >4%) were ihyro tgetone, Z-tgetone, Z- -oimene n Z-oimenone. DAP pplition inrese the mount of ihyrotgetone n Z-tgetone nt 12% of DAP, the mount of these ompouns rehe 76.4% n 11.19%, respetively. On the other hn, DAP erese Z- -oimene n Z-oimenone ontent, lthough ontrol plnts h the mximum ontent of these ompouns roughly 13.23% n 5.53% respetively. Other ompouns were in tgetes oil inluing sinene, -pinene oxie, E- tgetone, E- oimenone, trns-ryophyllene, -humulene, iylogermrene, ryophyllene oxie, neophytiene, isoeugenyl phenylette, verenone, methyl sliylte, is-thujone, 15

6 Meiinl n Aromti Plnt Siene n Biotehnology 7 (1), Glol Siene Books llo oimene n E- -oimene. The most effetive DAP levels were 7.2% n 9.6% for the proution of these importnt ompouns. Dihyro tgetone is the min ompoun of the EO of Mexin mrigol n rehe its highest mount t 12% of DAP. Previous report showe tht T. minut EO nlyze y GC-MS ws ompose minly y Z- -oimene (4.42%), ihyro tgetone (17.64%), Z-tgetone (9.96%), E-tgetone (1.57%), E-tgetenone (5.3%) (Lwrene et l. 1985). It is well oumente tht phosphorus is n essentil element in reproutive growth of plnts (Mrshner 1986) n thus, the vegettive growth n the numer of flowers inrese with folir sprying of DAP whih ws expete in our stuy. Phosphorus is lso known to hve multifrious ellulr funtions in plnts, inluing: signlling n trnsmemrne metoli flux n therefore, the seonry metolism is moulte y these mehnisms (Rm et l. 23). In onlusion, it ppers tht phosphorus is ruil nutrient element for Mexin mrigol ultivtion. DISCUSSION Effet of DAP on plnt height Results showe tht plnt height ws signifintly (P.5) ffete y folir sprying of DAP. Plnt height is one of the most importnt ftors, n the tllest height ws hieve t 7.2% DAP (2.9 m). It showe tht DAP oul hve positive effet on length of plnts. Aoring to reserh rrie out y Nssr et l. (24). The plnt height n the numer of rnhes of hmomile were signifintly inrese with inresing pplition of phosphorus. Singh et l. (28) revele the positive effet of phosphorous on the plnt height of Clenul offiinlis L. The effet of DAP phosphte on growth my e ue to the tivity of phosphte soluiliztion use y the strin n inrese further minerl vilility uptke. Effet of DAP on lef re Results showe tht mong the DAP tretments, the lrgest lef re ws reore t 7.2% DAP, rising to mm² t 7.2% DAP. The spry of immonium phosphte inrese lef re n the rte of photosynthesis in the leves of menthol mint (Menth rvensis), whih ultimtely fvoure the iosynthesis of monoterpenes in seonry metolite plnts (Rm et l. 23). Effet of DAP on the numer of xillry shoots DAP inrese the numer of xillry shoots, n t 9.6% DAP, the numer of shoots peke 166. Stuies of El-Ghnour et l. (29) emonstrte tht growth prmeters of Mjorn hortensis L. suh s the numer of rnhes were positively ffete y pplition of phosphorous. Some reserhers showe tht the inrese in growth hrters might e ue to the ft tht phosphte soluilizing teri inoulte plnts were le to sor nutrients from solution t fster rtes thn un-inoulte plnts (Hshemi et l. 212) Effet of DAP on the length of xillry shoots The length of xillry shoots t 2.4% (124.2 m), 4.8% (131 m), 7.2% (136.1 m) n 9.6% (119.5 m) DAP signifintly inrese in omprison to ontrol plnts (78 m). Effet of DAP on the numer of flowers The effet of folir sprying of DAP on the numer of ws remrkle. The lest numer of flowers went to ontrol plnts (212) though t 9.6% DAP, the numer of flowers lepe new pek (3287). It is well oumente tht phosphorus is n essentil element in reproutive n vegettive growth of plnts (Mrshner 1986) n thus, the vegettive growth n flower numers stimultion inrese y pplie DAP in ws expete in our stuy. Effet of DAP on hlorophyll ontent As regrs hlorophyll ontent, it vrie wiely. It ws out mg/g fresh weight lef, ut t 4.8% DAP it erese signifintly to mg/g fresh weight lef. However, the mount of hlorophyll peke t 32 mg/g fresh weight lef t 7.2% DAP. Effet of DAP on fresh weight of shoots The DAP pplition inrese signifint (P.5) shoot fresh weight, n t 4.8 n 7.2% DAP shoot fresh weight inrese to roun 31 g. However, the lowest shoot fresh weight ws otine in ontrol plnts (239.5 g). Effet of DAP on fresh weight of root Regring root fresh weight, ontrol plnts h the highest mount of fresh weight (79 g), lthough it erese with folir sprying of DAP, n ry weight hit the lowest point t 4.8% (35.25 g) DAP. Effet of DAP on ry weight of root Aoring to the results, the sme s root fresh weight, ontrol plnts h the highest root ry weight (42.75 g), n pplition of DAP erese root ry weight. In ft, root ry weight snk to out 17.5g t 9.6 n 12% DAP. Effet of DAP on EO The oil of T. minut hs een investigte y Meshktlst et l. (21), who ientifie Z- -oimene, ihyrotgetone, Z- n E-tgetone, n Z- n E-oimenone s the mjor omponents. The results of our nlysis show tht oil of T. minut ws prtiulrly rih in ihyro tgetone, Z- tgetone, Z- -oimene n Z-oimenone. However, there re reports showing tht perentge of minor onstituents vries. For exmple -phellnrene n o-ymene were the mjor oil omponents from Argentin (Gill et l. 2). The vritions in hemil omposition oul e ue nture of the soil, the mount of sunlight n temperture vritions n the ourrene of hemotypes (Meshktlst et l. 21). Thus, we elieve tht intrinsi n externl ftors oul hve ffete the ontent n omposition of the oil of T. minut. These results re similr to those of Slrini et l. (1994) with pyrethrum (Tnetum inerriifolium) who reporte tht pplition of DAP inrese signifintly henes n oil yiel for this rop (Slrini et l. 1994). They re lso in greement with the t of Nikolov et l. (1999) n with Nile et l. (25) who oserve inresing iomss of hmomile. Phosphorus is lso known to hve multifrious ellulr funtions in plnts, inluing: signling n trnsmemrne metoli flux n therefore, the seonry metolism ws moulte y these mehnisms (Rm et l. 23). Shrkhiz n Omiigi (28) reporte tht pplition of phosphorus in feverfew (Tnetum prthenium L.) inrese signifintly the growth suh s height, numer of flowers, shoot fresh weight n EO. Trivino n Johnson (2) reporte tht totl yiel of voltile oil of Orignum mjorn L. ws inrese y pplition of phosphorus. Moreover, shoot fresh weight ws inrese two-fol y phosphor tretment s ompre to the ontrol. Ihimur (1995) oserve tht DAP inrese signifintly the fresh weight n essentil oil onentrtion in Sweet sil. Similr results hve een note with lk umin (Nigell stiv) n oriner (Corinrum stivum) (Ds et l. 1991, Ughrej n Chunwt 1992). Folir pplition of DAP my filitte llevition of intrellulr inorgni phosphte efiieny fvoring fster 16

7 Folir pplition of immomium phosphte on Mexin mrigol. Neghn et l. flux through the norml glyolyti step, whih is energy onservtion n phosphoenolpyruvte vilility re highly onutive to iosyntheti proesses inluing seonry metolite nolism. In ft, phosphoenolpyruvte oupies key position in serving s n isoprenogeni sustrte for voltile oils in plstis (Mhmou n Croteu 22). Aoringly, it is speulte tht folir sprying DAP my help plnts to hve ner norml moe of metolism. This my e one of the working moels for future experimenttion. In onlusion, it ppers tht P is ruil nutrient element for Mexin mrigol ultivtion. Therefore, it is strongly reommene tht on sites low in ville P, the rop oul e supplie with equte P. Furthermore, we suggest tht the influene of DAP on the growth, hemil omposition n iohemil inies of Tgetes minut L. e thoroughly stuie on lotions with wie rnge of limtology, physil n hemil properties n minerlogil hrteristis. ACKNOWLEDGEMENTS This work is eite to the memory of Dr. Shrreh Njfin for proviing T. minut L. n her ollortion. We express our grtitue to Mr. Mehi Hmiin euse he let us to rry out this reserh t his greenhouse. Finlly, the uthors thnk Dr. Jime A. Teixeir Silv for improving the grmmr, style n figure qulity. REFERENCES Ael S, Tioni CA, Deltorr CA (22) Phosphte sensing in higher plnt. Physiologi Plntrum 115, 1-8 Ams RP (21) Ientifition of Essentil oil Components y Gs Chromtogrphy/Qurupole Mss Spetrosopy, Allur Pulishing Corp., Crol Strem, IL, USA, 455 pp Anonymous (1996) Europen Phrmopoei (3 r En), Pulisher, Lonon, pp Arif S, Alhmmi A (28) Allelopthi effet of Tgetes minut L. wter extrt on sees germintion n seeling root growth of Ai sk. The Assiut University Bulletin for Environmentl Reserhes 11 (1), Atkinson RE, Curtis RF, Phillips GT (1964) Bi-thienyl erivtives from Tgetes minut L. Tetrheron Letters 43, Azhr H, Mlik A, Khn A, Shh M, Muhmm P (24) Spinosie, new oumroyl flvone glyosie from Amrnthus spinosus. Journl of Chemil n Phrmeutil Reserh 27 (12), Bu KGD, Kul VK (27) Vritions in quntittive n qulittive hrteristis of wil mrigol (Tgetes minut L.) oils istille uner vuum n t NTP. Inustril Crops n Prouts 26, Bu GDK, Kul VK (25) Simple, portle mini istilltion pprtus for the proution of essentil oils n hyrosols. US Ptent Nº 6, 911, 119B2. CSIR, June 28 Btish DR, Aror K, Singh HP, Kohli KR (27) Potentil utiliztion of rie power of Tgetes minut s nturl heriie for mnging rie wees. Crop Protetion Mngement Plns 26, Btten KM, Kte-Sow M, Keni-Dvies F, Hrrison P (26) Two invsive plnts lter soil miroil ommunity omposition in serpentine grsslns. Journl of Biologil Invsions 8, Beerr J, Bittner M, Hernánez V, Brimtrup C, Silv M (21) Ativi e eites eseniles e nelo, queule ilhuen y ulén frente hongos fitoptógenos. Ltin Amerin n Crien Bulletin of Meiinl n Aromti Plnts 9, Buitrgo Diz A, Rojs Ver J, Rojs Fermin L, Morle Menez A, Apriio Zmrno R, Roriguez Contrers L (211) Composition of the essentil oil leves n roots of Allium shoenoprsum L. (Alliee). Ltin Amerin n Crien Bulletin of Meiinl n Aromti Plnts 1, Céspees CL, Avil JG, Mrtinez A, Serrto B, Clerón-Mugi JC, Slgo-Grigli R (26) Antifungl n Antiteril tivities of Mexin mrigol (Tgetes minut L.). Journl of Agriulturl n Foo Chemistry 54, Chlht JC, Grry RP, Muhyimn A (1995) Essentil oil of Tgetes minut from Rwn n Frne: hemil omposition oring to hrvesting lotion, growth stge n prt of plnt extrte. Journl of Essentil Oil Reserh 4 (4), Crveiro CC, Mtos FJA, Mho MIL, Alenr JW (1988) Essentil oils of Tgetes minut from Brzil. Perfum Flvor 13 (5), Ds AK, Shu MK, Som MG (1991) Effet of N n P levels on growth n yiel of lk umim (Nigell stiv L.). Journl of Hortiulture 4, 41-7 Dvies NW (199) Gs hromtogrphi retention inies of monoterpenes n sesquiterpenes on methyl silion n rowx 2M phses. Journl of Chromtogrphy A 53, 1-24 Defule RJJ, Rushing R, Hssell BM, Shepr G, Wr B (23) Influene of fertilizer on growth n mrker ompoun of fiel-grown Ehinee speies n feverfew. Sienti Hortiulture 98, Dole JM (1999) Floriulture: Priniples n Speies, Prentie-Hll In., Upper Sle River, New Jersey, pp El-Ghnour IA, Desouky EM, Gll YGM, Arf RA, Aou Seer AMM (29) Effet of phosphorus on growth n essentil oil of mrjorm (Mjorn hortensis L.). Egyptin Aemi Journl of Biologil Siene 1 (1), Gill A, Ghers CM, Leih S (2) Essentil oil yiel n omposition of Tgetes minut essions from Argentin. Biohemil Systemti n Eology 28, Gillij YG, Gleiser RM, Zyglo JA (28) Mosquito repellent tivity of essentil oils of romti plnts growing in Argentin. Journl of Bioresoure Tehnology 99, Hmwi MH, Youssef MMA, Zwm HS (24) Mngement of Meloiogyne inognit, the root-knot nemtoe, on soyen s ffete y Mexin mrigol n se mrosi (msis plnts). Journl of Pest Siene 77, Hrley JL (1971) Myorrhiz, Oxfor University Press, Lonon Hshemi D, Zreost F, Brri Ziyri M, Zrhini M, Kvini B, Ji Solimnri M, Torkshvn AM, Zrhini S (212) Influene of phosphte on quntity n qulity fetures of mrigol (Tgetes eret L.). Austrlin Journl of Crop Siene 6 (6), Hethelyi E, Dnos B, Tetenyi P (1986) GC-MS nlysis of the essentil oils of four Tgetes speies n the nti-miroil tivity of Tgetes minut. Flvour n Frgrne Journl 1, Hosni K, Ms K, Chhe T, Ben Trit M, Mrzouk B (211) Comprtive nlysis of the essentil oils of Hyperium triquetrifolium Turr. Extrte y ultrsoun, hyroistilltion n Soxhlet/ynmi hespe. Meiinl n Aromti Plnt Siene n Biotehnology 5 (1), -14 Huson JB (199) Antivirl Compouns from Plnts, CRC Press, INC, Bo Rton, FL Hulin N (28) Wil mrigol - Tgetes minut L., new wee on the Isln of Hvr, n new ontriution to the knowlege of its istriution in Dlmti (Croti). Agriulture Conspetus Sientifius 73, Ihimur M, Ikushim M, Miyzki T, Kimur M (1995) Effet of DAP on growth n onentrtion of minerl elements n essentil oils of sweet sil leves. At Hortiulture 396, Jeffrey C, Kereit JW (27) Composite: Introution with key to tries. Fmilies n gener of vsulr plnts. In: Kereit JW, Jeffrey C (Es) Flowering Plnts VIII, Springer, Berlin, pp Kákoniová D, Mú ková M, Mllrová M, Kuliková Z (26) Biotehnologil proution of seonry metolites y Txus t L. in vitro. In: Teixeir Silv JA (E) Floriulture, Ornmentl n Plnt Biotehnology: Avnes n Topil Issues (Vol 4, 1 st En), Glol Siene Books Lt, Isleworth, UK, pp Kterere D, Grzini G, Themo K, Nyzem N, Ritieni A (212) Antioxint tivity of some Afrin meiinl n ietry lefy Afrin vegetles. Afrin Journl of Biotehnology 11 (17), Kterere D, Eloff J (25) Mngement of ietes in Afrin tritionl meiine. Journl of Antiieti Plnts 6, Kéit SM, Vinent C, Shmit JP, Rmswmy S (2) Effet of vrious essentil oils on Cllosoruhus multus (Coleopter: Bruhie). Journl of Store Prouts Reserh 36, Krueger R, Dover KE, MSorley R, Wng KH (21) Mrigols (Tgetes spp.) for nemtoe mngement. Journl of Foo n Agriulturl Sienes 5, 1-8 Lwrene BM (1996) Tgetes oil. Perfumer n Flvorist 21 (126), Mgw ML, Guniz M, Gweru N, Humphrey G (26) Chemil omposition n iologil tivities of essentil oil from the leves of Sesuvium portulstrum. Journl of Ethnophrmology 13, Mhmou S, Croteu R (22) Strtegies for metoli engineering of monoterpene iosynthesis in plnts. Trens in Plnt Siene 7, Mrshner H (2) Minerl Nutrition of Higher Plnts, Aemi Press, Lonon, 195 pp MVugh R (1943) Botnil olletions of the L Plt expeition of Brittoni 5 (1), Meshktlst MH, Sfei-Ghomi J, Mohrrmipour S, Nseri M (21) Chemil hrteriztion of Tgetes minut L. ultivte in south west of Irn y nno sle injetion. Digest Journl of Nnomterils n Biostrutures 5 (1), Moreno J, Rojs LB, Apriio R, Mró L-M, Usuillg A (21) Chemil omposition of the essentil oil from the rk Burser tomentos (Jq) Tr & Plnh. Ltin Amerin n Crien Bulletin of Meiinl n Aromti Plnts 9, Ms K, Hosni K, Ben Trit M, Chhe T, Khouk Me, Mrzouk B (27) Chnges on essentil oil omposition of oriner (Corinrum stivum L.) fruits uring three stges of mturity. Foo Chemistry 12, Nssr AH, Hshim MF, Hssn NS, Aozi H (24) Effet of gmm irrition n phosphorus on growth n oil proution of hmomile (Chmo- 17

8 Meiinl n Aromti Plnt Siene n Biotehnology 7 (1), Glol Siene Books mill reutit L. Rushert). Interntionl Journl of Agriulture n Biology 6 (5), Nell M, V tsh M, Vierheilig H, Steinkellner S, Zitterl-Eglseer K, Frnz CH, Novk J (29) Effet of phosphorus uptke on growth n seonry metolites of gren sge (Slvi offiinlis L.). Journl of the Siene of Foo n Agriulture 89, Nikolov A, Kozhuhrov AK, Zheljzkov VD, Crker LE (1999) Minerl nutrition of hmomile (Chmomill reutit L.). At Hortiulture 52, Nile CM, Vieir CM, Néstor A, Herei Z, Siqueir JM (25) Biomss proution of hmomile pitul s result of nitrogen n phosphorus. Hortiulturl Biotehnology in Brzil 23 (1), Perkins J (1912) Beitrge Zur flor Von Boliv. Plnt Diversity n Evolution 49 (1), Pritekel C, Whittemore-Olson A, Snow N, Moore JC (26) Impts from invsive plnt speies n their ontrol on the plnt ommunity n elow groun eosystem t Roky Mountin Ntionl Prk, USA. Journl of Applie Soil Eology 32, Rm M, Singh R, Rm D, Sngwn RS (23) Folir pplition of phosphte inrese the yiel of essentil oil in menthol mint (Menth rvensis). Austrlin Journl of Experimentl Agriulture 463, Rees A (E) (1817) Tgetes. In: The Cylopei, Lonon, 35 (1), Reihe C (193) Estuios ritios sore l flor e Chile. Anles e l Universi e Chile, 112, Rios JL, Reio MC (25) Meiinl plnts n ntimiroil tivity. Journl of Ethnophrmology (2), 8-84 Roriguez E, Mry TJ (1977) Tgetee-hemil review. In: Heywoo VH, Hrorne JB, Turner BL (Es) The Biology n Chemistry of Composite, Aemi Press, Lonon, pp Shrkhiz MJ, Omiigi R (28) The effet of phosphorus on the proutivity of feverfew (Tnetum prthenium L. Shultz Bip). Avnes in Nturl n Applie Sienes 2 (2), Slrini AA, Chpmn KSR, Hollowy RJ (1994) Effet of sl n sie resse phosphorus on the hene yiel n pyrethrines onentrtion in the in the henes of pyrethrum (Tnetum inerrifolium) n on soil n plnt phosphorus. Austrlin Journl of Agriulturl Reserh 45, Sngwn NS, Frooqi AHA, Shih F, Sngwn RS (21) Regultion of essentil oil proution in plnts. Plnt Growth Regultion 34, 3-21 Sroglou V, Dorizs N, Kypriotkis Z, Sklts HD (26) Anlysis of the essentil oil omposition of eight Anthemis speies from Greee. Journl of Chromtogrphy A 114, Srivnti LR, Zunino MP, Zyglo JA (26) Tgetes minut L. n Shinus reir L. essentil oils s llelopthi gents. Ltin Amerin n Crien Bulletin of Meiinl n Aromti Plnts 7 (3), Sefikon F, Slehyr S, Mirz M, Diri M (24) The essentil oil of Tgetes eret L. ourring in Irn. Flvour n Frgrne Journl 19, Shimoto T (1987) Retention inies in essentil oil nlysis. In: Snr P, Bihi P (E) Cpillry Gs Chromtogrphy in Essentil Oil Anlysis, Huethig, New York, pp Shhzi I, Amj H, Ummr WKH, Mroof MSH (21) Evluting iologil tivities of the see extrts from Tgetes minut L. foun in Northern Pkistn. Journl of Meiinl Plnts Reserh 4 (2), Singh YP, Dwivei R, Dwivei SV (28) Effet of phosphorus n nitrogen on growth n flower yiel of lenul (Clenul offiinlis). Plnt Arhives Journl 8 (2), Singh B, Singh V (22) Crop proutivity n vrition in hemil omposition of Tgetes minut L. essentil oil n solute uring rop mturity in Mi-Hill of western Himlyn region. Journl of Essentil Oil-Bering Plnt 5, 3-37 Singh HP, Btish DR, Kohli RK (23) Allelopthi intertions n llelohemils: New possiilities for sustinle wee mngement. Critil Reviews in Plnt Sienes 22, Soule JA (1993) Tgetes minut: A potentil new her from South Ameri. In: Jnik J, Simon JE (Es) New Crops, Wiley, New York, pp Teixeir Silv JA (23) Anthemiee: Avnes in tissue ulture, genetis n trnsgeni iotehnology. Afrin Journl of Biotehnology 2 (12), Teixeir Silv JA (24) Mining the essentil oils of the Anthemiee: A review. Afrin Journl of Biotehnology 3 (12), Teixeir Silv JA, Yonekur L, Kgn J, Mooksnit J, Nhut DT, Afh G (25) Importnt seonry metolites n essentil oils of speies within the Anthemie (Asteree). Journl of Hers, Spies n Meiinl Plnts 11 (1/2), 1-46 Tomov BS, Wterhouse JS, Doerski J (25) The effet of frtionte Tgetes oil voltiles on phi reproution. Entomologi Experimentlis et Applit 115, Trivino MG, Johnson CB (2) Seson hs mjor effet on the essentil oil yiel response to nutrient supply in Orignum mjorn. Journl of Hortiulturl Siene n Biotehnology 75 (5), Ughrej PP, Chunwt BS (1992) Nutritionl stuies in oriner (Corinrum stivum L.). Effet on growth n proution. Gujrt Agriulturl University Reserh Journl 17, Urzuá A, Sntner R, Eheverriá J, Villloos C, Plios SM, Rossi Y (21) Insetiil properties of Peumus olus Mol. essentil oil on the house fly, Mus omesti L. Ltin Amerin n Crien Bulletin of Meiinl n Aromti Plnts 9, Vázquez AM, Demmel GI, Crio SG, Aimr ML, Cntero JJ, Rossi LI, Velso MI (211) Phytohemistry of Tgetes minut L. (Asteree) from Córo, Argentin: Comprtive stuy etween essentil oil n HS-SPME nlyses. Ltin Amerin n Crien Bulletin of Meiinl n Aromti Plnts 1 (4), Wng KH, Sipes BS, Shmitt DP (23) Mngement of Rotylenhulus reniformis in pinepple y interyle over rops. Journl of Nemtology 34,