Swiss Federal Research Station Agroscope Changins Wädenswil AC. CH-1260 Nyon (Switzerland).

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1 B A S E Biotechnol. Agron. Soc. Environ. 16 (4), Evlution of the llelopthic potentil of wter-solule compounds of rley (Hordeum vulgre L. susp. vulgre) nd gret rome (Bromus dindrus Roth.) using modified iossy Imen Bouhouel (1, 2), Aurélie Gfeller (1, 3), Mrie-Lure Fuconnier (4), Benjmin Delory (1), Hjer Slim Amr (2), Ptrick du Jrdin (1) (1) University of Liège - Gemloux Agro-Bio Tech. Plnt Biology Lortory. Pssge des Déportés, 2. BE-5 Gemloux (Belgium). E-mil: imenouhouel@gmil.com (2) University of Crthge. Ntionl Agronomic Institute of Tunisi. Deprtment of Agronomy nd Plnt Biotechnology. Genetics nd Cerel Breeding Lortory. Chrles Nicolle Street, 43. TN-82 Tunis Mhrjène (Tunisi). (3) Swiss Federl Reserch Sttion Agroscope Chngins Wädenswil AC. CH-126 Nyon (Switzerlnd). (4) University of Liège - Gemloux Agro-Bio Tech. Generl nd Orgnic Chemistry Lortory. Pssge des Déportés, 2. BE-5 Gemloux (Belgium). Received on Decemer 29, ; ccepted on June 16, 16. Description of the suject. The present study focuses on the description of the llelopthic interctions etween wild nd crop species tht my occur in given ecosystem. Ojectives. The ojective is the evlution of the llo- nd utoinhiition ctivity of root exudtes of rley (Hordeum vulgre L. susp. vulgre) nd gret rome (Bromus dindrus Roth.) seedlings y wter-solule llelochemicls. Method. The llelopthic ctivities of five Tunisin rley genotypes (modern vrieties nd lndrces), one Sudi Arin rley lndrce nd gret rome were ssessed using modified lortory iossy nmed seedling-fter-seedling gr method. Results. The rley or the gret rome reduced, to greter extent, the root growth compred to the shoot growth of receiver species. The response of the root system rchitecture of the gret rome towrds rley root exudtes ws studied in detil. All the mesured root trits were highly sensitive to the presence of rley. In our conditions, the llelopthic ctivity of rley root exudtes hd no pprent reltionship with the size of the root nd prominent ction of genetic determinnts in the llelopthic potentil etween genotypes is proposed. The lloinhiitory ctivity of rley or gret rome root exudtes deferred etween the receiver species ut ws lwys higher thn the utoinhiition potentil. The utoinhiition in rley proved to depend on whether the genotypes used s donor nd receiver re identicl or different, suggesting specific interction of llelochemicls with the receiver plnt. These molecules seem to e the min ctors in the llelopthic rley potentil s externl fctors such vritions of ph hve no evident relevnce in the inhiition process. Conclusions. Brley nd gret rome exude molecules in their surroundings. This ffects the growth of the receiver plnts, suggesting tht these compounds might contriute to the plnt community dynmics. Keywords. Allelopthy, Hordeum vulgre, Bromus dindrus, ph, root exudtes, root systems. Évlution du potentiel llélopthique des composés hydrosolules de l orge (Hordeum vulgre L. susp. vulgre) et du grnd rome (Bromus dindrus Roth.) moyennnt un io-essi modifié Description du sujet. L présente étude se foclise sur l description des interctions llélopthiques entre des espèces suvges et cultivées qui peuvent survenir dns un écosystème donné. Ojectifs. L ojectif est l évlution de l ctivité d uto- et d lloinhiition des exsudts rcinires de l orge (Hordeum vulgre L. susp. vulgre) et du grnd rome (Bromus dindrus Roth.) vi les llélochimiques hydrosolules. Méthode. Les ctivités llélopthiques de cinq génotypes tunisiens d orge (vriétés modernes et orges locles), d une orge locle d Arie soudite et du grnd rome ont été évluées moyennnt un io-essi modifié nommé «seedling-fter-seedling gr method».

2 Allelopthic potentil of rley nd gret rome 483 Résultts. L orge et le grnd rome ont réduit dns une grnde mesure l croissnce des rcines des espèces receveuses comprtivement à celle des pousses. L réponse de l rchitecture du système rcinire du grnd rome à l égrd des exsudts rcinires de l orge été étudiée en détil. Il s est véré que tous les trits des rcines nlysés ont été très sensiles en présence de l orge. Dns nos conditions, l ctivité llélopthique des exsudts rcinires de l orge n vit ucune reltion pprente vec l tille des rcines et une ction prépondérnte des déterminnts génétiques dns le potentiel llélopthique entre les génotypes est insi proposée. L ctivité d lloinhiition des exsudts rcinires de l orge ou du grnd rome étit différente entre les espèces receveuses mis toujours plus élevée pr rpport u potentiel d utoinhiition. L utoinhiition chez l orge été dépendnte des génotypes utilisés comme donneurs et receveurs qui étient identiques ou différents, suggérnt insi une interction spécifique des llélochimiques vec l plnte receveuse. Ces molécules semlent être les principux cteurs du potentiel llélopthique chez l orge, étnt donné que les fcteurs externes comme les vritions du ph n ont ucune pertinence évidente dns le processus d inhiition. Conclusions. L orge et le grnd rome relâchent dns leur environnement des molécules qui ffectent l croissnce des plntes receveuses, suggérnt insi que ces composés pourrient contriuer à l dynmique des communutés végétles. Mots-clés. Allélopthie, Hordeum vulgre, Bromus dindrus, ph, exsudt rcinire, système rcinire. 1. INTRODUCTION Negtive interctions mong plnts re medited y two iologicl phenomen: competition for limited resources, such s spce, light, wter, or nutrients, nd llelopthy, the chemicl interction etween plnts (Olofsdotter et l., 2). The llelopthy phenomenon includes oth hrmful nd eneficil iochemicl interctions etween ll types of plnts y the production of secondry metolites, commonly clled llelochemicls, into the surrounding environment (Rice, 1984). Allotoxicity nd utotoxicity refer to situtions where the donor plnt, relesing llelochemicl inhiitors, nd the receiver plnt, showing reduced seed germintion nd/or plnt growth s response to these llelochemicls, elong to different or the sme species, respectively (Putnm, 1985; Singh et l., 1999). These chemicl interctions re ssumed to ply significnt roles t the community nd ecosystem levels, prticipting to plnt fitness in oth nturl nd mnged ecosystems (Chou, 1999). In groecosystems, frming prctices, i.e. monoculture or overseeding (Chon et l., 6), were oserved to fvor utotoxicity, resulting in soil-sickness nd losses in plnt productivity nd crop yield (Singh et l., 1999). Regrding llotoxicity, the llelopthic potentil of crop plnts ws exploited y lterntive nd eco-friendly weed control strtegies (Belz, 7). Allelopthic weeds cn lso ffect crops in numer of wys, like delying or preventing seed germintion nd reducing seedling growth. Most llelopthic evidence hs seprtely focused on the effect of crops on weeds, crops on crops or weeds on crops (Chon et l., ), ut the scientific literture is rther poor concerning the description of ll the llelopthic interctions in given ecosystem, more especilly through the root exudtes. Furthermore, the uto- nd lloinhiition ctivities of root exudtes were never compred in wild or in crop species. Despite the ecologicl nd gronomic importnce of llelopthy, the mechnisms involved re still poorly understood. Roots hve the cpcity to lter oth physicl nd chemicl chrcteristics of the rhizosphere (Drrh, 1993), which in comintion my prticipte to the llelopthic effects. For exmple, the root exudtes-medited ph chnge is criticl chemicl prmeter tht cts on plnt growth y ffecting the vilility of nutrients nd toxic elements in the soil (Hinsinger et l., 3). Lower ph vlues tend to decrese the vilility of phosphorus, clcium, potssium or mgnesium (Fgeri et l., 1989; Rjn et l., 1997) nd to increse the soluility of luminum, cdmium or mngnese (Hue et l., 1; Shmsi et l., 7). Acidic environments my lso inhiit plnt growth y ffecting the plsmlemm of root cells (Brix et l., 2). In ddition, the ph hs een shown to ply significnt role in the uptke of phenolics which include mny llelochemicls nd in the expression of their toxicity (Blum et l., 1985). Similrly, the vrition of ph impcts enzymtic ctivity (Singh et l., 14). It is noteworthy tht most llelopthic studies descriing the toxicity of root exudtes filed to distinguish etween the effects of ph vrition nd the direct toxicity of the relesed compounds. Allelopthic properties hve een demonstrted in rley (Hordeum vulgre L. susp. vulgre), like in mny cerel crops, nd shown to involve diverse mechnisms such s residue decomposition from vrious plnt tissues (Guels et l., 1989), root exudtion (Bertholdsson, 4; Bouhouel et l., ), voltile orgnic compounds (VOCs) emissions from ove nd elowground prts of the plnt (Ninkovic, 3; Gfeller et l., 13). Brley residues re utotoxic, mking rley-rley cropping sequence inpproprite (Oueslti et l., 5). This species produces complex mixture of llelochemicls (phenolics, lkloids, cynoglucosides nd polymines)

3 484 Biotechnol. Agron. Soc. Environ. 16 (4), Bouhouel I., Gfeller A., Fuconnier M.-L. et l. tht seems to e specilly diversified, s compred with e.g. whet (Bghestni et l., 1999; Kremer et l., 9). Altogether, these oservtions mke rley n interesting model of llelopthy in crop plnts. In this pper, we hve evluted the chemicl interctions vi wter-solule compounds of root exudtes etween nd within cultivted rley (Hordeum vulgre L. susp. vulgre) nd gret rome (Bromus dindrus Roth., syn. Bromus rigidus Roth. susp. gussonii Prl.), troulesome grssy weed. This weed is lrgely distriuted in Tunisin cerel crops nd cuses significnt ( 5%) yield losses (Souissi et l., 1). The description of the llelopthic interctions etween these crop nd wild species would llow to determine the most efficient genotypes of rley tht might e recommended for frmers or considered in reeding progrms of Tunisin cultivted rley for iologicl weed control. This study imed lso to understnd some underlying llelopthic mechnisms, e.g. the role of the ph s influenced y root exudtes, or the vigor of root system in the llelopthic process. With this im, we hve dopted the seedlingfter-seedling gr method, n experimentl protocol modified from the equl-comptment-gr method of Wu et l. (), where the donor nd the receiver species of llelochemicls re grown sequentilly, to ssess the llelopthic potentil of wter-solule compounds nd to minimize resource competition. As reported y Nilsson (1994), it is indeed chllenging to distinguish llelopthic effects from resource competition. This choice ws sed on previous work which focused on the comprison of seed-toseed nd seed-fter-seed experimentl protocols (Bouhouel et l., ). The present method llows us: to determine the llelopthic potentil of wtersolule compounds of donor species on the receiver species in more dvnced stge of growth s compred to the seed-fter-seed protocol, to expose the receiver species for longer time to llelochemicls, to monitor root growth nd rchitecture of receiver species developing in the wter gr medium. In ddition, the seedling-fter-seedling gr method is suitle protocol for the common chickweed (Stellri medi L.) used s weed reference (Overlnd, 1966) nd receiver species of rley compounds. This test ws impossile using the seedto-seed (photodormnt seeds) nd hs not yielded conclusive results using the seed-fter-seed. In this work, we ssess: the llotoxicity potentil of rley (Hordeum vulgre L.) nd gret rome (Bromus dindrus Roth.) root exudtes on ech other medited y wter-solule llelochemicls using modified iossy, the vrition in root system rchitecture (RSA) of gret rome in response to rley root llelochemicls considering tht root rchitecture is highly plstic nd environmentlly responsive trit (Kellermeier et l., 13), the utotoxicity effect in oth species, the possile reltionship etween root vigor of different rley genotypes nd their llelopthic potentil, the importnce of ph on the expression of the llelopthic potentil. 2. MATERIALS AND METHODS 2.1. Plnt mterils Seeds of five Tunisin rley (Hordeum vulgre L. susp. vulgre) genotypes, nd one genotype introduced in Tunisi from Sudi Ari ( Sudi ) were otined from the Ntionl Agronomic Institute of Tunisi (Tle 1). The most cultivted modern vrieties, Rihne nd Mnel, were chosen in this study (El Felh, 11; Ghri et l., 13). In ddition, the modern vriety Tej nd rley lndrces etter dpted to locl environmentl constrints, including wter (El Fleh et l., 1985) nd sline stress (Hmmmi et l., 16), were used. Seeds of gret rome (Bromus dindrus Roth., syn. Bromus rigidus Roth. susp. gussonii Prl.) were collected from infested sites in the North of Tunisi, more specificlly in the region of Bej (etween N, E nd N, E). Seeds of the common chickweed (Stellri medi L.) were purchsed from the compny Ariotech (Rennes, Frnce) Steriliztion nd pre-germintion All the seeds were surfce-sterilized to void microil contmintion potentilly influencing the stility of llelochemicls (Inderjit, 5). Briefly, the rley seeds were incuted in H 2 SO 4 (5% v/v) for 1 h nd Tle 1. Chrcteristics nd origin of the used rley genotypes Crctéristiques et origine des génotypes d orge utilisés. Genotype Type Origin N row Mnel Modern vriety Tunisi 6 Rihne Modern vriety Tunisi 6 Tej Modern vriety Tunisi 2 Ardhoui Lndrce Tunisi 6 Ari Lndrce Tunisi 6 Sudi Lndrce Sudi Ari 2

4 Allelopthic potentil of rley nd gret rome 485 wshed five times in sterile idistilled wter. Seeds were susequently shken in AgNO 3 (1% w/v) t rpm for min nd rinsed successively with NCl (1% w/v), sterile idistilled wter, NCl (1% w/v) nd five times with sterile idistilled wter (Lnoue et l., ). Gret rome nd the common chickweed seeds were sterilized ccording to Wu et l. (7). The seeds were surfce-sterilized y soking them in ethnol (7% v/v) for 2.5 min nd were rinsed four times with sterile idistilled wter. Seeds were then soked in sodium hypochlorite (2.5% v/v) solution for min followed y five rinses in sterile idistilled wter. After steriliztion, the rley nd the gret rome seeds were pre-germinted on moist sterile filter pper nd were plced in drkness in growth chmer t 22 C for 72 nd 96 h, respectively. However, the common chickweed seeds were incuted for 7 dys with light/drk cycle of 16/8 h, t temperture of 22 C nd inflorescent light of 3.56 ±.16 x 3 lux. Germinted seeds were selected for iossy experiments Allelopthic ctivity of rley ginst weeds The llelopthic ctivity of rley ginst two weed species, nmely gret rome nd common chickweed ws tested ccording to the seedling-fter-seedling gr method. This method derived from the equlcomprtment-gr-method (ECAM) of Wu et l. () where seeds of the llelochemicls-donor species re grown together with the receiver species. However, in our experiments, seeds of the donor nd receiver species re grown sequentilly nd not synchronously, s follows. Pre-germinted seeds of the rley were uniformly selected nd septiclly sown on the entire gr surfce with three densities (5, 12 nd 16 seeds/eker), in 1.5 l glss eker (le Prfit, Villeurnne, Frnce) filled with 9 ml of.3% wter gr. The ekers were seled with plstic film nd plced in controlled growth chmer with dily light/drk cycle of 16/8 h, t temperture of 22 C nd influorescent light of 3.56 ±.16 x 3 lux. After 7 dys, the rley seedlings were removed nd replced y pre-germinted weed seeds (gret rome or common chickweed) nd septiclly sown on the gr surfce. Therefter, the ekers were gin seled nd plced ck in the growth chmer. A tretment without rley seeds ws used s control. This iossy ws rrnged s completely rndomized lock design with five replictes for ech tretment. After dys, the gretest root nd shoot lengths of weed seedlings were mesured. In order to ssess the effect of rley llelochemicls on the root rchitecture of the receiver species using the sme experimentl protocol, the Ardhoui genotype ws chosen sed on its high llelopthic potentil ginst the gret rome (Bouhouel et l., ). Ardhoui seeds were sown with one density (16 seeds/eker) on the surfce of gr medium. A eker without donor seeds ws used s control. This experiment ws lso performed with five replictes for ech tretment. After dys, root system rchitecture (RSA) of 5 gret rome seedlings per repetition ws nlyzed using rchitect softwre (Delory et l., 16). Cerel species re generlly chrcterized y the primry root tht emerges quickly from the seed fter germintion nd y the seminl roots tht emerge fter few dys forming the emryonic root system. Therefter, lterl roots rise on the primry or seminl roots nd cn produce successively higher order rnched roots, up to third or fourth order (e.g. the first-order lterl roots re directly locted on the primry or seminl roots nd those of second-order re locted on the first-order lterl roots (Ormn-Ligez et l., 13; Rich et l., 13). In this experiment, eight prmeters were considered: totl root length (cm), primry root length (cm), totl length of first-order lterl roots (cm), totl length of second-order lterl roots (cm), numer of first-order lterl roots, numer of second-order lterl roots, density of first-order lterl roots (cm -1 ) nd growth rte of root system (cm.dy -1 ). This lst prmeter ws clculted s the difference etween the totl root system length t time t nd t -1 divided y the difference etween the root system ge t time t nd t Allelopthic ctivity of rley ginst rley of the sme or different genotype The llelopthic ctivity of rley ginst itself ws tested ccording to the seedling-fter-seedling gr method. In this iossy, Mnel (low llelopthic potentil) nd Ardhoui (high llelopthic potentil) (Bouhouel et l., ) were used s donor genotypes of llelochemicls ginst either the sme genotype or the other one ( Mnel or Ardhoui ). Donor seeds were sown with one density (16 seeds/eker) on the surfce of gr medium. A eker without donor seeds ws used s control. The experiment ws rrnged s completely rndomized lock design with five replictes for ech tretment Allelopthic ctivity of gret rome ginst itself nd ginst rley The lloinhiition ctivity of the gret rome ws evluted ginst the two rley genotypes Mnel (low llelopthic potentil) nd Ardhoui (high llelopthic potentil) (Bouhouel et l., ). The sme provennce of gret rome ws used for nlyzing the utoinhiition ctivity of this weed. The

5 486 Biotechnol. Agron. Soc. Environ. 16 (4), Bouhouel I., Gfeller A., Fuconnier M.-L. et l. experimentl set-up ws identicl to the one mentioned ove Reltion etween root vigor nd toxicity of rley root exudtes According to the seedling-fter-seedling gr method, 16 pre-germinted seeds of the Tunisin rley genotypes ( Mnel, Rihne, Tej, Ardhoui nd Ari ) were sown on the gr medium. The experiment ws performed with five replictes for ech genotype. After 7 dys, the length of the longest root, presumly the primry root (Rich et l., 13) ws mesured for ech rley plnt. Therefter, correltion etween rley root length dt nd their inhiitory ction on the root of gret rome nd common chickweed seedlings ws done Effect of the growing medium ph on phytotoxicity This experiment ws mde to determine to wht extent the oserved inhiition effect of rley roots cn e explined y their ction on the ph of the gr growing medium, independently of the toxicity of relesed llelochemicls. In these conditions, Ardhoui genotype ws used s donor species using two densities (5 nd 16 seeds/eker). The ph of the medium ws mesured using ph meter (WTW 3i, Weilheim, Germny) fter the initil growth period of the donor species, nd efore the receiver species were introduced. Two tretments were pplied in the presence ( ph = 6.12) nd in the sence (ph = 6.) of potssium phosphte uffer, where quntities were djusted in ccordnce with rley densities. The uffer (1 M stock solution) ws dded in the gr medium efore utoclving (to get finl concentrtion in the medium of 3. mm nd 3.5 mm in the ekers receiving 5 nd 16 rley seeds, respectively). Bekers without rley seeds nd with uffer in the medium were used s controls. This iossy ws rrnged s completely rndomized lock design with four replictes for ech tretment nd repeted twice over time. After 7 dys, the rley seedlings were replced y pre-germinted seeds of gret rome. The gretest root nd shoot lengths of weed seedlings were recorded fter dys of growth. The impct of this uffer (3. nd 3.5 mm) on the growth of pre-germinted Ardhoui seeds ws tested. Bekers without uffer in the gr growing medium were used s controls nd the length of the root nd shoot were lso mesured fter 7 dys Sttisticl nlysis Experimentl dt were sujected to Anlysis of Vrince (ANOVA) using SAS pckge (SAS V9.1) with the Lest Significnt Difference (LSD) test t 5% level of proility. The percentge of inhiition on the growth of receiver plnt ws clculted s (Control Tretment)/Control*. Since interction is not significnt etween seed density nd genotypes for root nd shoot growth inhiition prmeter of the two weed species, the two fctors were illustrted seprtely into two grphs in figures 1 nd 2. Regressions were performed with MINITAB 17 softwre. The vectoriztion of the different roots ws mde with DART softwre (Dt Anlysis of Root Trcings). The nlysis of root rchitecture dt ws performed using the R softwre clled rchitect from the rchidart pckge (Delory et l., 16). This softwre clcultes common root rchitecture prmeters nd performs XY plotting of the vectorized root system. 3. RESULTS 3.1. Allelopthic ctivity of rley ginst weeds In oth weed species, gret rome nd common chickweed, growth inhiition ws oserved fter dys of seedling growth on the sme gr medium on which rley seedlings hd first developed (Figures 1 nd 2). The rley root llelochemicls hd significnt inhiitory ctivity from the lowest pplied density of rley on root (F 6;85 = 37.81; p <.1) nd shoot growth (F 6;85 =.39; p <.1) of common chickweed, nd on root growth (F 6;85 = 17.22; p <.1) of gret rome when compred to the control. However, no significnt difference ws oserved in the shoot growth inhiition of gret rome (F 6;85 =.87; p =.519) s compred to the control. The results lso showed tht this effect depends on the numer of rley seedlings: s rley density incresed from 5 to 12 nd 16 seeds/eker, the root growth inhiition of gret rome nd common chickweed incresed linerly. For ll densities, the inhiition rtes were higher on root growth thn on shoot growth. For exmple, the six rley genotypes reduced root growth of gret rome nd common chickweed y 45% nd 62% nd shoot y 5 nd 27%, respectively when using 16 rley seeds/eker. This effect ws lso higher in common chickweed thn in gret rome. When compring rley genotypes, oth weed species responded differentilly to the llelopthic compounds of rley (F 5;72 = 7.82; p <.1 for root growth inhiition of gret rome, nd F 5;72 = 26.86; p <.1; F 5;72 = 6.19; p <.1 for root nd shoot growth inhiition of common chickweed, respectively), except for the shoot growth of gret rome (F 5;72 =.82; p =.234) (Figures 1 nd 2). Overll, the inhiitory ctivity of Sudi nd Ardhoui ws higher thn tht of Mnel nd Tej.

6 Allelopthic potentil of rley nd gret rome 487 Root growth inhiition (%) c Seed density 5 seeds/eker 12 seeds/eker 16 seeds/eker Root growth inhiition (%) Genotypes c P Seed density <.1 P Genotypes <.1 P Interction =.1 Mnel Tej Rihne Ari Ardhoui Sudi Shoot growth inhiition (%) seeds/eker 12 seeds/eker 16 seeds/eker 5 Seed density Genotypes Shoot growth inhiition (%) 5 45 P Seed density =.246 P Genotypes =.54 P Interction = 1 Mnel Tej Rihne Ari Ardhoui Sudi Figure 1. Root nd shoot growth inhiition (%) of gret rome seedlings fter dys exposure to root llelochemicls of six rley genotypes grown with three densities Tux d inhiition de l croissnce des rcines et des pousses (%) des plntules du grnd rome près jours d exposition ux llélochimiques rcinires de six génotypes d orge cultivés vec trois densités. Brs represent stndrd errors of the men nd different letters indicte significnt differences t p <.5 ccording to LSD test les rres verticles indiquent les erreurs stndrds des moyennes et les différentes lettres indiquent une différence significtive à p <,5 selon le test de PPDS. The root llelopthic compounds relesed y Ardhoui reduced significntly the totl root length of gret rome seedlings s compred to the control (F 1;7 = 33.95; p <.1) (Tle 2). To lrge extent, this effect is cused y reduction of the primry root length (F 1;7 = 32.9; p <.1), first- (F 1;7 =.; p <.1) nd second-order (F 1;7 = 7.66; p =.8) lterl root length. Moreover, the treted seedlings of gret rome exhiited lower rnching degree. The numer of the first- (F 1;7 = 26.2; p <.1) nd second-order (F 1;7 = 13.83; p =.1) lterl roots ws significntly reduced (which explins lower density of secondorder lterl roots). These results reveled tht ll the root trits of gret rome re sensitive to rley llelochemicls Allelopthic ctivity of rley ginst rley of the sme or different genotype The self-inhiitory effect of rley root nd shoot ws significnt, except for root nd shoot growth in the Ardhoui - Ardhoui comintion (Tle 3). This effect ws more pronounced on root thn on shoot growth (Figure 3). By compring the effects within the sme genotype or etween two different rley genotypes, the results indicted tht the inhiition rte of root or shoot growth ws significntly different when compring the Ardhoui - Ardhoui comintion with the Ardhoui - Mnel comintion. This is not the cse when compring the Mnel - Mnel nd Mnel - Ardhoui comintions. In prticulr, root inhiition rtes oserved in the

7 488 Biotechnol. Agron. Soc. Environ. 16 (4), Bouhouel I., Gfeller A., Fuconnier M.-L. et l. Root growth inhiition (%) Seed density c 5 seeds/eker 12 seeds/eker 16 seeds/eker Root growth inhiition (%) c c c Genotypes d P Seed density <.1 P Genotypes <.1 P Interction =.622 Mnel Tej Rihne Ari Ardhoui Sudi Shoot growth inhiition (%) seeds/eker 12 seeds/eker 16 seeds/eker Seed density Shoot growth inhiition (%) Genotypes c P Seed density <.1 P Genotypes <.1 P Interction =.776 Mnel Tej Rihne Ari Ardhoui Sudi Figure 2. Root nd shoot growth inhiition (%) of common chickweed seedlings fter dys exposure to root llelochemicls of six rley genotypes grown with three densities Tux d inhiition de l croissnce des rcines et des pousses (%) des plntules de l stellire intermédiire près jours d exposition ux llélochimiques rcinires de six génotypes d orge cultivés vec trois densités. Brs represent stndrd errors of the men nd different letters indicte significnt differences t p <.5 ccording to LSD test les rres verticles indiquent les erreurs stndrds des moyennes et les différentes lettres indiquent une différence significtive à p <,5 selon le test de PPDS. Tle 2. Effect of rley root llelochemicls on the root system rchitecture (RSA) of gret rome seedlings Effet des llélochimiques rcinires d orge sur l rchitecture du système rcinire (ASR) des plntules du grnd rome. Trit Control Ardhoui Totl root length (cm) 5. ± ±.47 Primry root length (cm) 4.1 ± ±.38 Totl length of first-order lterl roots (cm) 1. ± ±.28 Totl length of second-order lterl roots (cm).2 ±.2.1 ±.1 Numer of first-order lterl roots 4.6 ± ±.73 Numer of second-order lterl roots.17 ±..2 ±.4 Density of first-order lterl roots (cm -1 ).67 ±..34 ±.27 Growth rte of the root system (cm.dy -1 ).51 ±.13. ±.5 Vlues re mens ± stndrd error nd different letters indicte significnt differences t p <.5 ccording to LSD test les vleurs sont des moyennes ± erreurs stndrds et les différentes lettres indiquent des différences significtives à p <,5 selon le test de PPDS. Mnel - Mnel (%) or Ardhoui - Mnel (24%) comintions were significntly higher thn in the Ardhoui - Ardhoui (7%) or Mnel - Ardhoui (12%) comintions Allelopthic ctivity of gret rome ginst itself nd ginst rley The results (Figure 4) showed tht compounds produced y the gret rome roots reduced significntly root growth (F 1;8 = 13.23; p <.1 for gret rome-gret rome, F 1;8 = ; p <.1 for gret rome- Mnel nd F 1;8 = 53.4;

8 Allelopthic potentil of rley nd gret rome 489 Tle 3. Anlysis of vrince (ANOVA) of root nd shoot length of two rley genotypes grown on medi pre-colonised y the sme genotype, or the lterntive genotype, nd with those erlier seedlings mnully removed Anlyse de l vrince (ANOVA) de l longueur des rcines et des pousses de deux génotypes d orge cultivés sur un milieu pré-colonisé pr le même génotype ou un génotype différent, dont les plntules ont été mnuellement retirées. Fctor Root length F vlues df pvlue Shoot length F vlues df pvlue Brley donor-receiver genotype comintion Mnel - Mnel Ardhoui - Mnel Ardhoui - Ardhoui Mnel - Ardhoui ; < ;.36 df: degrees of freedom degrés de lierté ; < ; < ; ; ; ;.6 Root nd shoot growth inhiition (%) 5 c Root Mnel - Mnel Ardhoui - Mnel Ardhoui - Ardhoui Mnel - Ardhoui Shoot Figure 3. Root nd shoot growth inhiition (%) of rley seedlings from two genotypes fter dys exposure to its own llelochemicls or those of the lterntive genotypes Tux d inhiition de l croissnce des rcines et des pousses (%) des plntules de deux génotypes d orge près jours d exposition ux llélochimiques du même ou de génotypes différents. Brs represent stndrd errors of the men nd different letters indicte significnt differences t p <.5 ccording to LSD test les rres verticles indiquent les erreurs stndrds des moyennes et les différentes lettres indiquent une différence significtive à p <,5 selon le test de PPDS. Root nd shoot growth inhiition (%) 5 Root Brome-Brome Brome- Mnel Brome- Ardhoui Shoot Figure 4. Root nd shoot growth inhiition (%) in gret rome nd two genotypes of rley seedlings fter dys exposure to root llelochemicls of gret rome Tux d inhiition de l croissnce des rcines et des pousses (%) des plntules du grnd rome et de deux génotypes d orge près jours d exposition ux llélochimiques du grnd rome. Brs represent stndrd errors of the men nd different letters indicte significnt differences t p <.5 ccording to LSD test les rres verticles indiquent les erreurs stndrds des moyennes et les différentes lettres indiquent une différence significtive à p <,5 selon le test de PPDS. p <.1 for gret rome- Ardhoui ) of the receiver plnts fter dys s compred to the control. The sme trend ws otined for shoot growth compred to the control (F 1;8 = 41.22; p <.1 for gret rome- Mnel nd F 1;8 = 22.93; p <.1 for gret rome- Ardhoui ), except for the shoot growth of gret rome-gret rome (F 1;8 = 3.49; p =.65). In these experiments, lloinhiition rtes were greter thn utoinhiition rtes, especilly in root, lthough oth (root nd shoot growth) were significnt. The results lso show tht growth inhiition of rley y gret rome root compounds ws similr etween the two genotypes Mnel nd Ardhoui.

9 49 Biotechnol. Agron. Soc. Environ. 16 (4), Bouhouel I., Gfeller A., Fuconnier M.-L. et l Reltion etween root vigor nd toxicity of rley root exudtes As root elongtion rte differed etween rley genotypes, the question ws rised concerning the possile correltion etween root vigor (s ssessed y root length) nd inhiitory ctivity. No significnt correltion ws otined etween rley root length fter 7 dys nd its inhiitory ction ginst the gret rome (r 2 =.5, p =.91) nd the common chickweed (r 2 =., p =.52) species (Figure 5). In conclusion, the oserved genotypic effect is not linked to differentil root growth rtes etween genotypes Effect of the growing medium ph on phytotoxicity The first ojective of this study is to show the ph-vrition of the wter gr medium fter 7 dys of the growth of donor species. Therey, the ph-medium fter the initil growth period of Ardhoui seeds, nd efore the gret rome seeds were introduced, ws determined in the sence nd in the presence of the potssium phosphte uffer. In the sence of this uffer, the culture medium hs initilly ph of 6. (control). After 7 dys, this ph reched vlue of 4.51 nd 4.12 respectively in ekers receiving 5 nd 16 Ardhoui seedlings. The ph of the medium ws significntly different (F 2;9 = 7.51; p <.1) in the presence of Ardhoui genotype compred to the control. However, in the presence of the potssium phosphte uffer, the ph vlues of the medium were 6.11 nd 6.13 efore sowing 5 nd 16 Ardhoui seedlings, respectively. After 7 dys of growth, ph reched respectively vlue of 6.9 nd In these conditions, the difference etween these ph vlues in dy nd dy 7 ws not significnt (F 1;5 = 5.2; p =.66 nd F 1;5 = 3.9; p =.96 using 5 nd 16 Ardhoui seedlings, respectively). Buffering effect of the potssium phosphte ws sufficient to mintin stle the cidity of the wter gr. The effect (neutrl, stimultory or inhiitory) of the ddition of the potssium phosphte uffer to the wter-gr medium on the growth of donor seedlings ws ssessed. Results showed tht this uffer hd no significnt influence on root nd shoot growth of Ardhoui seedlings when compred to the control (Figure 6). Root nd shoot length (cm) 5 Control Potssium phosphte uffer (3 mm) Potssium phosphte uffer (3.5 mm) Root Shoot Figure 6. Effect of the ddition of potssium phosphte uffer on the growth of rley Ardhoui seedlings Effet de l ddition du tmpon phosphte de potssium sur l croissnce des plntules d orge d Ardhoui. Brs represent stndrd errors of the men nd different letters indicte significnt differences t p <.5 ccording to LSD test les rres verticles indiquent les erreurs stndrds des moyennes et les différentes lettres indiquent une différence significtive à p <,5 selon le test de PPDS. A Root growth inhiition of gret rome seedlings (%) 55 y = -.48x R 2 =.5 ns Root length of rley genotypes (cm) B Root growth inhiition of common chickweed seedlings (%) y = -4.97x R 2 =. ns Root length of rley genotypes (cm) Figure 5. Correltion etween the root length of the five Tunisin rley genotypes nd their inhiitory ction on the root of gret rome (A) nd common chickweed (B) seedlings Corréltion entre l longueur des rcines des cinq génotypes d orge tunisienne et leur ction inhiitrice sur les rcines des plntules du grnd rome (A) et de l stellire intermédiire (B). ns : p >.5.

10 Allelopthic potentil of rley nd gret rome 491 In second experiment (Figure 7), growth inhiition of gret rome y the rley genotype Ardhoui ws studied in the presence or sence of potssium phosphte uffer in the medium. Regrding root growth fter dys, significnt inhiition ws oserved s compred to the control nd this is true in oth the presence nd sence of the uffer (F 4;58 = 96.71; p <.1). This effect ws however not significnt for shoot growth inhiition (F 4;58 =.89; p =.473). The inhiition rtes were slightly reduced y the presence of the uffer for the two rley seed densities (5 nd 16 seeds/eker), ut differences etween the two tretments (i.e. in the presence or in the sence of the uffer) were not significnt for the two morphologicl prmeters. 4. DISCUSSION The equl-comprtment-gr method (ECAM) developed y Wu et l. () is common tool for investigting plnt-to-plnt llelopthy nd these uthors initilly dopted this co-culture method to determine the llelopthic potentil of whet root exudtes towrds Lolium rigidum Gudin. They rgued tht ECAM limits competition effects e.g. for nutrient resources s donor nd receiver species re co-cultivted in nutrient-free wter gr medium nd for light y inserting pperord etween the two species (Wu et l., ) or y chnging seed density nd sowing ptterns (Asduzzmn et l., 14). However, other competition for resources my persist, e.g. for spce needed for root development (Cffro et l., 13), nd chnges in the tmosphere during co-culture my lso prticipte to the oserved chnges in plnt growth. Consequently, we cnnot prove the role of the root exudtes in the oserved interctions etween co-cultivted plnts. Indeed, the relese of VOCs y the ove nd elowground prts of the rley plnt hs een demonstrted nd their possile roles in iotic interctions hve een discussed (Ninkovic, 3; Gfeller et l., 13). Our work imed t ddressing these methodologicl limittions y growing the donor nd receiver plnts one fter the other, insted of together, in the sme eker. Bsed on previous work, we hve shown tht llelochemicls were incresingly toxic over time ginst gret rome nd ryegrss (Lolium rigidum) fter removl of rley seedlings (Bouhouel et l., ) indicting the persistence of the llelopthic effect. For this reson, our iossy nmed seedlingfter-seedling gr method presents the two following interests: the competition etween donor nd receiver plnts is reduced ecuse they re grown sequentilly nd do not compete concomitntly for resources, Root nd shoot growth inhiition (%) Ardhoui (5) Ardhoui (5) + uffer (3 mm) Ardhoui (16) Ardhoui (16) + uffer (3.5 mm) Root Shoot Figure 7. Root nd shoot growth inhiition (%) of gret rome seedlings fter dys growing in Ardhoui gr medium using two densities (5 nd 16 seeds/eker), mixed with or without potssium phosphte uffer Tux d inhiition de l croissnce des rcines et des pousses (%) des plntules du grnd rome près jours de culture sur un milieu gélosé d Ardhoui repiqué vec deux densités (5 et 16 grins/ocl), vec ou sns l ddition du tmpon phosphte de potssium. Brs represent stndrd errors of the men nd different letters indicte significnt differences t p <.5 ccording to LSD test les rres verticles indiquent les erreurs stndrds des moyennes et les différentes lettres indiquent une différence significtive à p <,5 selon le test de PPDS. the method is specific to wter-solule compounds relesed y roots or resulting from their degrdtion, due to the renewl of the tmosphere of the continer etween the two successive cultures. The seedlingfter-seedling gr method lso llowed to test the effect of rley llelochemicls on growth of gret rome nd vice-vers in more dvnced stge of growth s compred to the seed-fter seed (Bouhouel et l., ). Another dvntge of this method ws to test the effect of rley lloinhiition on common chickweed (S. medi), which ws not considered y our previous studies. However, it must e cknowledged tht the sequentil cultivtion of the donor nd receiver seedlings is cse of interction tht cnnot e fully trnsposed to field situtions where oth species cohit, potentilly influencing ech other in synchronous mnner. After dys, growth of gret rome nd common chickweed ws significntly reduced y the preceding cultivtion of rley in the sme eker, in densitydependent mnner (Figures 1 nd 2). The inhiitory

11 492 Biotechnol. Agron. Soc. Environ. 16 (4), Bouhouel I., Gfeller A., Fuconnier M.-L. et l. ction of rley roots showed more pronounced effect on the roots thn on the shoots of the receiver species. Similr findings were otined in severl tests (Ben-Hmmoud et l., 1; Oueslti et l., 5), nd it is ssumed tht this prt of the plnt is the primry trget of the exuded llelochemicls (Vird- Crétt et l., 9). In this pper, further ttention ws pyed to investigte the qulittive chnges of the root system, i.e. the effects on root rchitecture in gret rome exposed to rley root llelochemicls (Tle 2). A conclusion is tht llelochemicls not only inhiit root growth, ut lso hve n impct on root rchitecture, i.e. chnges in totl root length nd degrees of rnching re oserved s compred to the untreted seedlings. All the mesured root trits of gret rome were sensitive to rley llelochemicls. Allelopthic effects were expected to hve sutle consequences on nutrient use efficiency in receiver plnts. The rley genotypes exhiited differentil llelopthic ctivity ginst weeds (gret rome nd common chickweed), with rley lndrces Sudi nd Ardhoui eing more toxic thn rley vrieties Mnel nd Tej (Figures 1 nd 2). A vrition in llelopthic ctivity ws similrly reported in different rley germplsms (Bghestni et l., 1999; Bertholdsson, 4; Oveisi et l., 8). On the other hnd, oth weed species (gret rome nd common chickweed) do not rect in the sme wy ginst the rley root compounds. Indeed, the growth of common chickweed seedlings ws more ffected thn tht of gret rome (Figures 1 nd 2), confirming its sensitivity to rley root llelochemicls s reported y Overlnd (1966). The llelopthic potentil of rley root exudtes ginst the two weed species hs no pprent reltionship with their root sizes, suggesting tht longer roots my not relese higher mounts of, or more toxic, llelochemicls (Figure 5). These results re consistent with previous reports in rice (Jensen et l., 1) nd rley (Bertholdsson, 4), concluding tht llelopthy in rley is controlled y genetic fctors distinct from those controlling root development. The effect of the ph of the growing medium on rley phytotoxicity ws investigted y the ddition of the potssium phosphte uffer. This uffer hd no effect on the growth of rley seedlings (Figure 6), ut hd slightly reduced the inhiitory ction of the rley root llelochemicls (Figure 7), nevertheless the differences etween the two tretments (i.e. in the presence or in the sence of the uffer) were not significnt. Consequently, no cler conclusions cn e drwn concerning the role of ph vrition in the inhiitory ction. The molecules exuded in the culture medium seem to e the min ctors in the llelopthic rley potentil. The present reserch lso showed tht rley root exudtes cuse utotoxicity, ut interestingly the mgnitude of this effect depended on the comintion of the donor nd receiving genotypes, which my e of the sme or different genotypes (Figure 3). This confirmed previous oservtions in whet y Wu et l. (7) who distinguished etween vrietl llelopthy (interction etween two different vrieties) nd vrietl utotoxicity (interction within the sme vrieties). The root growth inhiition ws higher when Mnel (% nd 24% in Mnel - Mnel nd Ardhoui - Mnel respectively) ws the receiver species of llelochemicls compred to Ardhoui (7% nd 12% in Ardhoui - Ardhoui nd Mnel - Ardhoui respectively). This might indicte tht Mnel is sensitive vriety to root compounds ecuse it is less le to detoxify the llelochemicls produced y Ardhoui. This study provides lortory-sed evidence of the llelopthic ctivity of gret rome root exudtes ginst rley prticulrly on roots (Figure 4). At this level, Mnel (low llelopthic potentil) nd Ardhoui (high llelopthic potentil) genotypes seem to rect in similr wy to gret rome root llelochemicls, whilst noting slightly greter resistnt ction with Ardhoui. The root exudtes of this weed lso cuse lower utotoxicity compred to its lloinhiition ctivity. Similrly, the sme trend ws otined in rley y compring its uto- nd lloinhiition potentil (Figure 4). All these results indicted tht there ws n inter- nd intrspecies difference in llelochemicls recognition nd this discrimintion suggests tht this phenomenon could e implicted in the mechnisms of plnt defense. For exmple, the cinnmic cid n llelochemicl of cucumer roots exudtes dded to nutritive solution showed n utotoxic effect y inducing n oxidtive stress ccompnied y the deth of cucumer root cells, ut not in Cucurit ficifoli Bouché, species of the sme fmily (Ding et l., 7). Incresing the rtio etween llotoxicity nd utotoxicity, could e chieved y plnts using t lest three mechnisms: no or lower ffinity of the toxic compounds with the moleculr trgets, detoxiction y degrdtion of the toxic compounds, sequestrtion of the toxic compounds preventing contct with their moleculr trget site. Severl reserches lend support to the mechnisms of detoxiction (Berson et l., 5) or sequestrtion (Duke et l., 1994). To the est of our knowledge, there is no reserch demonstrting the two first hypotheses ut further work is clerly needed to unrvel the ction mechnisms of llelopthy nd their diversity mong the mny comintions of plnt species, prticulrly those etween weeds nd importnt crop species.

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