Molecules 2013, 18, ; doi: /molecules OPEN ACCESS molecules ISSN Article

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1 Moleules 2013, 18, ; oi: /moleules PE ACCESS moleules ISS Artile Effet of Sliyli Ai n Struturlly Relte Compouns in the Aumultion of Phytolexins in Cotyleons of Common Ben (Phseolus vulgris L.) Cultivrs Diego Durngo 1, *, tli Pulgrin 1, Fernno Eheverri 2, Gustvo Esor 2 n Winston Quiñones 2, * 1 Grupo e Quími e los Proutos turles y los Alimentos, Esuel e Quími, 2 Fult e Cienis, Universi ionl e Colomi-See Meellín, Clle 59ª Autopist orte, P.. Box 3840, Meellín, Colomi Quími rgáni e Proutos turles, Fult e Cienis Exts y turles, Universi e Antioqui, Clle , P.. Box 1226, Meellín, Colomi * Authors to whom orresponene shoul e resse; E-Mils: lurngo@unl.eu.o (D.D.); wquinone@quimy.ue.eu.o (W.Q.); Tel.: (D.D.); Fx: (D.D.); Tel.: (W.Q.); Fx: (W.Q.). Reeive: 26 July 2013; in revise form: 29 August 2013 / Aepte: 29 August 2013 / Pulishe: 2 Septemer 2013 Astrt: In the present work, isoflvonoi phytolexin proution in response to the pplition of sliyli i in otyleons of four ommon en (Phseolus vulgris) ultivrs (SA) ws evlute. The time-ourse n ose-response profiles of the inution proess were estlishe y quntifying the isoflvonois y HPLC. Cotyleons of nthrnose-resistnt ultivrs inue y SA proue sustntilly higher phytolexin ontents s ompre to the suseptile ones. In ition, mximum levels of phytolexins ( fol inreses) were rehe etween 96 n 144 h, n when onentrtion of SA from 3.62 to mm ws use. The oservtions lso inite tht there ws reltively goo orreltion etween the phytolexin ontents n the inhiitory effet ginst C. linemuthinum; the higher ntifungl tivity ws oserve uring the first 48 hours for extrts from otyleons trete with SA t 1.45 n 3.62 mm, n etween 96 n 144 h fter inution. Finlly, ompouns struturlly relte to SA (ihyro-quinzolinones n some imines) showe strong eliitor effet. Moreover, inue extrts from otyleons trete with these potentil eliitors, esies the properly eliitors, isplye wek to moerte ntifungl tivity. These ompouns my e

2 Moleules 2013, onsiere goo nites for eveloping of new phytoprotetnts. Furthermore, phytolexin-eliiting sustnes my ontriute for seleting isese resistnt ultivrs. Keywors: ultivrs; eliitor; phseollin; ose-response profiles; Colletotrihum linemuthinum; ntifungl tivity; ihyro-quinzolinones 1. Introution The ommon en (Phseolus vulgris L.) is the most importnt eile foo legume in the Worl; it represents 50% of the grin legumes onsume worlwie n provies 15% of the protein n 30% of the lori requirement to the Worl popultion [1]. The ommon en lso represents vlule soure of vitmins, minerls n fier, espeilly for the poorer people of Afri n Ltin Ameri [2]. Unfortuntely, this rop is seriously ffete y severl fungl pthogens tht n use severe yiel losses. Among these fungl iseses, the most wiespre n estrutive, espeilly in ool wethers in Ltin Ameri n Afri, is nthrnose (use y Colletotrihum linemuthinum, S. & Mgnus, Sriner) tht ffets yiel, see qulity, n mrketility of ens. Tritionlly, nthrnose hs een suessfully ontrolle through the pplition of syntheti fungiies, ut there is growing glol onern over the ontinuous use of non-seletive syntheti hemils on foo rops euse of their potentil eleterious effets on humn helth n the environment. Also, the evelopment of fungiie resistne y pthogens my inrese proution osts resulting of the nee for pply higher more frequent oses to the rops, n susequently using environmentl prolems ue to the presene of fungiie resiues in foos [3]. In n ttempt to moify this sitution, reserhers re tively working in lterntive methos of ontrol of fungl iseses. Within this ontext is the utiliztion of eliitors, ompouns tht re le to stimulte the nturl efense mehnisms in plnts, inluing the iosynthesis n umultion of fungitoxi ompouns (nme phytolexins) [4,5]. Due to their non-ioil hrter n seletive tion, eliitors offer eologil vntges over syntheti fungiies [6,7]. Moreover, eliitors n serve s verstile tools for seleting isese resistnt ultivrs n provie vlule informtion out the plnt-pthogen reltionship. Sliyli i (SA), n enogenous eliitor, plys ruil role in plnt growth n evelopment, n in the inution proesses of systemi quire resistne (SAR) [8,9]. SA is involve in signl trnsution systems, whih stimulte prtiulr enzymes tlyzing iosyntheti retions to proue efense ompouns [10], whih thus my provie protetion for plnts ginst pthogens. Exogenous pplition of SA n lso result in the inution of efense ompouns n onsequently resistne ginst pthogens [11,12]. Furthermore some erivtives n nlogues of SA, suh s 2,6-ihloroisoniotini i (IA) n ienzolr-s-methyl (BTH), resemle SA y ting s exogenous hemil inuers of SAR n proteting plnts from infetions y fungl, teril, or virl pthogens [13 17]. Previous works hve showe tht the pplition of some eliitors, inluing CuCl 2 [18], hitosn [18,19], rhioni n linolei i [20], essions of Pseuomons syringe pv. phseoliol [21], rhizoteri [21], Fusrium solni f. sp. phseoli [21] n plnt hormones [22] on

3 Moleules 2013, Phseolus vulgris inue the isoflvonoi phytolexin umultion like genistein, izein, 2-hyroxygenistein, lergioiin, phseollin, phseolliin, phseollinisoflvn, kievitone n oumestrol (Figure 1). The ims of this stuy were to investigte the phytolexin umultion n inhiitory effets ginst C. linemuthinum of extrts from ommon en otyleons of four ultivrs with ifferent phytopthologil ehvior towrs nthrnose, trete or not with SA. Aitionlly, the effet in the phytolexin umultion on otyleons of ommon en using some struturlly relte ompouns to SA, like ihyroquinzolinones n some imines ws evlute. Figure 1. Chemil strutures of some phytolexins in ommon en. H R H H R 1 R 2 H R 1 : H; R 2 : H Dizein R 1 : H; R 2 : H Genistein R 1 : H; R 2 : H 2'-Hyroxygenistein H H R: H Dlergioiin R: CH 2 CH=C(CH 3 ) 2 Kievitone Coumestrol H H H H H H Phseollin Phseollinisoflvn Phseolliin 2. Results n Disussion 2.1. Time-Course Phytolexin Aumultion Eliitors hve een reognize s n ttrtive lterntive to non-seletive fungiies in rop protetion. Exogenous pplition of eliitors n inue lolize or systemi resistne in suseptile plnts, whih eome resistnt to lter infetions [6,8]. In ition, eliitors n e use lone or in omintion with fungiies, reuing the mounts neessry to ontrol pthogeni miroorgnisms n the unesirle environmentl sie effets ssoite with the inisriminte use of fungiies. Besies their potentil use for rop protetion, eliitors n serve s verstile tools for seleting isese resistnt ultivrs. To investigte the effet of pplition of SA in the ourse-time, otyleons of four ommon en ultivrs (two nthrnose-resistnt: CRPICA 106 n ICA Quimy, n two ntrnose-suseptile: Crgmnto Moho n Crgmnto Rojo) were eliite with 1.45 mm SA uring 4 h. Then, otyleons were extrte every 24 h for six onseutive ys n the orresponing extrts nlyze y HPLC. Conentrtion of phytolexins in the ourse time is shown in Figure 2. As n e seen, exogenous eliittion of otyleons with SA resulte in rmti inrese in the

4 Moleules 2013, phytolexin levels, minly oumestrol, phseollin, 2'-hyroxygenistein, n kievitone, in reltion to the ontrols. Anlysis inites tht the phytolexin ontent vrie signifintly (p = 0.05) etween tretments, exept for lergioiin, genistein, n phseollinisoflvn in nthrnose-suseptile ultivrs. In generl, phytolexin umultion inrese progressively oring to the post-inution time; the highest mounts of phytolexins were umulte etween 96 n 144 h. therwise, ontents of genistein n izein, preursors of phseollin n kievitone respetively, long with lergioiin remine lmost onstnt over the whole perio of the evlution (144 h). Results show tht oumestrol ws the mjor metolite eliite in ll vrieties. Mximum onentrtions of this oumestn in nthrnose-suseptile ultivrs were out n µg/g for Crgmnto Rojo n Crgmnto Moho, respetively. Menwhile for ICA Quimy n CRPICA 106, oumestrol rehe in tht orer, levels of n µg/g. n the other hn, kievitone presente mximum vlues of n 2.36 µg/g for Crgmnto Rojo n Crgmnto Moho, wheres otyleons from resistnt ultivrs umulte mounts of (for ICA Quimy) n µg/g (for CRPICA 106). verll, the mount of phytolexins in resistnt ultivrs ws higher thn tht foun in otyleons of suseptile ones. Figure 2. Time-ourse umultion of phytolexins in ommon en otyleons trete with SA., genistein;, lergioiin;, phseollinisoflvn;, phseolliin;, izein;, 2 -hyroxygenistein;, kievitone;, oumestrol;, phseollin. Brs represent the men onentrtions of the isoflvonois ± stnr evition (n = 3). Cultivrs: CM, Crgmnto Moho; CR, Crgmnto Rojo; IQ, ICA Quimy; CI, CRPICA 106. For eh ompoun, rs with ifferent letters re signifintly ifferent (p = 0.05; Fisher s LSD test). Conentrtion (µg/g f. w.) Conentrtion (µg/g f. w.) CR Control Time (h) CM e Control Time (h)

5 Moleules 2013, Figure 2. Cont. Conentrtion (µg/g f. w.) Conentrtion (µg/g f. w.) IQ Control Time (h) CI e e e Control Time (h) 2.2. Dose Response of Eliitor Tretments The use of SA s plnt efense inuer is limite y its phytotoxi tivity. The effet of the onentrtion of SA on the synthesis of phytolexins on ommon en otyleons ws evlute in the mm rnge n fter 96 h of inution (Figure 3). All ultivrs showe signifint inreses in the phytolexin levels fter tretment with SA in reltion to the ontrol. Even t 0.36 mm SA, mounts of oumestrol were etween 2- n 6-fol ove tht in the orresponing ontrols. In generl, levels of phytolexins in otyleons inrese steily t 3.62 mm SA n elow, in ose-epenent mnner. As shown in Figure 3, phytolexin ontents progressively inrese to reh their mximum onentrtions t 3.62 mm SA for Crgmnto Rojo, Crgmnto Moho, n ICA Quimy. Then t 7.25 mm SA, phytolexin onentrtions eline slightly for Crgmnto Moho, n rpily for Crgmnto Rojo n ICA Quimy. Unlike these ultivrs, CRPICA 106 rehe the mximum level of phytolexins t mm SA. However fter 96 h n t onentrtions of 3.62 mm SA n ove, ommon en otyleons strte to show symptoms of nerosis, wheres ontrols n tretments elow 3.62 mm SA remine green for this perio (t not shown). Most of otyleons trete with 7.25 mm SA turne rownish n exhiite wilting. Aoringly, uner these onitions hypersensitive response ourre, n our results suggest tht SA t 3.62 mm n elow, is sfe n oul e use s eliitor in ommon en. onetheless, further stuies out the physiologil effets of SA in ommon en otyleons (n other tissues) re neee. n the other hn, otyleons from resistnt ultivrs (ICA Quimy n CRPICA 106) umulte signifintly higher mount of phytolexins s ompre to the suseptile ultivrs (Crgmnto Moho n Rojo). For exmple, oumestrol rehe mximl levels of out n

6 Moleules 2013, µg/g for Crgmnto Rojo n Crgmnto Moho, n ner n µg/g for CRPICA 106 n ICA Quimy respetively; lmost two- n three times higher phytolexin proution in the resistnt ultivrs in omprison with the suseptile ones. It is noteworthy tht otyleons of CRPICA 106 umulte high mounts of phseolliin n phseollinisoflvn, eing respetively out 13 n 20-fol higher tht in the other ultivrs. However, no sustntil ifferene ws oserve in the mount of izein n genistein etween resistnt n suseptile ultivrs. Figure 3. Aumultion of isoflvonoi phytolexins on otyleons of Phseolus vulgris y SA t ifferent onentrtions., genistein;, lergioiin;, phseollinisoflvn;, phseolliin;, izein;, 2 -hyroxygenistein;, kievitone;, oumestrol;, phseollin. Brs represent the men onentrtions of the isoflvonois ± stnr evition (n = 3). Cultivrs: CM, Crgmnto Moho; CR, Crgmnto Rojo; IQ, ICA Quimy; CI, CRPICA 106. For eh ompoun, rs with ifferent letters re signifintly ifferent (p = 0.05; Fisher s LSD test). Conentrtion (µg/g f. w.) Conentrtion (µg/g f. w.) CR Control Conentrtion Sliyli i (mm) CM e Control Conentrtion Sliyli i (mm)

7 Moleules 2013, Figure 3. Cont. Conentrtion (µg/g f. w.) CI e Control Conentrtion Sliyli i (mm) SA is reognize nturl eliitor involve in the isese resistne of plnts. It my improve the plnt efensive pity ginst ro rry of pthogens fter pproprite tretment. In this stuy, the pplition of SA on en otyleons resulte in strong inreses of phytolexin ontent n ntifungl tivity of the extrts in omprison to ontrols. The t inite tht SA tivte phytolexin iosynthesis in ll ultivrs. Coumestrol, phseollin, n 2'-hyroxygenistein were the mjor phytolexins inue in otyleons from Colomin en vrieties. This ontrsts with n erlier stuy, in whih kievitone ws the mjor phytolexin in otyleons, wheres phseollin preomintes in hypootyls [18]. In ition, it ws foun tht nthrnose-resistnt ultivrs trete with SA umulte signifintly higher levels of phytolexins s ompre to the suseptile ones. These results re in orne with other results reporte for ommon en eliite y CuCl 2 [18] n some other rops [23 26]. Hene, the inution of en otyleons with SA my ontriute to the evelopment of rpi n low-ost tehniques for seleting resistnt ultivrs to nthrnose se on the iosynthesis of phytolexins. Aoring to ourse-time experiments, s result of inution with SA, there ws grul inrese in phytolexin ontent minly for oumestrol, phseollin, kievitone, 2'-hyroxygenistein, phseolliin n phseollinisoflvn. Menwhile, genistein, izein, n lergioiin ontents, preursors of phytolexins kievitone, oumestrol n pterorpns, remine lmost onstnt over the whole perio of the evlution. therwise, ose-response experiments showe tht when otyleons were immerse on SA t 3.62 mm n elow, the phytolexin proution ws inrese in ose-epenent mnner; this ws ppreily higher for nthrnoseresistnt ultivrs. However, the pplition of SA t high onentrtions (7.25 n mm) resulte in fst eline of phytolexin ontents, exept for the ultivr CRPICA 106. We hypothesize tht the ereses in the iosynthesis of efense seonry metolites n result in some effets relte with the phytotoxi hrter of SA, whih hs een well oumente [27,28]. The ove results re in greement with the nerosis symptoms oserve in otyleons trete with SA t 7.25 n mm. evertheless, the moleulr mehnism y whih SA t high onentrtion inhiite the phytolexin formtion is unler. Previously, Wr et l. [29] foun tht hikpe plnts trete with SA t 2 mm showe phytotoxiity symptoms n lower peroxise n polyphenol oxise tivity, n phenol ontent. Aoringly, they suggeste tht SA t 1.5 mm is sfe to these plnts n oul e utilize s plnt efense inuer. Thus, it ws oserve tht the highest levels of phytolexins in ommon en otyleons were hieve fter 96 h of post-inution n using SA t 3.62 mm (for v. Crgmnto Rojo, Crgmnto

8 Moleules 2013, Moho, n ICA Quimy) n t mm (for CRPICA 106). Interestingly, the phytolexin mounts etete in our stuy from Colomin ommon en ultivrs re sustntilly lower ompre with those reporte for orth Amerin n Europen ultivrs. Hynes et l. [21] reporte umultions of kievitone rehing 850 ± 251 µg/g f.w in woune otyleons (white en v. AC Seforth) following inoultion with Fusrium solni f. sp. phseoli Antifungl Ativity The ntifungl tivity of extrts otine from otyleons (ourse-time experiments t 48, 96, n 144 h; ose-response ssys t 0.72, 1.45, y 3.62 mm) of ommon en ultivrs Crgmnto Rojo (suseptile to nthrnose) n ICA Quimy (resistnt to nthrnose), non-trete n trete with SA, in terms of ril growth inhiition of C. linemuthinum re summrize in Figure 4. The inhiition of C. linemuthinum growth ws epenent to the onentrtion of SA use in tretments n the inution (post-inution) time. In generl, the ril growth ws inhiite in higher proportion uring the first 24 h. As shown in Figure 4 (top), inhiitions of C. linemuthinum using extrts from oth ultivrs trete with SA t 1.45 n 3.62 mm were signifintly higher ompre to the untrete otyleons. At 24 h, it n e notie tht the extrt otine from otyleons of ICA Quimy trete with 1.45 mm SA ws more tive (inhiition out 50%) towr C. linemuthinum thn the extrt proeeing from Crgmnto Rojo (inhiition 33%) uner the sme onitions. onetheless, the ntifungl tivity of otyleons trete with 3.62 mm SA ws similr for oth ultivrs. n the other hn, otyleons of oth ultivrs eliite y 0.72 mm SA showe no sustntil ifferenes with respet to untrete otyleons n solvent ontrol. Thus, the inrese in the ril growth inhiition of C. linemuthinum seems to e relte with the upper phytolexin levels presents in the extrts. Even so, the omprle ntifungl effet ginst C. linemuthinum shown y Crgmnto Rojo n ICA Quimy otyleons inue with SA t 3.62 mm results strnge given their so ifferent hemil profiles. From Figure 4 (ottom), it n e seen tht extrts from ICA Quimy otyleons inue y 1.45 mm SA n inute uring 48 n 96 h showe ril growth inhiitions of 33.3 n 50.0% respetively. However, extrts otine fter 144 h presente less ntifungl tivity n no signifint ifferenes were foun etween this extrt, untrete otyleons, n the solvent ontrol. This ehvior in the ntifungl tivity orreltes with the inrese mounts of isoflvonoi phytollexins etete in ICA Quimy otyleons 96 h post-inution. Menwhile, it n e foun tht extrts from Crgmnto Rojo otyleons trete with 1.45 mm SA n inute for 96 n 144 h showe inhiitions of out 33.3%. The ove is in greement with the higher levels of phytolexins etete for these extrts s ompre to untrete otyleons n tht otine 48 h post-inution. In ition, Figure 4 showe tht inhiitory effets of extrts rpily erese with time, ft tht suggests tht the fungus h rpi pttion to the meium. The ove is onsistent with the known pity of phytopthogeni fungi, inluing C. linemuthinum, to irumvent some of the plnt hemil efenses through metolism n etoxifition [30]. However, further stuies re neessry to estlish the reltionship etween phytolexin level n the inhiitory effets ginst C. linemuthinum.

9 Moleules 2013, Figure 4. Antifungl tivity ginst C. linemuthinum of extrts from ommon en otyleons inue y SA t ifferent onentrtion (up) n post-inution time (own). Cultivrs: CR, Crgmnto Rojo; IQ, ICA Quimy. Figure 4:, solvent ontrol;, CR-untrete otyleons;, CR-0.72 mm SA;, CR-1.45 mm SA;, CR-3.62 mm SA;, IQ-untrete otyleons;, IQ-0.72 mm SA;, IQ-1.45 mm SA;, IQ-3.62 mm SA. Figure 4:, solvent ontrol;, CR-untrete otyleons;, CR-48 h post-inution;, CR-96 h post-inution;, CR-144 h post-inution;, IQ-untrete otyleons;, IQ-48 h post-inution;, IQ-96 h post-inution;, IQ-144 h post-inution. For eh time point, the rs hee y the sme letter o not iffer t p = 0.05 (Fisher s LSD test). ur stuy lso inites tht the tivity of extrts from en otyleons trete with SA ginst C. linemuthinum ws only slightly enhne. onetheless, it is importnt to note tht the extrts were evlute without n itionl purifition n t reltively low onentrtion (<300 µg/ml), n in stti experimentl system tht not llow genertion of new mounts of phytolexins. In generl, there were no signifint ifferenes in the fungitoxiity ginst C. linemuthinum etween untrete otyleons n those trete t 0.62 mm SA, t lest uring the first 24 h of evlution. Likewise, extrts otine fter 48 h (for Crgmnto Rojo) n 144 h (for ICA Quimy) of inution showe similr inhiitory effet thn untrete otyleons (out 16%). The results lso emonstrte tht the pplition of 1.45 n 3.62 mm SA on otyleons or s post-inution time of 96 n 144 h for Crgmnto Rojo n 48 n 96 h for ICA Quimy le to more tive extrts (inhiitions etween 33 n 50%). These finings re in greement with the greter phytolexin ontents estlishe for otyleons eliite y 1.45 n 3.62 mm SA n post-inution times longer

10 Moleules 2013, thn 96 h, in reltion to untrete ontrols. Uner these onitions, otyleons of Crgmnto Rojo n ICA Quimy were foun to umulte the higher levels of oumestrol, 2 -hyroxygenistein, phseollin, n kievitone, mong other. onetheless, no mrke ifferenes in the ntifungl tivity were foun etween extrts from nthrnose-resistnt n nthrnose-suseptile ultivrs. Moreover, experimentl t lso suggest rpi pttion of phytopthogeni miroorgnisms to the meium ontining the ifferent extrts; the inhiitory effet for ll extrts ginst C. linemuthinum ws nerly the sme fter 72 h of evlution (<10% inhiition). It seems tht the use of extrts without further purifition n t low onentrtions (<300 µg/ml) oul e responsile for the quik etoxifition of the meium y C. linemuthinum, esies the moest ntifungl tivity fter 72 h. Extrts t these onentrtions my ontin fungitoxi ompouns t very low levels, eing rpily trnsforme into innouous metolites y iotrnsformtion fter 72 h. Thus, the lk of ntifungl tivity of extrts fter 72 h my e ue to metolism of the phytolexins y the fungus Inuer n Antifungl Effets of Struturlly Relte Compouns to SA Currently, there is n inresing interest in the serh for new eliitors for ontrolling importnt plnt iseses. Here, the inuer effet of phytolexins in en otyleons of some ihyroquinzolinones n imines (Figure 5), long with ABZ, IA, BTH, n 2-BA ws evlute. Ben phytolexins were groupe in three lsses: isoflvones n isoflvnones (genistein, izein, lergioiin, 2 -hyroxygenistein, n kievitone), oumestn (oumestrol), n pterorpns n isoflvns (phseolliin, phseollin, n phseollinisoflvn). As n e seen in Tle 1, ihyro-quinzolinones n imines possess strong eliittion effet, eing even higher thn tht shown y the plnt hormone, SA, n the struturlly relte ompoun, 2-BA. The upper isoflvones/isoflvnones umultion ws foun to e inue y 1, followe y 10 n 9. The isoflvones/isoflvnones ontent etete in response to 1 ws ner twie tht foun in otyleons trete with SA. In ition, 1 inue high levels of oumestrol (73.55 µg/g f.w., the highest mount) n pterorpns/isoflvn. In ft, oumestrol ontent using 1, 6 n 10 s eliitors ws inrese in tht orer y out six, five, n three-fol in omprison to the otyleons inue y SA. It is noteworthy tht oumestrol ws not etete in ppreile mounts in untrete otyleons. verll, pterorpns/isoflvn levels of ommon en otyleons in response to ihyroquinzolinones n imines were lwys higher thn tht etete when SA ws use s eliitor; only 2, 3, 5, n 6 showe slightly higher inreses in pterorpns/isoflvn ontent. Menwhile, 10 h stronger inuer effet on the pterorpns/isoflvn ontent euse these inrese lmost ten n five-fol the levels etete in untrete (ontrol) n SA-trete otyleons respetively. It is noteworthy tht extrts proeeing from ommon en otyleons v. Crgmnto Moho trete with 10 lso showe the higher inhiitory effets ginst C. linemuthinum uring the first 48 h. Furthermore, lthough en otyleons trete with 6 resulte in mrke inrese of oumestrol, the ntifungl tivity ws reltively wek (55.9 ± 8.8 n 44.2 ± 20.9 fter 24 n 48 h, respetively). This result suggests low fungitoxi effet of oumestrol ginst C. linemuthinum. In generl,

11 Moleules 2013, inhiitory effet of extrts ws rpily erese; t 24 h, fungl inhiitions were lmost twie tht foun fter 48 h exept for 6 n 7. Aitionlly, we lso evlute the iret ntifungl properties of these potentil eliitors. In generl, ihyroquinzolinones n imines isplye moerte to wek fungistti tivity ginst C. linemuthinum. As n e seen in Tle 1, the highest ril growth inhiition ws exhiite y 10 (76.9% fter 48 h t 200 μg/ml), followe y 8 n 6. Remrkly, 10, 6, n 1, whih showe higher phytolexin-inuing effet, lso isplye inhiitions of 76.9 ± 0.0, 57.7 ± 13.3, n 38.5 ± 6.7, so ihyroquinzolinones n imines my hve ul moe of tion for ontrolling of fungl iseses; elevting host resistne n reuing pthogen inoulum. Figure 5. Chemil strutures of ompouns evlute s eliitors. H H H 2 H 2 H H 2 H CH 3 H SA 2-BA 1 2 H H H H 2 H H H H H H H H H H H CH H H S S S Cl IA Cl Aienzolr i (ABZ) Aienzolr-S-methyl (BTH)

12 Moleules 2013, Tle 1. Eliitor effet of some ihyro-quinzolinones n imines relte struturlly to SA (t 1.45 mm n fter 96 h post-inution) in otyleons of ommon en v. Crgmnto Moho, n ntifungl tivity of the extrts from inue otyleons. Compoun Isoflvones/Isoflvnones * (µg/g f.w.) Coumestn (µg/g f.w.) Pterorpns/Isoflvn (µg/g f.w.) Ril growth inhiition (%) of C. linemuthinum Eliite-otyleon extrts 24 h 48 h 48 h SA ± ± ± 1.42 n ± BA ± 2.33,, ± ± 5.45 n.. n ± 9.36,,, ± 22.02,,, ± 10.52,,, 82.4 ± ± ± ± ± ± ± ± ± ± 4.06, ± ± 9.89, 38.2 ± ± ± ± 6.84,, ± ± 16.37,, 47.1 ± ± ± ± ± ± ± ± ± ± 14.19,,, ± 18.89,,, ± ± ± ± ± ± ± 9.33,, 64.7 ± ± ± ± 2.86,, ± ± 5.00,, 64.7 ± ± ± ǂ ± 16.75,,, ± ± 4.08,, 47.1 ± ± ± ǂ ± 11.98,,, ± 4.40,,, ± 17.05,,, 70.6 ± ± ± ± ± ± 6.11,, 47.1 ± ± ± 0.0 ABZ ± ± 3.27, 9.32 ± 0.41 n.. n.. BHT 7.90 ± ± ± 1.57 n.. n.. IA ± ± ± 6.51 n.. n.. Control 9.01 ± 0.52 Tres 5.02 ± 0.33 n.. n.. Dt orrespon to the men onentrtions of the groupe isoflvonois ± stnr esvition (n = 3). * Dlergioiin, 2'-hyroxygenistein, izein, genistein, kievitone. Phseolliin, phseollinisoflvn, phseollin. n.. not etermine. Dihyro-quinzolinones; imines; ǂ yl hyrzones. n..: not etermine. Signifint ifferene (p = 0.05) etween tretment n ontrol ( ), IA ( ), BHT ( ), n ABZ ( ). Eliitor

13 Moleules 2013, The results onerning the ility to stimulte the phytolexin iosynthesis of ompouns struturlly relte to SA showe tht ihyroquinzolinones n imines exhiit strong eliitor effet. Ltely, new syntheti ompouns hve een ssye s eliitors in ifferent plnt tissues [31,32]. Thus, some hemils suh s 2,6-ihloroisoniotini i (IA) n S-methyl enzo-1,2,3-thiizole-7- rothioi i (ienzolr-s-methyl or BTH) hve een foun to e effetive inuers of plnt efenses [13 17]. Both ompouns were isovere s result of sreening ssys of eliitors of ro-spetrum resistne in uumer (Cuumis stivus L.) [15,33]. However, only BTH is ommerilly ville (from Syngent) uner the nmes Atigr n Bion. The present stuy lerly inite tht 2-BA, 2, 3, 5 inue phytolexin levels lmost similr to tht etete when en otyleons were trete with SA, well-known eliitor; prtiulrly, pterorpns/isoflvn ontents were nerly twie tht foun in untrete ontrols. Remrkly, phytolexin umultion in response to some ihyroquinzolinones n imines (for exmple, 1, 4, 6, n 10) ws even higher thn tht foun for SA n some reognize syntheti eliitors, suh s IA, ABZ, n BHT. In ft, pterorpns/isoflvn levels on otyleons were inrese y s muh s 106 n 169% y 1 n 10 respetively, ompre with IA. Aitionlly, the mounts of oumestrol n pterorpns/isoflvn for otyleons trete with ihyroquinzolinones n imines were higher thn for SA. The presene of nitrogen-ontining funtionl groups (suh s n ryl mie n nitrogente sustituent in position 2 resemling the struture of SA) in 2-BA n ihyro-quinzolinones my e n importnt struturl feture for their phytolexin-inuing tivity. Some of the potentil syntheti eliitors tht hve reently een reporte re nitrogen-rih ompouns. For instne, syntheti pyrzine-2- roxmie erivtives were foun to t s potentil eliitors in tissue ultures of nonis rvensis, Silyum mrinum, n Genist tintori [34,35]. Thus, the pplition of 5-tert-utyl-6-hloro--(3- ioo-4-methylphenyl)pyrzine-2-roxmie in llus ulture of Genist tintori enhne the genistin proution out 57 times ompre to untrete ontrol [35]. Furthermore, the pplition of 1 µm 2-(2-fluoro-6-nitroenzylsulfnyl) pyriine-4-rothiomie signifintly inrese the proution of the isoflvonois genistin (11.60 mg/g ry weight), izein (8.31 mg/g ry weight), n genistein (1.50 mg/g ry weight) on Trifolium pretense L. suspension ulture fter 48 h of pplition s ompre to the ontrol y 152, 151 n 400% respetively, whih ws reently reporte y Kšprová et l. [36]. Similrly, 3,5-ihloronthrnili i (DCA), effiiently inue efense retions in Ariopsis (Ariopsis thlin) plnts to the phytopthogens Hyloperonospor prsiti n Pseuomons syringe [37]. Authors lso inite tht the removl of the mino group from DCA signifintly reues its iologil tivity. Moreover, the syntheti sustnes 2-pyrzineroxyli i, piolini i, 2,6-pyriineiroxyli i, 2,3-pyriineiroxyli i, pyrrole-2-roxyli i, oxoni i, mong others, inrese the phytolexin(phytossnes n momiltone A) ontents in rie plnts [38]. Although SA, 2-BA, AI, ABZ n BTH re funtionlly ifferent ompouns, they shre some ommon struturl fetures. Thus, ll ompouns hve the enzoyl frgment, n n jent eletronegtive group (sustitute t the 2-position, exept for IA). Also, ll ompouns present n eletron withrwing group one to the ronyl group (forming roxyli i for SA, ABZ n IA, n mie for 2-BA, n thioester for BTH). evertheless, while SA is n i ompoun n 2-BA is si, oth inue similr phytolexin ontents. Hene, the ove results suggest tht the iity/siity of ompouns hs no effet on the phytolexin-inuing hrter.

14 Moleules 2013, The otyleons expose to ihyroquinzolinones 2 n 3 proue similr phytolexin levels thn those foun when 2-BA (the syntheti preursor) ws use. It inites tht the presene of the -nitrophenyl group in these ompouns h no effet on the phytolexin umultion. In ontrst, ihyroquinzolinones hving -methoxyphenyl (1) n -enzoioxoyl (6) group improve the synthesis of oumestrol s ompre to 2-BA. Also, 1 eliite in en otyleons high ontents of pterorpns/isoflvn. These results suggest tht the eliiting effets n e relte with the funtionl fetures presents in the ihyroquinzolinones. The presene of eletron-onting groups (like methoxy-, n methylienioxy- group) in the C-phenyl moiety of the teste ihyroquinzolinones seems to e n importnt requirement for the oumestrol-eliiting effet. Furthermore, it ws oserve tht the pterorpns/isoflvn umulte in greter onentrtion when otyleons were trete with 4 n 7. Both ompouns hve nitrogen-ontining heteroyli ring, pyriine n pyrrole respetively. Interestingly, when the ihyroquinzolinones presente furn ring inste nitrogen-ontining heteroyli system (5 vs. 7) there ws sustntil loss of eliiting tivity of pterorpns/isoflvn. n the other hn, yl hyrzones 9 n 10 h strong effet on isoflvones/isoflvnones ontent. Also, 10 enhne the proution of oumestrol n pterorpns/isoflvn. In ition, ihyroquinzolinones n imines isplye moerte to wek iret ntifungl tivity. Besies their promissing phytolexin-inuing tivity, ihyroquinzolinones n imines 1, 6, n 10 lso inhiit the ril growth of C. linemuthinum etween 38.5 n 76.9%. These results re in greement with previous reports tht estlish tht Shiff ses n ihyroquinzolinones hve ntifungl properties [39,40]. Therey, the iret fungistti properties of ihyroquinzolinones n imines offer n itionl vntge over SA, n other eliitors. Therefore, these ompouns hve the potentil to offer ul moe of tion with oth iret inhiitory effets gin C. linemuthinum n the pity of enhne the phytolexin ontent, n onsequently the plnt resistne. These results inite tht the ihyroquinzolinones n imines re promissing eliitors of phytolexins in ommon en otyleons. To the est of our knowlege, this is the first report out the phytolexin-inuer effet of ihyroquinzolinones n some imines. 3. Experimentl 3.1. Regents Genistein n izein were purhse from Sigm (St. Louis, M, USA). Dlergioiin, 2 -hyroxygenistein, oumestrol, phseolliin, phseollin isoflvn, n phseollin were otine uring previous work n ientifie s esrie in elsewhere [18]. Sliyli i (SA) ws quire from Merk (Drmstt, Germny), while 2-minoenzmie (2-BA), enzoi hyrzie n isoniotini hyrzie were from Sigm-Alrih Co. Dihyroquinzolinones 1 to 7 were prepre y onventionl retion etween 2-minoenzmie n lehyes, followe y further heting with n i tlyst (AH). Imine 11 ws otine from enzoi hyrzie wheres imines 8 to 10 were from proue from isoniotini hyrzie. Syntheti SA nlogs suh s 2,6-ihloroisoniotini i (IA), enzo-1,2,3-thiizole-7-rothioi i (ABZ), n the ommerilly ville eliitor enzo- 1,2,3-thiizole-7-rothioi i S-methyl ester (BTH) were lso purhse from Sigm-Alrih Co.

15 Moleules 2013, Plnt Mteril Anthrnose-suseptile en ultivrs Crgmnto Moho n Crgmnto Rojo were otine from Semills & Semills Lt (Meellín, Colomi). Anthrnose-resistnt ultivrs ICA Quimy n CRPICA 106 were quire from Semiol Lt (Bogotá, Colomi) n Corpoi (Corporión Colomin e Investigión Agropeuri, L Selv, Antioqui, Colomi). Sees from eh Colomin en (Phseolus vulgris L.) ultivr were surfe-sterilize for 15 min in Cl (2.0%), wshe with tp wter, n sown in moist vermiulite. Sees were llowe to germinte for seven ys in the rkness n t room onitions. Cotyleons hrveste from seelings were refully wshe with tp wter in orer to preserve their integrity Tretments Dose-Response Experiments Cotyleons (10 g) of eh en ultivr were immerse for 4 h in solutions of SA t ifferent onentrtion (0.36, 0.72, 1.45, 3.62, 7.25, n mm). Before prepring ll solutions, SA ws issolve in ethnol (0.2%). Ben otyleons sumerge into sterile istille wter were use s ontrols. Then, otyleons were ple on moist filter pper in polystyrene trys n overe with streth film. Mterils were store t room temperture n in the rk uring 96 h. Experiments were one t lest three times Time-Course Experiments Cotyleons (10 g) of eh en ultivr were ippe into the solution of 1.45 mm SA for 4 h. Susequently, plnt mterils were eposite on moist filter pper in sterile polystyrene ox, overe with streth film n store t room temperture in the rkness uring 24, 48, 72, 96, 120, n 144 h. Ben otyleons trete with istillte wter inste SA solution, n store uring 144 h were use s ontrols. All ssys were rrie out t lest three times Inuer Effet of Struturlly Relte Compouns to SA Some ihyroquinzolinones, otine from 2-minoenzmie n lehyes, n imines (imines n yl hyrzones) were evlute for their inuer tivity on en otyleons. Evlutions were rrie out using otyleons (10 g) of ultivr Crgmnto Moho, whih were ippe into solutions of eh imine t 1.45 mm n uring 4 h. Solutions were prepre issolving the ihyro-quinzolinone or Shiff se in 0.2% ethnol. Compouns 2,6-ihloroisoniotini i (IA), enzo (1,2,3) thiizole-7-rothioi i (ABZ), n enzo-1,2,3-thiizole-7-rothioi i S-methyl ester (BTH) were use s positive ontrols, wheres sterile istille wter ws use s the negtive ontrol. Then, otyleons were eposite on moist filter pper in sterile polystyrene ox, overe with streth film n store t room temperture in the rkness for 96 h Smple Preprtion Cotyleons were ut n further groun in mortr with 20 ml of 95% ethnol. Then, the solutions were filtrte through Whtmn o. 1 filter pper n entrifuge for 6 min (10,000 rpm). The

16 Moleules 2013, superntnt solutions were evporte to ryness uner reue pressure t 40 C (Rotvpor Buhi R-210 with vuum ontroller V-850) n the resiue ws extrte three times with ethyl ette (EtA, 3 20 ml). The orgni phses were omine n rought to ryness uner reue pressure. Susequently, the resiue ws reissolve in methnol (HPLC-gre MeH, 5.0 ml), n filtere through syringe sterile filter with 0.45-mm pore size (Srtorius Bioteh GmH, Goettingen, Germny). The resulting solution (0.5 ml) ws use without further purifition for HPLC nlysis. The smples were kept in mer glss vils n store t 4 C until HPLC nlysis ws rrie out. The remining solutions (4.5 ml) from eh replite were omine, evporte to ryness t 40 C uner vuum, n use in ntifungl ssys HPLC Anlysis The phytolexin nlysis ws rrie out on Gilson hromtogrph equippe with Gilson moel 170 ioe rry etetor, using Phenomenex Seurity Gur rtrige C18 ( mm) followe y Phenomenex Lun 5 μ C18 (2) reverse-phse olumn (150 mm 4.6 mm i.., 5 μm) (Phenomenex, Torrne, CA, USA). The metolites were elute t flow rte of 0.7 ml/min with the solvents A = methnol, n B = 0.5% eti i in wter, s follows: from 10% A to 70% A in 40 min, then 70% A to 90% A in 20 min, n susequently y holing for 8 min to reequilirte the olumn, for the next injetion. Injetion volume ws 20 µl. Phytolexins were monitore t the wvelengths of 248, 254, 270, 286 n 310 nm, lthough ioe-rry etetion ws use over wvelength rnge of 200 to 500 nm to ollet spetrl t Ientifition n Quntifition of Phytolexins Isoflvonoi phytolexins were hrterize y ompring the retention times (Rt) of the uthenti smples of lergioiin, 2 -hyroxygenistein, izein, genistein, oumestrol, n phseollin with those in the extrts, n y o-elution. Aitionlly, retention times of these isoflvonois, long with kievitone, phseolliin n phseollin isoflvno were onfirme y liqui hromtogrphy with mss spetrometry etetion (LC-MSD) on n HP 1100 series HPLC pprtus (Agilent Tehnologies, Wlronn, Germny) interfe to n HP series 1100 mss seletive etetor equippe with n API-ES hmer, using positive ion moe, n the sme hromtogrphi onitions s esrie ove. MSD onitions were progrmme s follows: pillry voltge, 3 kv; neulizing pressure, 60 psi; rying gs temperture, 350 C; rying gs flow, 12 L/min. Retention times of lergioiin, 2'-hyroxygenistein, izein, genistein, oumestrol, kievitone, phseolliin, phseollin isoflvn, n phseollin were respetively 28.85, 30.50, 31.45, 34.48, 37.50, 40.89, 43.51, 44.15, n min. Quntifition of phytolexins ws rrie out using stnr lirtion urves (pek res vs. ompoun onentrtion for ifferent onentrtions). Five working solutions were prepre for eh stnr in methnol ontining genistein, izein, lergioiin, 2'-hyroxygenistein, oumestrol, n phseollin in 1, 10, 25, 50, n 100 mg/l onentrtions. For phytolexins without pure stnr phseollinisoflvno n phseolliin, n kievitone, onentrtions were respetively estimte from the lirtion urves for phseollin, n lergioiin, n juste on the sis of ifferenes in moleulr weight. Dt for eh pek were ollete using the wvelength tht provies mximum

17 Moleules 2013, response. The results were expresse s μg phytolexin/g fresh mteril n presente s men vlues ± stnr evition Antifungl Assys The toxiity of extrts proeeing from SA-trete n untrete otyleons ginst C. linemuthinum ws evlute through the poisone foo tehnique [41]. The fungus ws isolte from isese P. vulgris pos, hrterize y morphologil nlysis, n mintine on potto extrose gr (PDA) t 4 C. Resulting extrts from inution experiments (without further purifition) otine s esrie ove were issolve in imethylsulfoxie (DMS, 70 μl) n ilute in Petri ishes (mesuring 5 m in imeter) with PDA (20 ml; onentrtions pprox. etween 100 to 300 μg/ml). Petri ishes with n without DMS were use s ontrols (solvent n solute ontrol, respetively). The Petri ishes were inute t room temperture n the imeter of myelil growth ws mesure eh 24 hours. The inution ws stoppe when the myelil mss of ontrol Petri ishes h lmost fille it (fter 96 h). The reltive growth inhiition of the tretments (SA-trete n untrete hypootyls-roots) ompre to the ontrols ws lulte s perentge, using the following formul: Inhiition (%) = {1 ril growth of tretment (mm)/ril growth of ontrol (mm)} 100 (1) The results re expresse s men vlues of three replites [± stnr evition (SD)]. Aitionlly, the ntifungl tivity ginst C. linemuthinum of potentil eliitors (ihyro-quinzolinones n imines) ws lso evlute. Assys were performe t 200 μg/ml n uner the onitions esrie ove Sttistil Anlysis Results were nlyze y one-wy AVA n men vlues were ompre with the Fisher s lest signifint ifferenes (LSD) t the 0.05 proility level. 4. Conlusions It is ler from the ove isussions tht SA sustntilly inreses the phytolexin ontent in ommon en otyleons. As result of inution, nthrnose-resistnt ultivrs umulte higher phytolexin levels in omprison to nthrnose-suseptile ultivrs. Therefore, the nlysis of the phytolexin proution in en otyleons in response to SA might serve s tool for seleting isese-resistnt ultivrs in ommon en reeing progrms. ur results lso suggeste tht the phytolexin-inuing effets epen on the onentrtion, the inution perio, n the hemil nture of the eliitor. Mxim levels of phytolexins were hieve when SA ws pplie t 3.62 mm (for Crgmnto Moho, Crgmnto Rojo, n ICA Quimy) n mm (for CRPICA 106), n etween 96 to 144 h fter eliittion. However, SA t higher onentrtions thn 3.62 mm use rpi erese of the phytolexin ontents for nthrnose-suseptile ultivrs n ICA Quimy, n nerosis symptoms were evient on otyleons. Therefore, SA t 3.62 mm n elow, oul e sfely use s eliitor in ommon en. In ition, ihyroquinzolinones n imines were foun to possess promissory phytolexin-eliiting tivity; they enhne the phytolexin mounts n

18 Moleules 2013, fungistti properties on extrts from eliite otyleons. In ition, ihyroquinzolinones n imines isplye moerte to wek iret ntifungl tivity, so these ompouns offer ul moe of tion with oth iret inhiitory effet gin C. linemuthinum n the pity of enhne the phytolexin ontent, n onsequently the plnt resistne. Aknowlegments This stuy ws supporte y the Universi ionl e Colomi-See Meellín (projets: DIME n y Dotorl fellowship to D.D.) n y Universi e Antioqui (projets: CDI n Progrm Sosteniili ). Conflits of Interest The uthors elre no onflit of interest. Referenes 1. MConnell, M.; Mmii, S.; Lee, R.; Chikr, S.; Rossi, M.; Pp, R.; MClen, P. Synteni reltionship mong legumes revele using gene-se geneti linkge mp of ommon en (Phseolus vulgris L.). Theor. Appl. Genet. 2010, 121, Prees, M.; Beerr, V.; Ty, J. Inorgni nutritionl omposition of ommon en (Phseolus vulgris L.) genotypes re Chile. Chil. J. Agri. Res. 2009, 69, Tripthi, P.; Duey,.K. Exploittion of nturl prouts s lterntive strtegy to ontrol post-hrvest fungl rotting of fruits n vegetles. Posthrvest Biol. Tehnol. 2004, 32, Hmmershmit, R. Inue isese resistne: How o inue plnts stop pthogens? Physiol. Mol. Plnt Pthol. 1999, 55, Erev, A. A novel strtegy for plnt protetion: Inue resistne. J. Cell Mol. Biol. 2004, 3, Thkur, M.; Sohl, B.S. Role of eliitors in inuing resistne in plnts ginst pthogen infetion: A review. ISR Biohem. 2013, 1, Holopinen, J.K.; Heijri, J.; erg, A.M.; Vourinen, M.; Kinulinen, P. Potentil for the use of exogenous hemil eliitors in isese n inset. pen For. S. J. 2009, 2, Heil, M.; Bostok, R.M. Inue systemi resistne (ISR) ginst pthogens in the ontext of inue plnt efenses. Ann. Bot.-Lonon 2002, 89, Ryls, J.A.; euenshwner, U.H.; Willits, M.G.; Molin, A.; Steiner, H.Y.; Hunt, M.D. Systemi quire resistne. Plnt Cell. 1996, 8, Chen, Z.; Zheng, Z.; Hung, J.; Li, Z.; Fn, B. Biosynthesis of sliyli i in plnts. Plnt Signl Behv. 2009, 4, Viml, R.; Surihnrselvn, M. Inue resistne in hei ginst powery milew y folir pplition of sliyli i. J. Biopest. 2009, 2, Mnl, S. Inution of phenolis, lignin n key efense enzymes in eggplnt (Solnum melongen L.) roots in response to eliitors. Afr. J. Biotehnol. 2010, 9,

19 Moleules 2013, Vernooij, B.; Frierih, L.; Goy, P.A.; Stu, T.; Kessmnn, H.; Ryls, J. 2,6-Diloroisoniotini i-inue resistne to pthogens without the umultion of sliyli i. Mol. Plnt Miroe Intert. 1995, 8, Kogel, K.H.; Bkhove, U.; Romme, Y. Aquire resistne in rley: The resistne mehnisms inue y 2,6 ihloro-isoniotini i is phenoopy of genetilly se mehnism governing re speifi powery milew resistne. Plnt Physiol. 1994, 106, Metrux J.P.; Ahl-Goy, P.; Stu, T.; Speih, J.; Steinemnn, A.; Ryls, J.; Wr, E. Inue resistne in uumer in response to 2, 6-ihloroisoniotini i n pthogens. In Avnes in Moleulr Genetis of Plnt-Miroe Intertions, 1st e.; Henneke, H., Verm, D.P.S., Es.; Kluwer Aemi Pulishers: Dorreht, The etherlns, 1991; Volume 1, pp Cole, D.L. The effiy of ienzolr-s-methyl, n inuer of systemi quire resistne, ginst teril n fungl iseses of too. Crop Prot. 1999, 18, Anfok, G.H. Benzo-(1,2,3)-thiizole-7-rothioi i S-methyl ester inues systemi resistne in tomto (Lyopersion esulentum Mill. v. Volleung) to uumer mosi virus. Crop Prot. 2000, 19, Durngo, D.; Quiñones, W.; Torres, F.; Rosero, Y.; Gil, J.; Eheverri, F. Phytolexin umultion in olomin en vrieties n minosugrs s eliitors. Moleules 2002, 7, Young, D.H.; Kohle, H.; Kuss, H. Effet of hitosn on memrne permeility of suspensionulture Glyine mx n Phseolus vulgris ells. Plnt Physiol. 1982, 70, Longln, A.C.; Slusrenko, A.J.; Frien, J. Arhioni n linolei is eliit isoflvonoi phytolexin umultion in Phseolus vulgris (Frenh en). J. Phytopthol. 1987, 120, Hynes, R.K.; Hill, J.; Rey, M.S.; Lzrovits, G. Phytolexin proution y woune white en (Phseolus vulgris) otyleons n hypootyls in response to inoultion with rhizoteri. Cn. J. Miroiol. 1994, 40, Goossens, J.F.V.; Venrig, J.C. Effets of sisi i, ytokinins, n light on isoflvonoi phytolexin umultion in Phseolus vulgris L. Plnt 1982, 154, Peliie, F.M.; Dietrih, S.M.; Brg, M.R. Phytolexin response of fifteen rzilin soyen ultivrs. Rev. Brs. Fisiol. Veg. 2000, 12, Kessmnn, H.; Brz, W. Eliittion n suppression of phytolexin n isoflvone umultion in otyleons of Cier rietinum L., s use y wouning n y polymeri omponents from the fungus Asohyt riei. J. Phytopthol. 1986, 117, Christophe, S.; Zunzzi, J.; El-Turk, J.; Leymrie, J.; Bre, C.; Buffr, D.; e Kozk, I.; Rtet, P.; Husson, P.; Konorosi, A.; Esnult, R. Gene expression is not systemilly linke to phytolexin proution uring lflf lef intertion with pthogeni teri. Mol. Pln-Miroe Intert. 1997, 10, iholson, R.L.; Kollipr, S.S.; Vinent, J.R.; Lyons, P.C.; Cen-Gomez, G. Phytolexin synthesis y the sorghum mesootyl in response to infetion y pthogeni n nonpthogeni fungi. Pro. tl. A. Si. USA 1987, 84, Shettel,.L.; Blke,.E. Plnt growth response to severl llelopthi hemils. Wee Si. 1983, 31, Pnhev, T.V.; Popov, L.P.; Uzunov, A.. Effets of sliyli i on growth n photosynthesis in rley plnts. J. Plnt Physiol. 1996, 149,

20 Moleules 2013, Wr, A.R.; Pulrj, M.G.; Wr, M.Y.; Ignimuthu, S. Role of sliyli i in inution of plnt efense system in hikpe (Cier rietinum L.). Plnt Signl Behv. 2011, 6, Pers, M.S.; Ahihonu, P.W.K. Metolism n etoxifition of phytolexins n nlogs y phytopthogeni fungi. Phytohemistry 2005, 66, Qin, Z.G.; Zho, Z.J.; Xu, Y.; Qin, X.; Zhong, J.J. ovel syntheti 2,6-ihloroisoniotinte erivtives s effetive eliitors for inuing the iosynthesis of plnt seonry metolites. Appl. Miroiol. Biot. 2006, 71, Zho, Z.; Xu, Y.; Qin, Z.; Tin, W.; Qin, X.; Zhong, J.J. ovel fluoro- n hyroxyl-ontining jsmonte erivtives s highly effiient eliitors in suspension ultures of Txus hinensis. Bioorg. Me. Chem. Lett. 2004, 14, Görlh, J.; Volrth, S.; Knuf-Beiter, G.; Hengy, G.; Bekhove, U.; Kogel, K.H.; ostenorp, M.; Stu, T.; Wr, E.; Kessmn, H.; Ryls, J. Benzothiizole, novel lss of inuers of systemi quire resistne, Ativtes gene expression n isese resistne in rley. Plnt Cell 1996, 8, Dolezl, M.; Tumov, L.; Kesetoviová, D.; Tum, J.; Králová, K. Sustitute -phenylpyrzine- 2-roxmies, their synthesis n evlution s heriies n ioti eliitors. Moleules 2007, 12, Lenk, T.; Tum, J.; Dolezl, M. Pyrzineroxmies s potentil eliitors of flvonolignn n flvonoi proution in Silyum mrinum n nonis rvensis ultures in vitro. Moleules 2011, 16, Ksprová, M.; Sitk, T.; Klimezová, V.; Dusek, J. ew syntheti pyriine erivte s potentil eliitor in proution of isoflvonois n flvonois in Trifolium prtense L. suspension ulture. Si. Worl J. 2012, 74, Knoth, C.; Slus, M.S.; Girke, T.; Eulgem, T. The syntheti eliitor 3,5-ihloronthrnili i inues PR1-epenent n PR1-inepenent mehnisms of isese resistne in Ariopsis. Plnt Physiol. 2009, 150, Jinihiro, K.; Toyozo, Y.; Umenur, K.; Iwt, M. Methos of sreening eliitor inuing the proution of phytolexin in rie n rie isese ontrolling gent ontining eliitor s the tive ingreient. U.S. Ptent 6,146,893, file 21 April 1997, n issue 14 ovemer Shu, R.; Thkur, D.J.; Kshyp, P. Shiff se: An overview of its meiinl hemistry potentil for new rug moleules. Int. J. Phrm. Si. noteh. 2012, 5, Rkhi, R.; Prsson, M.A. A review on iologil tivity of quinzolinones. Int. J. Phrm. Phrm. Si. 2012, 4, Pine, R.; Vizíno, S.; Grí C.M.; Gil, J.H.; Durngo, D.L. Chemil omposition n ntifungl tivity of Piper uritum Kunth n Piper holtonii C. DC. ginst phytopthogeni fungi. Chil. J. Agri. Res. 2012, 72, Smple Avilility: Smples of the ompouns 1 to 11 re ville from the uthors y the uthors; liensee MDPI, Bsel, Switzerln. This rtile is n open ess rtile istriute uner the terms n onitions of the Cretive Commons Attriution liense (