DETECCIÓN DE FACTORES NOD EN. B. elkanii ICA INFLUENCIA DEL MEDIO DE CULTIVO

Transcription

1 Cultivos Tropiles, 014, vol. 35, no. 1, p enero-mrzo ISSN impreso: ISSN digitl: Ministerio de Eduión Superior. Cu Instituto Nionl de Cienis Agríols DETECCIÓN DE FACTORES NOD EN. B. elknii ICA INFLUENCIA DEL MEDIO DE CULTIVO Nod ftor detetion in B. elknii ICA Culture medium influene Mrí C. Nápoles Grí ), Jun C. Crer, Ellen Luyten, Bruno Domreht, Jos Vnderleyden, Josef Hormz y Armndo Rodríguez Alfonso ABSTRACT. Nodultion ftors, synthesized y different speies from Rhizoiee Fmily, hve een desried s key signls in leguminous intertion. They onstitute morphogens tht eliit the nodulr progrm in the plnt, llowing the teri to enter the roots nd showing positive influene on teroids development nd nitrogen fixtion effiieny. This study is relted to the detetion, y mens of two hromtogrphi tehniques, of Nod ftors prodution y Brdyrhizoium elknii ICA 8001 strin, ultivted in different ulture medi, nd the iologil effet of inoul in the intertion with plnt. Both methods demonstrted tht the ulture medium omposition indue in higher or lower quntity the Nod ftors produed y the terium. Also, the inoulnts from different medi eliit differentited nodultion on soyen plnt. RESUMEN. Los ftores de nodulión, sintetizdos por diferentes espeies de l fmili Rizoiee, hn sido desritos omo señles eseniles en l interión on plnts leguminoss. Ellos onstituyen morfógenos que induen el desrrollo de nódulos en l plnt, permitiendo l entrd de ls teris ls ríes y hn demostrdo tener un influeni positiv en el posterior desrrollo de los teroides y l efiieni de l fijión del nitrógeno. Este trjo está reliondo on l deteión, medinte dos ténis romtográfis, de l produión de ftores Nod por l ep Brdyrhizoium elknii ICA 8001, ultivd en diferentes medios; sí omo el efeto iológio de los inóulos otenidos en interión on l plnt. Amos métodos romtográfios demostrron que l omposiión del medio de ultivo indue, en myor o menor ntidd, el número de estruturs de ftores Nod produidos por est teri. Además, que inoulntes otenidos prtir de diferentes omposiiones de medio, induen un nodulión diferenid sore plnts de soy. Key words: Brdyrhizoium, nodultion, hromtogrphy, legumes Plrs lve: Brdyrhizoium, nodulión, romtogrfí, leguminoss INTRODUCTION It hs een demonstrted tht Nod ftors indue severl responses in the plnt in period from seonds to dys (1), inluding deformtion nd urling of rdil hirs, memrne depolriztion, ortex ell division, new flvonoids formtion, nodulin genes indution, the tivtion of lium spiking, erly nodulin (ENOD) Dr.C. Mrí C. Nápoles Grí, Investigdor Titulr del deprtmento de Fisiologí y Bioquími Vegetl, Instituto Nionl de Cienis Agríols, gvet postl 1, Sn José de ls Ljs, Myeque, Cu; Dr.C. Jun C. Crer, Mteri Nov, Unité de Biotehnologie (UBT). Belgique; Dr.C. Ellen Luyten; Dr.C. Bruno Domreht y Dr.C. Jos Vnderleyden, F. A. Jnssens Lortory of Genetis. Ctholi University of Leuven. Belgium; Dr.C. Josef Hormz, Instituto de Investigiones Científis del Azúr, Hn, Cu; Dr.C. Armndo Rodríguez Alfonso, Centro de Produtos Mrinos, Hn, Cu. ) tere@in.edu.u gene indution, nd infetion thred initition (, 3, 4). Nod ftors re ertinly key trigger for legume symioti signling nd nodule orgnogenesis (5). Lipohitinoligoshrids, hrterized in Brdyrhizoium elknii, vries in the size of their si struture, in the type of ftty id, nd in some modifitions of the si struture, suh s etyl nd rmoil group or N-methyl on the non reduing terminl nd -O-metylfuose or fuose nd glyerol presene on the end reduer. Ten different strutures of these moleules in Brdyrhizoium elknii USDA 61 from severl frtions otined y high pressure liquid hromtogrphy were identified (6). In 1996 (7) ompleted the hemil hrteriztion studies of Nod ftors produed y this strin, purifying the peks with smller sorne. Similr strutures were identified, ut with singulr omintions of the sustitutes. 9

2 Cultivos Tropiles, 014, vol. 35, no. 1, p enero-mrzo This vriility llows the potentil formtion of t lest 96 different strutures, from whih 3 hve lredy een identified, demonstrting the metoli diversity of this speies. In this pper, we studied the Nod ftors prodution y B. elknii ICA 8001 strin in different ulture medi, using two hromtogrphi tehniques nd the iologil effet of the inoulnts on the intertion with soyen plnts ws vlidted. MATERIAL AND METHODS Miroorgnism, medi nd ulture onditions Brdyrhizoium elknii ICA 8001 strin, from the Institute of Animl Sienes olletion ws used. The strin ws ultivted in the different ulture medi in gittion onditions t 30 rpm nd 30 C of temperture. The following ulture medi were used:yest Extrt Mnitol medium (YEM) (8), Propgton medium (9) nd Modified Propgtion medium A. In vitro nodultion ssys Experiments for nodultion studies were rried out in totlly rndomized design, using 10 plnts per tretment, under lortory onditions. The dt ws proessed y using the normlity test (Brtlett s test) nd vrine homogeneity (Kolmogorov-Smirnov s test) nd nlysis of vrine of simple lssifition ws pplied, using Dunn test of multiple rnges (p<0,05) to disriminte differenes etween medi. Soyen seeds, vriety Willim 8, were used. They were disinfeted in 70 % ethnol during 30 seonds nd lter in merury dihloride 0, % (v/v) during 90 seonds. They were rinsed ten times with sterile distilled wter nd then llowed to rest for one hour in sterile distilled wter to stimulte the germintion. Ten seeds were pled in eh Petri dish on wter-gr; they were overed with luminium foil nd inuted during four dys t 30 C. Ten dishes were used per experiment. The germinted seeds, with pproximtely 0 mm of emergent roots, were pled in flsks of 50 ml ontining 150 ml of Norris nd dte semisolid medium (10), one seed per flsk. The plnt roots were inoulted y dishrging 00 µl of the inoul otined with the strin B. jponium ICA 8001 in the different ulture medi, in ellulr onentrtion of 1.5 x 10 8 CFU.mL -1, orresponding to OD = 0,1 t 0 nm. A Nápoles, M.C. Induión de l nodulión en soy (Glyine mx (L.) Merrill) por Brdyrhizoium sp. Influeni del medio de ultivo. [Tesis de Dotordo]. Universidd de L Hn, L Hn, Cu p. A tretment of plnts without inoultion ws used s ontrol in eh ssy. Plnts were ultivted in growth hmer with photoperiod of 1 h light/1 h drkness, t dy/night temperture of 6/ºC nd reltive humidity of 70 %. Four weeks fter the inoultion, the numer of nodules per plnt nd the fresh nd dry mss of the nodules were determined. Also the ility to fix tmospheri nitrogen ws mesured y mens of the etylene redution ssy, using gs hromtogrph (5890 A; Hewlett-Pkrd, equipped with olumn of CHROMPACK "PLOT" (Porous Lyer Open Tuulr), 50 meters x 0,3 mm with Al O 3 /KCl 5,0 µm to C) s it is desried in (11). The temperture of the oven ws 130 C, the flow of N of 50 ml.min -1 nd ir nd hydrogen ws of 300 ml. min -1. The plnts were ut only leving the rdil system with the nodules. The flsks were seled with ruer hermeti tps through whih 10 ml of etylene were injeted. They were inuted during 15 minutes to mesure the quntity of formed ethylene, then extrting 100 µl of gs nd injeting it in the hromtogrph. An internl stndrd of 100 µl of propne gs ws used s referene in eh flsk. The otined vlues were expressed s ethylene miromoles produed y plnt per hour (µmol C H 4. pl -1.h -1 ). Detetion of Nod ftors The Nod ftors prodution were isolted, purified nd identified following the methodology desried y some uthors (1). For the hrteriztion y thin lyer hromtogrphy, Brdyrhizoium ultures were rdiotively lelled dding 15 µl of 14 C [- 14 C] eti id s sodium slt nd for high performne liquid dhromtogrphy determintion; the ells were ultivted in 1 L of ulture medium without isotopi lel. Determintion of Nod ftors prodution y thin lyer hromtogrphy (TLC): The Nod ftor profile produed nd exreted y B. jponium ICA 8001 strin in YEM, propgtion nd modified propgtion ulture medi ws studied. Genistein 10 µm ws lso used s mplifier in ll the extrts. The smples were pplied on reverse-phse TLC pltes (RP-18 F 54, Merk). H O/etonitrile (1:1, vol./vol.) ws used s the moile phse. Determintion of Nod ftors prodution y norml phse high performne liquid hromtogrphy (HPLC): The prodution of these moleules in YEM, propgtion nd modified propgtion medi ws determined, s well s in the lst one with genistein ddition s mplifier t 10 µm onentrtion. The Nod ftors purified were dissolved in 100 % etonitrile nd then injeted into norml phse olumn Ultrop TSK OH-10 5um LKB with dimensions of 4,6 x 93

3 Cultivos Tropiles, 014, vol. 35, no. 1, p enero-mrzo 50 mm. The flow rte 1 ml.min -1 ; s solvent were used: etonitrile (A): wter (B); the detetor: n UV spetrophotometer t 06 nm nd ell of 10 mm, the Grdient in Knuer pump: 0/0 10/0 70/0 t/%b nd with n Injetion of 50 µl in 100 % etonitrile. The running time in ll ses ws 70 minutes. The hromtogrphi profile of the Nod ftors ws evluted ording to the numer, distriution nd reltive intensity of the otined peks. RESULTS AND DISCUSSION Influene of different ulture medi on the soyen nodultion Nodules.pl -1 T1: (YEM) T: (YEM+G/10) T3: (YEM+Sm/10) T4: (YEM+Sm/10+Ez) T5: (YEM+M/10) T6:Propgtion T7: Modified Prop d T8: Control The evlution of the influene of different ulture medi on the soyen nodultion ws rried out on the se of previous results (Figure 1,, 3, 4). A new ulture medium ws designed from propgtion medium, in whih the milled soyen seed ws inorported up to the onentrtion of 10 g.l -1, the molsses onentrtion ws ut down to 5 g.l -1 nd the yest extrt ws removed. For the numer of nodules (Figure 1), it ws oserved tht the tretments presented signifint differenes. The est tretments were T (YEM + genistein), T3 (YEM + milled soyen) nd T4 (YEM + soyen treted enzymtilly), followed y T7 (modified propgtion medium) tht did not differ signifintly from T nd T4. The tretments T5: (YEM+molsses) nd T6: (propgtion) were inferior nd sttistilly similr to the T1 (YEM) whih only overme the ontrol without inoultion. The smllest numer of nodules in the tretments T1 nd T6 ould e ssoited to the sene in them of the induers ehved y the soyen. The ddition of milled soyen shows similr ehviour to the genistein ddition to the ulture medium, rehing similr vlues of nodultion. Figure shows the influene of the tretments on the nodulr fresh weight. The est results were rehed in the tretments with genistein, milled soyen without enzymti tretment nd the modified propgtion medium, whih did not differ signifintly. The enzymti tretment to the soyen diminished its effet on this vrile. Regrdless the milled soyen without enzymti treted (T3), the other tretments neither differed from the T4 (YEM + milled soyen tried enzymtilly) nd T5 (YEM + molsse 10). The lst one, were sttistilly similr, lso, to the YEM nd propgtion medi whih only overme to the solute ontrol. T1: yest extrt mnnitol medium; T: medium YEM with genistein 10 µm; T3: medium YEM with milled soyen t the onentrtion of 10 g.l -1 ; T4: medium YEM with milled soyen t the onentrtion of 10 g.l -1 nd treted enzymtilly; T5: medium YEM with molsses to the onentrtion of 10 g.l -1 ; T6: propgtion medium; T7: modified propgtion medium; T8: ontrol Common letters do not differ signifintly (Dunn p<0,05) SE*** =,1870 n=10 Figure 1. Effet of different ulture medi on the soyen nodultion, vriety Willim 8 FWnodules.pl -1 0,4 0,35 0,3 0,5 0, 0,15 0,1 0,05 0 T1: (YEM) T: (YEM+G/10) T3: (YEM+Sm/10) T4: (YEM+Sm/10+Ez) T5: (YEM+M/10) T6:Propgtion T7: Modified Prop T8: Control T1: yest extrt mnnitol medium; T: medium YEM with genistein 10 µm; T3: medium YEM with milled soyen t the onentrtion of 10 g.l -1 ; T4: medium YEM with milled soyen t the onentrtion of 10 g.l -1 nd treted enzymtilly; T5: medium YEM with molsses to the onentrtion of 10 g.l -1 ; T6: propgtion medium; T7: modified propgtion medium; T8: ontrol Common letters do not differ signifintly (Dunn p<0,05) SE*** = 0,017 n=10 Figure. Effet of different ulture medi on the fresh weight of the nodules developed on the soyen vriety Willim 8 d 94

4 Cultivos Tropiles, 014, vol. 35, no. 1, p enero-mrzo Although the nodules developed y the inoulum in the modified propgtion medium went inferior in numer to those produed y the tretment with milled soyen (Figure 1), they showed igger size, wht mkes them sttistilly similr when nlyzing the vrile nodulr fresh weight (Figure ). When the vrile nodule dry weight ws nlyzed (Figure 3), the effets of the genistein ddition were higher, long with the soyen without enzymti tretment, the molsses nd the Modified propgtion medi. The tretments T4 (soyen treted enzymtilly) nd T6 (propgtion medium) did not differ from the ontrol YEM (T1) nd they only surpss the ontrol without inoultion. The enzymti tretment ffeted negtively the nodulr dry weight. DWnodules.pl -1 0,1 0,08 0,06 0,04 0,0 0 T1: (YEM) T: (YEM+G/10) T3: (YEM+Sm/10) T4: (YEM+Sm/10+Ez) T5: (YEM+M/10) T6:Propgtion T7: Modified Prop T8: Control T1: yest extrt mnnitol medium; T: medium YEM with genistein 10 µm; T3: medium YEM with milled soyen t the onentrtion of 10 g.l -1 ; T4: medium YEM with milled soyen t the onentrtion of 10 g.l -1 nd treted enzymtilly; T5: medium YEM with molsses to the onentrtion of 10 g.l -1 ; T6: propgtion medium; T7: modified propgtion medium ; T8: ontrol Common letters do not differ signifintly (Dunn, p<0,05) SE*** = 0,0054 n=10 Figure 3. Effet of different ulture medi on the nodules dry weight developed on soyen vriety Willim 8 As for the tivity of etylene redution (Figure 4), the induers genistein, non treted soyen, molsses nd the modified propgtion medium, ll showed superior results nd sttistilly different to the rest of the tretments. One gin, the effet of the ddition of soyen nd/or molsses in the ulture medium stnds out, nd is omprle to the tion of genistein, nd the pity of the teri to fixing nitrogen, with regrd to other ulture medi. Soyen enzymti digestion efore dding it to the ulture medium influened negtively the pity of iologil fixtion, mking it signifintly different to the effet of the soyen without tretment nd similr to YEM nd propgtion medi. ARA (µmoles ethilene.h-1.pl -1 ) T1: (YEM) T: (YEM+G/10) T3: (YEM+Sm/10) T4: (YEM+Sm/10+Ez) T5: (YEM+M/10) T6:Propgtion T7: Modified Prop T8: Control T1: yest extrt mnnitol medium; T: medium YEM with genistein 10 µm; T3: medium YEM with milled soyen t the onentrtion of 10 g.l -1 the teril strin; T4: medium YEM with milled soyen t the onentrtion of 10 g.l -1 nd treted enzymtilly; T5: medium YEM with molsses to the onentrtion of 10 g.l -1 ; T6: propgtion medium; T7: modified propgtion medium; T8: ontrol Common letters do not differ signifintly (Dunn, p<0,05) SE *** = 0,3464 n=10 Figure 4. Influene of different ulture medi on the nitrogen fixtion in the nodules developed on soyen vriety Willim 8 Compring the results with enzymti digestion of soyen, it ws verified tht it didn't ffet the numer of nodules, while it signifintly diminished the nodulr weight nd their effiieny. These results ould e due to the hydrolysis ws on those induers or sensitive preursors to this type of enzymes, eing degrded some omponents from soyen seed tht prtiipte in the estlishment of the teroids, while other omponents of the seed tht were not degrded, n lso influene in the oloniztion nd effetiveness of the teril strin. Compring the influene of the tretments T5 (YEM with molsses) nd T6 (propgtion medium tht ontins molsses in sme proportion) it ws ppreited tht on the numer nd nodulr weight, the ehviour ws very similr, ut on the nitrogen fixtion (ARA), YEM medium with molsses overme the propgtion. This indites tht not only the molsses hve influene on, ut lso to keep in mind the role of the other omponents from the ulture medium nd the possile synergi or ntgonisti effet tht they n exert. The yests, for exmple, possess in their wll polymers of N-etylgluosmine (13), those during the hydrolysis proess for the extrt 95

5 Cultivos Tropiles, 014, vol. 35, no. 1, p enero-mrzo otinment, n e lierted s oligomers nd so s they re ontined in the ulture medium, they ould t s preursors of the nodultion ftors synthesis in Brdyrhizoium. Then, inrements in the prodution of ertin strutures of these iomoleules would led to igger nodulr effiieny. In the propgtion medium, these ompounds re in onentrtion five times higher thn in YEM medium, wht ould produe inhiition for exess of this ompound. An importnt influene ws demonstrted y molsses on the tivity of etylene redution, similr to tht hppened in previous experiment, where it didn't exerise n outstnding funtion s for the numer neither nodules fresh weight, ut in the dry weight of the sme ones nd in their fixtion tivity. This suggests tht this ompound influenes less in the indution of the nodules formtion, ut more in the teroids estlishment nd in its funtion inside the nodule. In generl, the results suggest tht the integrtion of the soyen seed milled nd the molsses in the omposition of modified propgtion ulture medium, led the teri to physiologi stte tht ws trnslted in signifintly superior results of their iologil tivity on the soyen plnt, in omprison with the trditionl medi. Detetion of the prodution of nodultion ftors in the strin B. elknii ICA 8001 y thin lyer hromtogrphy (TLC) The profiles of the nodultion ftors produed y this strin in presene of different ompounds nd ulture medi evluted in the nodultion ssy re shown in Figure 5. The strin exhiited vriety of profiles ording to the induer used for the tivtion of its nod genes. YEM medium indued the prodution of 8 hromtogrphi spots. When genistein ws used s induer it showed 6 spots nd those with smller moility ording to TLC, stood out for its intensity. The soyen smple without eing enzymtilly treted overme in numer nd intensity the hromtogrphi spots of the soyen treted, the sme wy on some nodultion hrters (Figures, 3 nd 4). There were six spots in the rw soyen, ll in the sme position tht those whih pper in genistein, ut this time the higher intensity orresponding to those of higher moility. The molsses, on the other hnd, showed four ompounds or group of ompounds, most of them in gret onentrtion nd lthough only two in similr position to those of the soyen, oinidently, those of more intensity orresponded to the more moility. YEM Y + G Y + S Y + S + E Y + M Propgtion Modif. Prop -: Non indued extrts +: Extrts indued with genistein 10 µm YEM: Yest Extrt Mnnitol medium Y+G: medium YEM with genistein 10 µm Y+S: medium YEM with milled soyen t the onentrtion of 10 g.l -1 Y+S+E: medium YEM with milled soyen t the onentrtion of 10 g.l -1 nd treted enzymtilly Y+M: medium YEM with molsses to the onentrtion of 10 g.l -1 Propgtion: Propgtion medium Modif Prop.: Modified Propgtion medium Figure 5. Profile of Nod ftors produed y B. elknii ICA 8001 in front of different induer ompounds 96

6 Cultivos Tropiles, 014, vol. 35, no. 1, p enero-mrzo Similr to the results otined in the nodultion ssys, the soyen nd the molsses onstituted ompounds tht stood out in the Nod metolites prodution with the synthesis of gret numer of ompounds, with the highest intensity. The modified propgtion medium overme the Propgtion medium with the prodution of eight hromtogrphi entities, some of them in high onentrtion. This medium uses the prodution of most of the Nod ftors shown y the other induers y seprte, wht is logil euse its hemil omposition, in whih ll of them re inluded. Anlyzing YEM nd propgtion medi, it ws oserved tht the first one overme the ltter one s for the prodution of these ompounds; however, they did not show signifint differenes in the nodultion results. The iggest prodution of Nod ftors (evidened in the TLC) in YEM medium ws not trnslted in igger effiieny of the nodultion, when these results were ompred with the nodultion in vitro ssys, wht suggests tht not ll the represented moleules hd identil iologil tivity. It is known tht not ll the Nod ftors produed nd exreted, possess signifint iologil tivity, other rhizoil systems suh s exopolyshride exretion (14), ethylene iosynthesis regultion (15) nd protein seretion systems (16) re often required for the estlishment of symiosis with legumes, proly euse they re involved in teril relese into the host ytoplsm nd teroid development. The literture indites the use of genistein s mplifier of the indution for Brdyrhizoium, onstituting the est informed induer for this genus. However, the results of this study demonstrted tht their pplition to onentrtion of 10 µm s mplifier of the synthesis of these iomoleules hd negtive effet on 70 % of the ssyed smples, wht is the result of prodution inhiition of these ompounds. Only in those ses tht did not show n ppreile prodution of Nod ftors (soyen enzymtilly digested nd Propgtion medium) the mplifier effet of genistein ws present. The rest of the smples tht reveled ler synthesis of these iomoleules without the help of the genistein exhiited repression in its prodution when this isoflvonoid ws present. This is due; pprently, to n inhiition phenomenon for induer exess (similr to wht hppens in the inhiition for sustrte exess) exerised y the ddition of the induers ontined in the ulture medium plus the pplied genistein. They hve not een referenes of this phenomenon in the onsulted literture, in whih these induers re lwys used in sl medi, free of ny other indution. In generl wy, the results of the Nod ftors prodution for this strin were relted diretly with the nodultion response. Detetion of the prodution of nodultion ftors in the strin B. jponium ICA 8001 y mens of high pressure liquid hromtogrphy (HPLC) in norml phse The nlysis of the profiles otined y mens of liquid hromtogrphy reveled mrked differenes etween the numer of hromtogrphi peks nd its reltive intensity for the different ulture medi (Figure 6). In ll the profiles, less resolved peks were presented in the smllest retention times (rt), whih orrespond to moleules more hydrophoi. The smllest numer of peks ws in the Propgtion medium (Figure 6B), followed y YEM medium (Figure 6A). There is smll differene etween these medi onerning the distriution of peks nd the re under the urve, with four peks in the propgtion medium nd six in YEM, in whih three oinide to eh other. The iggest quntity in peks nd with more re under the urve ws ppreited in the Modified Propgtion medium (Figure 6C). In eh one of the three mjority peks, the re is igger tht the totl re of ll the peks in eh one of the other smples. This indites higher prodution of Nod ftors in this medium. When genistein ws dded to this medium omposition, the depression of the hromtogrphi res ws oserved, it men tht the prodution of these Nod ftors ws repressed, esides tht hnge in the distriution of the peks hppened (Figure 6D). Under these onditions, some peks of the mjority ones disppered nd one of them (rt.= 7,63 min) diminished to third, while other (rt.= 8,8 min), inresed ten times. Given the similrity in the retention time for oth smples, they should orrespond to similr strutures of Nod ftors present in them, lthough in front of the genistein they were presented in smller onentrtion. Different results were found y the other uthors (17), where profile of these moleules with igger numer nd intensity of peks, hrterized the smples indued with genistein nd nringenine, respetively. The smples they studied, however, me from sl medium tht did not ontin induers, so the repression effet did not hppen. Although it is not possile to estlish n ext reltionship mong the spots numer otined y TLC nd the numer of peks found y mens of HPLC, euse the first method is muh more speifi, lelling rdio-tive nd seletively the Nod ftors, while HPLC is more potent to the seprtion of the produts, the otined results y the ltter one, onfirmed in generl, those found with the thin lyer hromtogrphy. 97

7 Cultivos Tropiles, 014, vol. 35, no. 1, p enero-mrzo A mv 130,5-1, ,00 minutes 70,0 Peks rt re % áre 1 17,93 11,84 9,69 0,85 88,34 7,9 3,67 5,43 4,44 4 7,36 10,54 8,6 5 30,05 3,79 3,10 6 3,67,8 1,86 B mv -18,49 130, ,00 minutes 61,59 Peks rt re % áre 1 17,89 83,74 77,63 4,77 14,76 13,68 3 7,69 6,3 5, ,43 3,14,91 C mv 1041,64-1, ,00 minutes,0 Peks rt re % áre 1 19,49 18,85 1,8 19,93 417, ,09 3,0,3 4 4,13 9,54 0,91 5 6,5 7,76 6,97 6 7,58 44,79 3,44 7 8,6 16,11 1,54 8 9,80 11,9 1, ,41 3,9 0, ,11 9,64 0, ,57 14,79 1,4 D mv 1049,5-6, ,00 minutes,03 Peks rt re % áre 1 17,46 11,74,05 0,04 3,1 4,04 3 0,95 48,96 8,57 4,01 47,06 8,3 5 3,47 146,59 5,65 6 5,34 34,35 6,01 7 7,63 56,57 9,90 8 8,8 149,01 6, ,84 5,3 0,9 10 3,7 48,84 8,55 Figure 6. Elution profile in norml phse of Nod Ftors produe y B. elknii ICA 8001 in ulture medi YEM (A), Propgtion (B), Modified Propgtion (C) nd Modified Propgtion with genistein 10 µm (D) 98

8 Cultivos Tropiles, 014, vol. 35, no. 1, p enero-mrzo By mens of oth tehniques ould e possile to verify the influene of the ulture medium on the nodultion ftors profiles. The tendeny to the inhiition or repression of its synthesis in presene of n induer exess in ulture medium ws lso oserved. REFERENCES 1. Cooper, J. E. Erly intertions etween legumes nd rhizoi: dislosing omplexity in moleulr dilogue. Journl of Applied Miroiology, 007, vol. 103, no. 5, p Jones, K. M.; Koyshi, K.; Dvies, B. W.; Tg, M. E. nd Wlker, G. C. How rhizoil symionts invde plnts: the Sinorhizoium-Medigo model. Nture Reviews, 007, vol. 5, p Oldroyd, G. E. nd Downie, J. A. Coordinting nodule morphogenesis with rhizoil infetion in legumes. Annu. Rev. Plnt Biol., 008, vol. 59, p Ito, S.; Ohtke, N.; Sueyoshi, K. y Ohym, T. The utoregultion of nodultion mehnism is relted to lef development. Plnt Cell Physiol., 008, vol. 49, p Kouhi, H.; Imizumi-Anrku, H.; Hyshi, M.; Hkoym, T.; Nkgw, T.; Umehr, Y.; Sugnum, N. nd Kwguhi, M. How mny pes in pod? Legume genes responsile for mutulisti symioses underground. Plnt Cell Physiol., 010, vol. 51, p Crlson, R. W.; Snjuán, J.; Bht, U. R.; Glushk, J.; Spink, H. P.; Wijfjes, H. W.; vn Brussel, A. N.; Stokkermns, T. J. W.; Peters, N. K. nd Stey, G. The strutures nd iologil tivities of the lipooligoshride nodultion signls produed y Type I nd Type II strins of Brdyrhizoium jponium. J. Biol. Chem., 1993, vol. 68, p Stokkermns, T. J. W.; Ron, O.; Kumr, V. S.; Rusell, C. W.; nd Peters, N. K. Biologil tivities nd strutures of Brdyrhizoium elknii low undne lipo-hitinoligoshrides. Mol. Plnt-Miroe Intert., 1996, vol. 9, p Vinent, J. M. A mnul for the prtil study of rootnodule teri/j.m. Vinent. En: Interntionl Biologil Progrmme Hndook. No. 15. Blkwele sientifi pulitions, Oxford, Englnd López, M. Mnul de Tenologís de Produión del iofertiliznte Bio-Rhizo. L Hn: Empres Cun de Produtos Veterinrios Cu-Vet. Instituto de Cieni Animl, Instituto de Investigiones Pstos y Forrjes Norris, D. O. nd Dte, R. A. Legume teriology Tropil Psteur Reserh. Priniples nd Methods. C.A.B. Bill., 1976, vol. 51, p Domreht, B. The omplex regultion nd role of the Rhizoium etli Rpon regulon in the symioti intertion with the ommon en plnt (Phseolus vulgris L.). Disserttiones de Agriultur, Deemer 001, 151 p. 1. Leremns, T. The role of the Nod ftor sulphtion genes in the Rhizoium-Phseolus vulgris (L.) intertion. Disserttiones de Agriultur, April p. 13. Konopk, J. N-Aetylgluosmine Funtions in Cell Signling. Sientifi, 01, 15 p. doi org/10.64/01/ Downie, J. A. The roles of extrellulr proteins, polyshrides nd signls in the intertions of rhizoi with legume roots. FEMS Miroiol. Rev., 010, vol. 34, p Gresshoff, P. M.; Lohr, D.; Chn, P.; Bisws, B.; Jing, Q. nd Reid, D. Geneti nlysis of ethylene regultion of legume nodultion. Plnt Signl, 009, vol. 4, p Dekin, W. J. nd Broughton, W. J. Symioti use of pthogeni strtegies: rhizoil protein seretion systems. Nt. Rev. Miroiol., 009, vol Cárdens, L.; Domínguez, J.; Quinto, C.; López-Lr, I.; Lugtenerg, B.; Spink, H.; Rdemker, G.; Hverkmp, J. nd Thoms-Otes, J. Isoltion, hemil strutures nd iologil tivity of lipo-hitin oligoshride nodultion signls from Rhizoium etli. Plnt Moleulr Biology, 1995, vol. 9, p Reiido: de septiemre de 011 Aeptdo: 4 de mrzo de 013 Cómo itr? Nápoles Grí, Mrí C.; Crer, Jun C.; Luyten, Ellen; Domreht, Bruno; Vnderleyden, Jos; Hormz, Josef y Rodríguez Alfonso, Armndo. Deteión de ftores Nod In. B. Elknii ICA Influeni del medio de ultivo. Cultivos Tropiles, 014, vol. 35, no. 1, p