Changes in growth and concentration of amino acids in Chinese cabbage seedlings using bacterial fermented broth

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1 A. ARTÍCULOS DE INVESTIGACIÓN / RESEARCH PAPERS Volumen 6, Nº. Págins 7- IDESIA (Chile) Enero, 8 Chnges in growth nd concentrtion of mino cids in Chinese cge seedlings using cteril fermented roth Cmios en el crecimiento y concentrción de minoácidos en ls plántuls de col chin usndo cldo cterino fermentdo Luiz Griel Gemin, Átil Frncisco Mógor *, Gild Mógor, Cinthi Röder, Vivin Jskiw Szilgyi-Zecchin ABSTRACT The iofertilizer derived from the fermenttion of sugrcne molsses y the cterium Corynecterium glutmicum is roth contining L-glutmic mino cid. To evlute the iofertiliztion effect of this roth on the orgnic production of Chinese cge seedlings (Brssic cmpestris vr. Pekinensis), n experiment ws performed in seedling nursery in the orgnic re t the Federl University of Prná (UFPR). Aqueous solutions of the fermented roth were pplied to leves t concentrtions of. ml L - nd.8 ml L - in ddition to the control with wter ppliction. The results indicted tht folir ppliction promoted the development of the eril prt nd roots nd incresed the concentrtion of totl free mino cids in Chinese cge seedlings. These results demonstrted the iofertilizing ction of the cteril fermented roth, promoting seedling growth nd stimulting mino cid synthesis, y incresing the concentrtion of totl free mino cids. Key words: Brssic cmpestris vr. pekinensis. Biofertilizer. Biostimulnt. Orgnic production. RESUMEN El iofertiliznte de l cñ de zúcr derivdo de l fermentción por l cteri Corynecterium glutmicum es un cldo que contiene minoácido L-glutámico. Pr evlur el efecto iofertilizción de este cldo en l producción orgánic de ls plántuls de col chin (Brssic cmpestris vr. Pekinensis), se relizó un experimento en un vivero de plántuls en l zon orgánic de l Universidd Federl de Prná (UFPR). Ls soluciones cuoss del cldo fermentdo se plicron hojs concentrciones de, ml L - y,8 ml L - demás del control con plicción de gu. Los resultdos indicron que l plicción folir promovió el desrrollo de l prte ére y ls ríces y umentó l concentrción de minoácidos lires totles en ls plántuls de col chin. Estos resultdos demuestrn l cción iofertiliznte del cldo cterino fermentdo, promoviendo el crecimiento de ls plántuls y estimulndo l síntesis de minoácidos, umentndo l concentrción de minoácidos lires totles. Plrs clve: Brssic cmpestris vr. pekinensis. Biofertiliznte. Biostimulnte. Producción orgánic. Introduction The fmily Brssiccee hs the gretest diversity mong the vegetle species grown in Brzil (Filgueir, ), which includes Chinese cge (Brssic cmpestris vr. Pekinensis). This species stnds out for its high nutritionl vlue, eing n excellent source of folic cid, vitmin A, vitmins B nd C, s well s clcium nd potssium (Emrp, ). The production of seedlings is n importnt step in the cultivtion of vegetles, phse in which the development nd dequte growth of plnts contriute to the success nd estlishment of the crop (Alvreng, ; Borcioni et l., 6). In ddition, it contriutes to the reduction of the cycle in the field, since the seedlings hve the root system nd eril prt growing compred to direct sowing in eds (Guerr et l., 6). Studies focusing on the use of promoting effect products derived from fermenttive processes on plnt growth re rre; however, recent studies hve shown tht cteril fermenttion of cne Universidde Federl do Prná (UFPR), Deprtmento de Fitotecni e Fitossnitrismo, Curiti, PR, Brsil. * Corresponding uthor: til.mogor@ufpr.r Fech de Recepción: Junio, 7. Fech de Aceptción: 8 Agosto, 7.

2 8 IDESIA (Chile) Volumen 6, Nº, Enero, 8 molsses contining the mino cid L-glutmic cid promoted increses in the iomss of the eril prt nd the growth of the roots of the seedlings, chrcterizing it s iofertilizer (Bettoni et l., ; Röder et l., 5). Biofertilizers re defined in Normtive Ruling N º 6 (Mp, ) s products tht contin ctive components or iologicl gents cple of cting, directly or indirectly, on the complete or on prts of the cultivted plnts, improving the performnce of the production system The use of iofertilizers in the production of seedlings my e considered s sustinle lterntive, especilly in n orgnic system. The ojective of this study ws to determine the iometric chnges in Chinese cge seedlings sumitted to folir pplictions of roth contining the mino cid L-glutmic cid resulting from the fermenttion of sugrcne molsses y the cterium Corynecterium glutmicum (Dreyer et l., ). In ddition, the totl free mino cid nd chlorophyll content in the leves of the seedlings were determined s possile iochemicl indictors of the iofertilizer ction. Mteril nd Methods The experiment ws crried out in protected cultivtion, in ench trditionlly used for the commercil production of seedlings with timed micro-sprinkling irrigtion, in the Orgnic Are of Cnguirí Frm - Federl University of Prná, locted in the city of Pinhis, Prná stte (5º 5 S, 9º 6 W t n ltitude of 9 m). Seeds of Yuuki (Tkii Seeds), Chinese cge hyrid with 95% germintion were used. Sowing ws crried out in expnded polystyrene trys with cells, filled with sustrte composed of composted poultry litter (Provso ), comined with composted pine rk in rtio of :.5, respectively (Bettoni et l., ). The tretments consisted of folir pplictions of queous solutions t the concentrtions of. ml.l nd.8 ml.l of sugrcne molsses roth fermented y the cterium Corynecterium glutmicum (AG), which presents % (m\v) of the mino cid L-glutmic cid nd 6% orgnic cron (Microquímic Indústris Químics Ltd.), s well s control with ppliction of distilled wter. The ppliction ws mde 5 dys fter sowing, when the first true leves ppered. A pressurized spry with CO nd constnt pressure of 5 pounds pol ws used with spry volume of ml per tretment (Röder et l., 5). The experimentl design ws completely rndomized, composed of three tretments with replictes, using five plnts per replicte for the evlutions. At dys fter sowing (DAS), when the seedlings hd produced the fifth true lef, the plnts were collected nd the following vriles mesured: eril prt fresh mss, eril prt dry mss, root fresh mss, eril prt dry mss nd root dry mss rtio, root volume, lef re, totl free mino cid content nd totl chlorophyll in lef tissue. The roots of the seedlings were wshed to remove the dhered sustrte. Then the plnts were sectioned, seprting the eril prt from the root system for the evlution of fresh mss. Lef re nd root volume were nlyzed using WinRhizo softwre coupled to Scner LA6 (Régent Instruments Inc. ); the leves were red t 5 DPI resolution nd the roots t 5 DPI (Szilgyi-Zecchin et l., 5). The dry mss ws otined y drying the plnts in n oven t temperture of 65 ºC ± 5 ºC nd forced ir circultion until constnt weight, nd then weighing on n nlyticl scle. Totl free mino cids were extrcted in distilled wter (Winters et l., ) nd quntified y rection of the smple with. M citrte uffer ph.6 nd ninhydrin solution (% ninhydrin,.% scoric cid in -methoxyethnol) (Mgné nd Lrher, 99). The redings were performed t 57 nm (Mgné nd Lrher, 99) in spectrophotometer (BELuv ). The stndrd curve ws performed with mm glutmine with vlues rnging from 8 to μg/ml, yielding the eqution: y =. x +.7, with R =.998. Pigments were extrcted ccording to Lichtenthler (987) with 8% cetone in distilled wter plus.% CCO (w/v) (Pompelli et l., ). The redings were crried out t 66 nd 67 nm in the sme spectrophotometer previously mentioned. The following formuls were pplied (Lichtenthler nd Buschmnn, ): [Cl ] =.5 x A66 nm -.79 x A67 nm () [Cl ] =.5 x A67 nm - 5. x A66 nm () [Cl + ] = 8.7 x A67 nm A66 nm () Dt were sumitted to nlysis of vrince nd for the vriles tht presented significnt differences,

3 Chnges in growth nd concentrtion of mino cids in Chinese cge seedlings using cteril fermented roth 9 the Scott-Knott test ws performed using significnce level of.. Dt were processed y ASSISTAT softwre, version 7.7 Bet (Silv nd Azevedo, ). Results nd Discussion The fresh mss of the eril prt nd roots of Chinese cge seedlings showed significnt increse on the order of 58% nd %, respectively, when sumitted to the ppliction of the cteril fermented roth contining % L-glutmic cid (AG) t concentrtion of. ml.l (Figure ). A similr result ws otined y Bettoni et l. () with the use of cteril fermented contining L-glutmic cid, which produced n increse in the eril prt nd the root system of Orignum vulgre seedlings. Effects of the exogenous ppliction of L-glutmic cid on growth promotion nd on iochemicl chnges relted to N metolism in plnts were reported y Yu et l. (), relting plnt growth to the sorption of the mino cid tht converted to glutmte provides greter cell division. Seedlings treted with. ml.l AG showed significnt gin of 78% in the dry mss of the eril prt compred to the control (Figure ). Fresh Mss g. Concentrtion ml.l.8 Shoot Root Figure. Effect of folir ppliction of different concentrtions of cteril fermented roth on fresh shoot nd root mss of Chinese cge seedlings cv. Yuuki. Brs topped y the sme letter do not differ significntly y the Scott-Knott test t P <.. (Shoot F = **; Root F =.78**). Dry Mss.. g. c.. Concentrtion ml.l.8 Shoot Root Figure. Effect of folir ppliction of different concentrtions of cteril fermented roth on dry shoot nd root mss of Chinese cge seedlings cv. Yuuki. Brs topped y the sme letter do not differ significntly y the Scott-Knott test t P <.. (Shoot F = 9.5 **; Root F =.96**).

4 IDESIA (Chile) Volumen 6, Nº, Enero, 8 Similrly, Frin et l. () verified n increse in the dry mss of the eril prt of Cichorium intyus L seedlings fter the ppliction of AG t concentrtion of.8 ml.l. Crdoso et l. () lso oserved ccumultion of % of dry mtter per hectre fter pplying iofertilizer contining L-glutmic cid in the cultivtion of Aven stiv. The dry mss of the roots in the seedlings sumitted to the tretment with.8 ml.l showed n increse of out 55% compred to the control (Figure ). Coll et l. () treting tomto stkes with iofertilizer sed on mino cids t concentrtion of 6 ml.l, found greter ccumultion of dry mss of the eril prt nd roots compred to the control; this result ws relted to the sorption nd metoliztion of mino cids present in the iofertilizer. The iomss prtition of plnts my e represented y the rtio etween the dry mss of the eril prt nd the dry mss of the roots. Guzmán-Antonio et l. () discussed the ehvior of this rtio fter fertilizing (N, P, K) hnero pepper seedlings since there is n increse in nitrogen supply; this rtio ws greter compred to the tretments with no ddition of N. We verified tht the seedlings sumitted to AG t the concentrtion of. ml.l, exhiited higher index (.6) compred to the other tretments (Figure ). This result indictes tht the ppliction of the iofertilizer led to greter growth of the eril prt of the seedlings in reltion to the roots. Bettoni et l. (), pplied 8 ml.l of protein hydrolyzte contining mino cids to leves of roccoli, finding greter increses of the fresh nd dry mss of the eril prt of the plnts. The uthors relted the result to the ction of mino cids in plnts ecuse they involved in the synthesis of enzymes nd plnt hormones. Appliction of. ml.l AG incresed lef re y 7% compred to the control (Figure ). Röder et l. (5) found similr results using cteril fermented contining L-glutmic cid in cge seedlings. However, t higher concentrtion (.8 ml.l ), lef re incresed y % compred to the control with no ppliction. These results re expected, considering tht L-glutmic cid is precursor of glutmine nd glutmte in nitrogen metolism, nd my ct to increse the lef re nd eril prt growth (Keruy, 8). Root volume of the Chinese cge seedlings showed significnt increse t the concentrtion of.8 ml.l of AG compred to the other tretments (Figure 5). This result is similr to tht found y Frin et l. () in the cultivtion of chicory seedlings nd y Bettoni et l. () in oregno seedlings sumitted to the ppliction of cteril fermented. This effect is desirle, since the increse of root volume in seedlings is directly ssocited with greter soil explortion nd nutrient sorption (Niu et l., 8). Appliction of AG did not chnge the concentrtion of chlorophyll A, B or the totl Dry Mss Aeril Prt/Dry Mss Roots 6 5 c Rtio. Concentrtion ml.l Figure. Effect of folir ppliction of different concentrtions of cteril fermented roth on the rtio of dry mss of shoots to dry roots of Chinese cge seedlings cv. Yuuki. Brs topped y the sme letter do not differ significntly ccording to Scott- Knott test t P <.. (Rtio F = 7.68**)..8

5 Chnges in growth nd concentrtion of mino cids in Chinese cge seedlings using cteril fermented roth Lef Are 8 cm 6 Concentrtion ml.l Figure. Effect of folir ppliction of different concentrtions of cteril fermented roth on lef re of Chinese cge seedlings cv. Yuuki. Brs topped y the sme letter do not differ significntly ccording to Scott-Knott test t P <.. (Lef re F = 7.6**). Root Volume c cm. Concentrtion ml.l Figure 5. Effect of folir ppliction of different concentrtions of cteril fermented roth on root volume of Chinese cge seedlings cv. Yuuki. Brs topped y the sme letter do not differ significntly ccording to Scott-Knott test t P <.. (Root Volume F = 5.86**).8 in the lef tissue of Chinese cge seedlings. The verges of chlorophyll A, chlorophyll B nd totl chlorophyll of tretments were.9;. nd.5 mg.g for the control,.79;. nd. mg.g for the tretment with. ml.l nd.8,.6 nd.9 mg.g for the tretment with.8 ml.l of the cteril fermented. Although glutmic cid is precursor to chlorophyll synthesis in developing leves (Yronsky et l., 6), no effect of the mino cid on ltering chlorophyll concentrtion hs een found. However, significnt gin ws oserved in the reltive chlorophyll index compred to the control in seedlings of Brssic olerce vr. cpitt sumitted to the ppliction of cteril fermented contining L-glutmic cid (Röder et l., 5). The ppliction of AG promoted significnt increse in the concentrtion of free mino cids in the leves of Chinese cge seedlings. In the tretment with the concentrtion of. ml.l AG, fivefold increse in totl free mino cids of the leves ws found compred to the control (Figure 6). Ertni et l. () reported tht the ppliction of iofertilizer contining mino cids in the cultivtion of corn t concentrtion of. ml.l incresed the mino cid content in the leves nd roots in reltion to the control, indicting the increse in mino cid synthesis

6 IDESIA (Chile) Volumen 6, Nº, Enero, 8 Totl Free Amino Acids µg.g c. Concentrtion ml.l Figure 6. Effect of folir ppliction of different concentrtions of cteril fermented roth on totl free mino cids in Chinese cge seedlings cv. Yuuki. Brs topped y the sme letter do not differ significntly ccording to Scott-Knott test t P <.. (Amino F = 5.55**)..8 due to the low concentrtion pplied. According to Brndão (7), increses in productivity otined y the use of mino cids vi leves in crops re justified ecuse once sored nd metolized, mino cids my prticipte in the synthesis of severl compounds with direct effect on plnt growth, justifying the results of growth promotion otined in this study. Our results show tht AG folir ppliction ws efficient in promoting development of the eril prt, roots nd the concentrtion of totl free mino cids in lef tissue of Chinese cge seedlings conducted in n orgnic system, relted to the increse in free mino cid content. Conclusions The use of the iofertilizer derived from cteril fermenttion contining % of the mino cid L-glutmic cid promoted significnt growth of the eril prt, roots nd totl free mino cid concentrtion of Chinese cge seedlings. The tretment t the concentrtion of.8 ml.l presented greter increses in the root system without reduction of the eril prt, which is desirle effect. The free mino cids in the leves my e iochemicl indictors of the iofertilizer ction s function of their increse in the leves treted with the fermented roth. Literture Cited Alvreng, M.A.R.. Tomte: Produção em cmpo, em cs de vegetção e em hidroponi. th ed. Editor Universitári de Lvrs. Lvrs, Minhs Geris, 5 p. Bettoni, M.M.; dos S.E.G. Frin, J.R. Olinik nd A.F. Mógor. Efeito d plicção folir de hidrolisdo protéico so produtividde de cultivres de rócolis. Revist Agro@ miente On-line, 7 (): Bettoni, M.M.; dos S.E.G. Frin, M. Procopiuk nd A.F. Mógor.. Crescimento de muds de orégno sumetids doses e frequêncis de plicção de ácido L-glutâmico em sistem orgânico. Revist Brsileir de Plnts Medicinis, 6 (): Borcioni, E.; A.F. Mógor nd F. Pinto 6. Aplicção de ácido fúlvico em muds influencindo o crescimento rdiculr e produtividde de lfce mericn. Revist Ciênci Agronômic, 7 (): Brndão, R.P. 7. Importânci dos Aminoácidos n gricultur sustentável. Informtivo BioSoj, 5, 6-8. Crdoso, A.M.; A.R.C. De Lim, R. Pretto, L.G. Arenhrdt, D.R. D Silv, J.A.G. D Silv nd A.M. Crdoso. A produção de iomss em vei rnc pelo uso de ioestimulntes em diferentes estádios de desenvolvimento. XIX Jornd de pesquis. Slão do conhecimento - ciênci, tecnologi, desenvolvimento socil.. Universidde Regionl do Noroeste do Estdo do Rio Grnde do Sul. Coll, G.; Y. Rouphel, R. Cnguier, E. Svecov nd M. Crdrelli. Biostimulnt ction of plnt-derived protein hydrolyste produced through enzymtic hydrolysis. Frontiers in Plnt Science, 5: 8. DOI:.89/ fpls..8.

7 Chnges in growth nd concentrtion of mino cids in Chinese cge seedlings using cteril fermented roth Dreyer, A.; N. Coello nd E. Mondiel.. Utilizción de l metodologí de superfície de respuest de l optimizción de um medio de cultivo pr l producción de L-lisin por Corynecterium glutmicum. Agronomí Tropicl, 5 (): Empres Brsileir de Pesquis Agropecuári - EMBRAPA. Ctálogo Brsileiro de Hortliçs, th ed. EMBRAPA- SEBRAE. Brsili, Brzil. 6 p. Ertni, A.; D. Pizzeghello, A. Altissimo nd S. Nrdi. Use of met hydrolyzte derived from tnning residues s plnt iostimulnt for hydroponiclly grown mize. Journl of Plnt Nutrition nd Soil Science, 76 (): Frin, E.G.; dos S., Á.F. Mógor, G. Mrgoti, J.G. Fowler nd M.M. Bettoni. Purple chicory pll ross seedlings growth ccording to the folir ppliction of l-glutmic cid. Scienti Agrri, (): 9-9. Filgueir, F.A.R.. Mnul de olericultur: cultur e comercilizção de hortliçs.. ed. Agronômic Ceres. São Pulo, Brzil, 6 p. Guerr, N.; É.S.B. d Silv, A.M. Tvres, A. Crlet nd A.M. de Oliveir Neto 6. Interferênci de plnts dninhs n cultur d eterr em semedur diret e trnsplntd. Revist gro@miente on-line, (): 5-. Guzmán-Antonio, A.; L. Borges-Gómez, L. Pinzón-López, E. Ruiz-Sánchez nd J. Zúñig-Aguilr. Efecto del ácido slicílico y l nutrición minerl sore l clidd de plántuls de chile hnero. Agronomí Mesomericn, (): Keruy, G. 8. Fisiologi Vegetl. In: SODEK, L. Metolismo do Nitrogênio.. ed. Gunr Koogn. Rio de Jneiro, Brzil. pp Lichtenthler, H.K Chlorophylls nd crotenoids: pigments of photosynthetic iomemrnes. In: Pcker, L.; Douce, R. Eds. Methods in Enzimology. Vol. 8. Acdemic Press. New York, US. pp Lichtenthler, H.K. nd C. Buschmnn. Chlorophylls nd crotenoids: Mesurement nd chrcteriztion y UV-VIS spectroscopy. Current Protocols in Food Anlyticl Chemistry. John Wiley nd Sons. New York, US. pp. F..-F..8. Mgné, C.; F. Lrher. 99. High sugr content interferes with colorimetric determintion of mino cids nd free proline. Anlyticl Biochemistry, (): 5-8. Mp. Ministério d gricultur, pecuári e stecimento-. Instrução normtiv Nº 6, de 6 de outuro de. MAPA. Brsíli, Brzil, 6 p. Niu, C.; D.J. Gis nd J.C. Cotes 8. Brnching out in new directions: the control of root rchitecture y lterl root formtion. New Phytologist, 79 (): Pompelli, M.F.; S.C. Frnç, R.C. Tigre, M.T. de Oliveir, M. Scilot nd E.C. Pereir. Spectrophotometric determintions of chloroplstidic pigments in cetone, ethnol nd dimethylsulphoxide. Brzilin Journl of Biosciences, (): Röder, C.; Á.F. Mógor, V.J. Szilgyi-Zecchin, E.G. dos S. Frin nd L.G. Gemin 5. Uso de iofertiliznte n produção de muds de repolho. Revist Ceres 6 (5): Silv, F.A.S. nd C.A.V. Azevedo. Versão do progrm computcionl Assistt pr o sistem opercionl Windows. Revist Brsileir de Produtos Agroindustriis, (): Szilgyi-Zecchin, V.J.; Á.F. Mógor, L. Ruro nd C. Röder. 5. Crescimento de muds de tomteiro (Solnum lycopersicum) estimuldo pel ctéri Bcillus myloliquefciens susp. plntrum FZB em cultur orgânic. Revist de Ciêncis Agráris, 8 (): 6-. Winters, A.L.; J.D. Lloyd, R. Jones nd R.J. Merry. Evlution of rpid method for estimting free mino cids in silges. Animl Feed Science nd Technology, 99 (): Yronsky, E.; I. Vershilovsky, Y. Poers, A.E. Alwdy, N. Averin nd B. Grimm 6. Cytokinin effects on tetrpyrrole iosynthesis nd photosynthetic ctivity in rley seedlings. Plnt (): Yu, C.; D.G. Lv, S.J. Qin, L. Yng, H.Y. M nd G.C. Liu. Chnges in photosynthesis, fluorescence, nd nitrogen metolism f hwthorn (Crtegus pinntifid) in response to exogenous glutmic cid. Photosynthetic, (8): 9-7.

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