Chitosans as bioactive macromolecules to protect economically relevant crops from their main pathogens

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1 Chitosns s iotiv mromoluls to prott onomilly rlvnt rops rom thir min pthogns Aljnro Flón 1, Ai T Roríguz 2, Migul A Rmírz 2, Dynir Rivro 2, Bnito Mrtínz 3, Jun C Crr 4, Dimy Costls 1, Aril Cruz 2, Luis G Gonzálz 5, Mrí C Jiménz 5, Lonl Jiménz 5, Iln Hrnánz 1, Dinvys Gonzáls 1, Rmon Márquz 1 1 Dprtmnto Fisiologí y Bioquími Vgtl Instituto Nionl Cinis Agríols, INCA L Hn, Cu 2 Estión xprimntl l Arroz Instituto Nionl Cinis Agríols Los Plios, Pinr l Río, Cu 3 Dprtmnto Protión Plnts Cntro Nionl Sni Agropuri, CENSA L Hn, Cu 4 Dprtmnto Biologí Clulr Vgtl Fult Agriultur, Univrsi Notr Dmm l Pix Nmur, Bélgi 5 Fult Agronomí Univrsi Grnm, Cu E- mil: llon@in.u.u ABSTRACT Stuis wr rri out s prt o th Agriultur Biothnology progrm, to prpr n hrtriz hmilly n iologilly irnt hitosns otin rom Cun lostr hitin. Chitosn polymrs wr sujt to i n nzymti hyrolysis y using low-ost ommril nzymti prprtions, n th rsulting oligoshri mixturs wr urthr hrtriz. Thir potntil ntimiroil tivitis wr lso vlut vrsus ungi n oomyts, lso tsting thir ility to inu nsiv n prottiv rsponss in too n ri plnts ginst two onomilly rlvnt psts, Phytophthor niotin n Pyriulri gris, rsptivly. With th i o intrntionl ollortion, irnt oligohitosns mixturs wr ompr or tivting nsiv rsponss in suspnsion ulturs o Ariopsis thlin lls. Ths rsults ring knowlg on th physil-hmil proprtis o th hitosns otin, suh s molr mss n tyltion gr, n thir inlun on tivting nsiv rsponss, th inhiition o growth in pthogns n th inution o rsistn in too n ri plnts. Som o ths hitosn rivtivs wr slt s possil tiv omponnts to prott oth typ o ultivrs, ing ppli t il-sl to vlut thir ts or th min nturl pthogns n ringing vry promising rsults. This rsrh llow us to stlish mthoology or prpring oligohitosns, n rsults shown inhr wr prt o BS, MS n PhD thss, n wr lso pulish in mor thn 20 sintii pprs n prsnt in mor thn 40 sintii onrns. Kywors: hitosn, nzymti hyrolysis, ntiungl tivity, inu rsistn, Phytophtor niotin REPORT Introution Highr plnts r l to initit nsiv rtions in rspons to pthogn s intions [1]. Thos rsponss r triggr whn th plnt rognizs svrl signls whih r rls s prouts o th mutul nzymti grtion o ll wlls o oth, th plnts n th pthogns [2]. Th rsulting oligoshris, lso known s oligoshrins, not only srv s primry signls o th nsiv rsponss, ut lso inlun othr iologil rsponss rgring vgtl growth n vlopmnt [3, 4]. Among oligoshrins, th ll wll ptins oligoglturonis, β-gluns, n th ungi ll wll hitin n hitosn rgmnts r potntil inurs o rsistn n prottion rom ivrs pthogns in plnts. Tht s th rson why thy r urrntly inlu s tiv omponnts in svrl prottiv prouts or griultur [3-6]. Chitin is linr polymr o N-tyl-gluosmin, n th son mor unnt nturl polymr tr llulos. It is oun on ungi ll wlls n xoskltons o rthropos s th min proution sour Corrsponing uthor worlwi, with mor thn 10 gigtons (1 x kg) prou pr yr. It is minly otin rom shrimp n r xoskltons, with vry smll proution rom lostr xoskltons [7]. Th lttr r isr in lrg mounts s yprout to th s to voi nvironmntl prolms, whih oul us to otin mromoluls highly pplil in miin, inustry n griultur [7]. Chitosn, th min hitin rivtiv, is linr polymr o gluosmin monomrs link y β 1-4 ons n it is otin y lklin tyltion. This pross prous polymr solul in ilut is, whih is vntgous ovr hitin or griulturl us. On this il, hitosns n thir rivtivs o lowr molulr wights oul ppli, s on thir provn iologil potntilitis, suh s signiint ntimiroil tivity on th growth n vlopmnt o ungi, tri n oomyts [5, 8]; prottion o plnts rom potntil pthogns n promotion o ultivr growth n vlopmnt [5, 6]. 1. Agrios GN How plnts n thmslvs ginst pthogns (Chptr six). In: Plnt Pthology. 5th., Nw York, USA: Ami Prss, 2005, p Esqurré-Tugyé MT, Bourt G, Dums B. Cll wll gring nzyms, inhiitory protins, n oligoshris prtiipt in th molulr ilogu twn plnts n pthogn. Plnt Physiol Biohm 2000;38: Rily BL, O Nill MA, Mohnn D. Ptins: strutur, iosynthsis, n oligoglturonis-rlt signling. Phytohmistry 2001;57: Shiuy N, Minmi E. Oligoshri signlling or nss rsponss in plnt. Physiol Mol Plnt Pthol 2001;59: Butist-Bños S, Hrnánz-Luzro AN, Vlázquz-l Vll MG, Hrnánz- Lópz M, Ait Brk E, Bosquz-Molin E, t l. Chitosn s potntil nturl ompoun to ontrol pr n posthrvst isss o hortiulturl ommoitis. Crop Prot 2006;25:

2 Aljnro Flón Roríguz t l. Rport Both, th irt ntimiroil tivity n th inution o nsiv rsponss ginst pthogns o hitosns, r inlun y th physil-hmil proprtis o th molul, inluing th tyltion gr (AD) n molulr wight (MW)[5, 9]. Th lowr AD o th givn hitosn polymr th highr inhiitory tivity on miroil growth is otin [5]. On th ontrry, rs rs in th MW o th givn hitosn my inrs or rs its inhiitory tivity on growth, pning on th spi [5, 10]. Ths typs o stuis r sr on oomyts, n thr r no stuis in litrtur nlyzing th inlun o ths two vrils on th vlopmnt o this gnus. Ths proprtis lso inlun th tivtion o nsiv rsponss in plnts. Kuss n oworkrs [11] monstrt tht prtil tyltion n rgmnttion o hitosns wr rquir or inuing H 2 in uumr. On th othr hn, rsults rom Vnr t l. [9] init tht hitosn polymrs with AD ovr 35% inu th highst Phnyllnin mmoni lys (PAL) n proxis (POD) lvls in wht lvs. Prvious works on th rol o AD n MW or hitosn-s inution o nsiv rsponss wr rri out only in isolt prts o pr- [9] or nononition [11] plnts ut nithr in omplt plnts nor in th prsn o pthogns. Evn whn thr r rports o th prottion o ulturs ginst pthogns y ministring irnt typs o hitosns [5, 12], th t o this ompoun on th Niotin tum-phytophthor niotin intrtion rmins to luit, ing only rport th prtil prottion ginst th Too Mosi Virus (TMV) y using oligohitosns mixturs [13]. Thror, thr ws no inormtion out th inution o rsistn ginst pthogns othr thn th TMV in too plnts, nor in th vlution o hitosn or inuing rsponss in Cun too n ri spis. Givn th urrnt ontxt o Cun griultur, it is highly rlvnt to hv non-toxi nturl prout in origin, prou rom ntionl sours n y orl mns, s rplmnt ltrntiv or th xpnsiv hmil pstiis ing import. Th Group o Biotiv Prouts (GPB) t th Ntionl Institut o Agriulturl Sins (INCA) hs xprin in vloping mthoologis to prpr oligoshrins rom ntionl rw mtrils n vluting thm s hormon sustituts or in vitro ultur, to promot root nhoring, soyn symiosis improvmnt n inution o plnt rsistn ginst isss. Svrl o ths rsults hv n lry ptnt. Bs on wht mntion ov, our rsrh o hitosns ous on th ollowing ojtivs: vlopmnt n pttion o mthoologis to otin hitosns polymrs n oligomrs rom ntionllyprou lostr hitin, whih show potntil iologil tivity in plnts; to vlut th t o hitosns o irnt AD n MW in th in vitro vlopmnt o pthogns o onomilly rlvnt rops; to vlut th t o hitosns in inuing rsistn in suspnsion ulturs o Ariopsis lls, n in too n ri plnts inoult with pthogns n; to tst promising ompouns on il xprimnts or oth rops. Mtrils n mthos Prprtion o hitosn poly- n oligoshris Chitosn polymrs wr otin y si tyltion with NOH t rtor sl, rom ntionlly otin lostr hitin. Th mthoology ormrly sri to otin shrimp hitin ws pt or lostr hitin prossing t th GBP (Group o iotiv prouts) o INCA. Stuis wr rri out y pplying hitosn hyrolysis with ommril nzymti mixs n using typil nzymology thniqus, suh s: th inlun o irnt sustrts t irnt onntrtions, ph, tmprtur n othrs. Chitosn mixturs wr hrtriz using mss sptromtry thniqu (MALDI-TOF) or th irst tim to trmin oligoshri AD n MW simultnously. Ai hyrolysis [14] ws lso tst to otin oligohitosns. Anlysis o th t o hitosns on pthogn growth Th t o hitosn-s ompouns on mylil growth, sporultion n spors viility wr nlyz. Pthogns wr isolt rom ri n too plnts, n ultur in ptri ishs ill with soli ultur mi (PDA, PDA-V8 n/or Czpk Dox ultur mi) ontining th tst hitosn ompouns. Spors wr inut with hitosns prior to ulturing thm on hitosn-r mi or y irt grmintion o spors in hitosn-ontining quous solutions. Evlution o hitosn-inu plnt rsistn Th tivtion o nsiv rsponss ws stui in suspnsion ulturs o Ariopsis thlin lls, n in lvs n roots o too n ri plnts. PAL inution n th inrs o H 2 proution in rspons to hitosn oligoshris wr trmin. Aoring to h xprimnt, β 1-3 glyns, hitins, PAL n POD nzymti tivitis wr ssss in lvs n roots o too n ri plnts. Aitionlly, th inution o rsistn y pplying hitosns o irnt AD n MW ws trmin in too slings ginst P. niotin t iossy sl. It ws lso tst in ri, y immrsing th ss into solution ontining hitosn polymrs n oligomrs prior to sowing, n urthr inoulting th lvs o th rsulting slings with Pyriulri gris. Fil-sl pplition o hitosns Fil xprimnts wr rri out in too (vrity Hn 92) n ri (vrity J-104) ultivrs unr proutiv onitions. Too plnts wr ppli with ompouns, y olir sprsion o hitosn po-lymr ollowing plnt trnsplnttion, n ri ultivrs wr stlish y immrsing th ss into solution ontining hitosn polymr n hitosn hyrolyst prior to sowing. Th nturl intion y mjor pthogns or ths two rops ws vlut s init on th instrutivs or intion sls n nlyss. Rsults n isussion A mthoology similr to tht sri or otining hitosn rom shrimp hitin ws us to otin 6. Shrthhnr RG, Nirnjn Rj S, Shtty NP, Amruthsh KN, Shtty HS. A Chitosn ormultion ElxTM inus owny milw iss rsistn n growth promotion in prl millt. Crop Prot 2004;23: Prshnth KVH, Thrnthn RN. Chitin/ hitosn: moiitions n thir unlimit pplition potntil-n ovrviw. Trns Foo Si Thnol 2007;18: Xu J, Zho X, Hn X, Du Y. Antiungl tivity o oligohitosn ginst Phytophthor psii n othr plnt pthogni ungi in vitro. Pst Biohm Physiol 2007;87: Vnr P, Vrum KM, Domr A, El Guri NE, Morshhr BM. Comprison o th ility o prtilly N-tylt hitosns n hitooligoshris to liit rsistn rtions in wht lvs. Plnt Physiol 1998;118: Jon Y-J, Shhii F, Kim SK. Prprtion o hitin n hitosn oligomrs n thir pplitions in physiologil untionl oos. Foo Rv Int 2000;16: Kuss H, Jlik W, Domr A, Sigrist J. Prtil tyltion o hitosn n onitioning prio r ssntil or liittion o H 2 in sur-r tissus rom vrious plnts. In: Domr A, Rorts GAF, Vårum KM (s), Avns in Chitin Sin II, Lyon, Frn,: J Anré Pulishr, 1997, p Trotl-Aziz P, Courht M, Vrnt G, Aziz A. Chitosn stimults ns rtions in grpvin lvs n inhiits vlopmnt o Botrytis inr. Eur J Plnt Pthol 2006;114: Zho XM, Sh XP, Du YG, Ling XM. Inution o ntivirl rsistn n stimulry t y oligohitosn in too. Pst Biohm Physiol 2007;87: Biotnologí Apli 2010; Vol.27, No.4

3 Aljnro Flón Roríguz t l. Rport hitosn polymrs t nk sl. Th thniqu ws moii oring to prvious stuy o th inlun o hitin/lkli rtio, tmprtur n rtion tim on th proprtis o th hitosn otin. Rsults show tht it is possil to gnrt high molr mss hitosns unr homognous prprtion onitions (low tmprtur n n lmost quivlnt lkli/hitin rtio), with minor moiition o its sonry strutur n with yils twn 74 n 80% o th sour hitin. All ths r onomilly rlvnt or n iint us in griultur. On th othr hn, oth ii n nzymti hyrolyss wr stui to gnrt low MW hitosn ompouns. Currntly, th nzymti hyrolysis is prrr ovr th ii on, u to its squn spiiity or lysis n th rsptiv ormtion o prouts [7]. Nvrthlss, th osts o th nzyms us must low nough to mk it orl or griulturl pplition n to otin prouts with th propr iologil tivity. Th irst stuis using this strtgy show tht it is possil to gnrt hitosn hyrolyzts hving prottiv tivity in plnts, y using low ost, ommril nzym mixs, to improv th iologil tivity o th otin hitosn rivtivs. In our stuy, iv nzymti omplxs wr ssy; llowing us to slt th most qut n it ws possil to optimiz th hyrolysis onitions (Figur 1). Hyrolyzts n thir mixs wr otin with high ontnt o iotiv oligoshris n ontining th slt omplx (Ptinx Ultr SPL) [14]. Th iologil tivity o hitosn oligoshris pns on its strutur. Othrwis, prvious rports Tl 1. Physil-hmil proprtis o th hitosn polymrs n oligomrs us in iologil ssys ginst Phytophthor niotin, n th inution o too slings rsistn ginst th sm pthogn Typ o Chitosn Polymr Polymr Oligomrs Arvition Prntg o tyltion gr 14. Crr JC, Vn Cutsm P. Prprtion o hitooligo-shris with gr o polymriztion highr thn 6 y i or nzymti grtion o hitosn. Biohm Eng J 2005;25: Molr mss (g/mol -1 ) Polymriztion gr Q x Q x OLG Dtrmin y inrr sptrosopy Dtrmin y MALDI-TOF. Mn molr mss s trmin y visosimtry. Avrg gr o polymriztion, lult rom th mn molr mss trmin y visosimtry n th monomr molr mss. A Enzym tivity (U/mL) i not rss th inlun o AD on th tivity o quito-oligoshris whil in othrs th AD ws not trmin or miguous mthos wr us to hrtriz it. In this work, hitosn ws polymriz y ii or nzymti hyrolysis, y using th Ptinx Ultr SPL omplx. Th rsulting oligoshris wr sltivly pripitt in mthnol solutions n rigorously hrtriz y mss sptromtry (MALDI-TOF). Th irns in th polymriztion gr (PD) n AD wr wll stlish (Tl 1). Th ii hyrolysis prou rgmnts with PD up to 16 gluosmin rsius, mostly monotylt. Mor signiintly, th nzymti hyrolysis rnr shortr rgmnts with high rt o ully tylt hitooligomrs, showing highr oligo siz/yil rtio. Bs on ths vins, w hos th nzymti mtho to prpr oligohitosns [14]. Among th iologil ssys, th t o irnt hitosns on growth n sxul rproution o tog C Enzym tivity (%) ph B Enzym tivity (U/mL) Tim (min) g Tmprtur ( 0 C) Figur 1. Optimiztion o nzymti hyrolysis with th nzym omplx Ptinx Ultr SPL. Inlun o ph (A), tmprtur (B), n thrml stility ovr tim (C) o th omplx s hitosns tivity on th Q-63 polymr s sustrt C 40 0 C 45 0 C 50 0 C 55 0 C 307 Biotnologí Apli 2010; Vol.27, No.4

Aljnro Flón Roríguz t l. Rport o n ri pthogns ws vlut in vitro. Signiintly novl rsults wr otin rgring th t o hitosns t irnt liyl stgs o th Srolium n Bipolris gnr.

4 Aljnro Flón Roríguz t l. Rport o n ri pthogns ws vlut in vitro. Signiintly novl rsults wr otin rgring th t o hitosns t irnt liyl stgs o th Srolium n Bipolris gnr. Similrly, it ws vin th inlun o hitosn MW n AD on th irnt stgs o th P. niotin too pthogn, hiving th highst inhiition y rsing MW own to oligomrs n t th lowst AD. (Figur 2, Tl 2). This is highly signiint rsult, u to its prtil rlvn whn prpring hitosn ompouns with inhiitory tivity. It ws lso vlut th inlun o hitosn polymr (Q-88) on othr pthogns growth (Phytium phnirmtum, Slrotium rolsii n Rizotoni solni) whih wr isolt rom too n othr soln, with th irst two pthogns s th most snsitiv to tht polymr, n stuying Q-88 or th vry irst tim in os-rspons xprimnts. Bs on ths rsults, n lso onsiring th nturl origin n iogrl ntur o hitosn, ths polymrs n thir rivtivs wr rommn to us or ontrolling soil phytopthogns in too ss n to ru th spott grin iss in ri. It ws lso monstrt tht it is possil to rtylt th high PD polymrs without ting thir PD, y working with two sts o hitooligoshris o in PD. Thir pity to inu rsistn ws vlut in suspnsions o Ariopsis thlin lls, y msuring th inution o two wll known nsiv mrkrs: PAL tivity n proution o hyrogn proxi (H 2 ). In this s, th ompltly tylt hitooligoshris inu th tivtion o oth PAL tivity n H 2 proution, n lso ll th, whih vri with thir PD n onntrtion. Th ility o th oligoshris to inrs H 2 proution n ll th ws progrssivly inhiit y rtyltion, ut th PAL tivity ws unt. This vin th rol o PD or gnrting nsiv rsponss on vgtl lls. Exprimnts run in too plnts show, or th vry irst tim, th inution o systmi rsponss ginst P. niotin y olir sprsion, sustrt pplition n prvious trtmnt o ss with th hitosn ompouns. All ths monstrt th potntility o ths ompouns to prott too rom its min iss t s lvl. Th inution o rsistn, ithr y tivting nsiv rsponss or y ruing th intion o P. niotin ws inlun y th onntrtion, MW n AD o th ppli ompouns, n lso y th ministrtion prours [15]. This stuy vin th nits o protting too t il sl unr smi-ontroll onitions, n lso th prtil rlvn o prpring hitosns with th qut physil-hmil proprtis, to nhn th prottiv t. In ri, th trtmnt o ss o th ommril vrity J-104 snsitiv to Piriulriosis (Pyriulri gris) with hitosn polymrs or hyrolizts prior to sowing, tivt nsiv rsponss in plnts ithr or not inoult with th pthogn, protting th plnts ginst intion. Ths ssys wr rri out unr smi-ontroll onitions n monstrt tht it is possil to prott this rop rom this signiint iss t il sl with oth typs o ompouns. Th most promising mong ll th hitosn-s ompouns stui in this work t il sl n unr ontroll or smi-ontroll onitions wr ssy t opn il sl in oth rops. In ri, oth th polymr n th hyrolizt wr stui, unr onitions voring th ourrn o Piriulriosis, with prottion ing vin only or th hitosn hyrolizt t th onntrtion ssy. Tl 2. Ets o hitosns n oligohitosns on in vitro viility o P. niotin zoospors (2.1 x 10 4 spors/ml) Chitosn Compoun onntrtion (g/l) ompouns Q Q OLG Th positiv sign inits myli growth in th plt or th svn ys nxt to soking zoospors with th trtmnt solutions (soking or 6 hrs). 15. Flón AB. Chitosns n l inhiiión in vitro Phytophthor niotin Br Hn y n l inuión rsistni n plnts to ontr st ptógno. Dotorl Thsis in Biologil Sins. Fulty o Biology, Univrsity o Hvn, July A Inhiition o ril growth (%) Q-63 Q-88 OLG Chitosn onntrtion (g/l) Figur 2. Ets o hitosns (Q-63, Q-88) n oligohitosns (OLG) on in vitro growth o P. niotin myli (A) in PDA-V8 mium, n o th Q-88 polymr on zoospor viility (1.9 x 10 4 spors/ml) o P. niotin t irnt soking tims (2, 4 n 6 hrs) with th hitosn polymr (B). Th rsult o th itoril ANOVA in th nti-mylil ssy, with 8 rplits pr trtmnt, init th intrtion mong th tors t p 0.05 (stnr rror = 0.06). Th prntgs o inhiition orrspon to th originl t. Dirnt lttrs rprsnt signiint irns mong mns o trtmnts, s trmin y ompring thm y th Tuky s tst (p 0.05). B 2 hrs 4 hrs 6 hrs Control 0.5 Chitosn onntrtion Biotnologí Apli 2010; Vol.27, No.4

5 Aljnro Flón Roríguz t l. Rport In too, tking togthr th rsults o rsistn inution n ntimiroil tivity, it ws i to vlut hitosn polymr (Q-88) y olir sprsion to plnts (vrity Hn 92), svrl ys tr trnsplnttion n without ltring th prottion shul us y th lol privt prour. In this sns, plnts riv th hitosn (thr oss) n lso th shul hmil trtmnt, ing inlly vlut or nturl intion y viruss, ungi n oomyts. A signiint prottion ws otin ginst ll th vlut pthogns s ompr to th ontrol, showing os-pnnt rspons or th ppli hitosn n lso positiv t o th ompoun on proution yils. Du to its ul iologil tion, hitosn-mit prottion o plnts n mit oth y irtly ting pthogn growth n y tivting th systmilly inu rsistn (SIR) in plnts. In ri, whr th ss wr trt or sowing, th prottion oun ws th son in mgnitu. But in too, whr th ompoun ws issmint y sprsion, oth ts n tking pl, th ntimiroil tion (spilly ginst ir-orn pthogns whih n gt into ontt th hitosn hr to th lv) n th SIR. Th lttr vin y th ru intion with root n stm pthogns tr sprsion. Both mhnisms o tion r vitl to implmnt prottiv strtgy in rops. Conlusions In this work, mthoologis wr pt to gnrt irnt hitosn ompouns rom lostr hitin. Th rsulting n promising polymrs, n thir mixs n rivtivs, wr vlut or iologil tivity n tivtion o rsistn n prottion rom pthogns in too n ri plnts. It ws monstrt, or th vry irst tim, th inlun o rtin physil-hmil proprtis o hitosn rivtivs on inhiiting pthogns growth n vlopmnt n lso in th iology o prottion o ully grown plnts ginst thm. Our rsults vin th struturtivity rltionship mong th rivtivs prpr n hrtriz n lso th iologil untions vlut. Th irst rsults on prottion o ri n too ultivrs t il sl r lso shown, vining thir rlvn to sign n prpr nw nturl, non-toxi n ntionlly prou io-pstiis to us in griultur. Th inlusion o hitosn s prt o th intgrl mngmnt o pthogns woul llow ruing or sustituting th pplition o hmils in som onomilly rlvnt rops, with th susqunt prottion o th nvironmnt. Sintii rlvn o th stuy This work provis vins on th potntilitis o hitosn polymrs, prtilly hyrolyz hitosn n oligohitosns vlop in Cu, to prott too n ri ultivrs rom rlvnt isss whih limit proution yils. This supports thir potntil introution or th ologil ontrol o pthogns in oth rops. By ths mns, signiint mount o lostr xosklton is us s sour or hitin n hitosn proution, two ompouns o high ggrgt vlu, inst o isring it into th nvironmnt with th ssoit ontmintion. Aknowlgmnts Th uthors wnt to thnk th ontriution n support o th ollowing ollortors: Th thnil tm t th INCA involv in this work; Dr. Pirr Vn Cutsm (Nmur Univrsity, Blgium), Dr. Silvi Butist-Bños (IPNY, Morlos, Mxio); Dr. Migul A Mrtínz-Téllz (CIAD, Sonor, Mxio); Dr. Frnno Gurii (UNAH); Dr. Euro Ortg (Univrsity o Hvn, Cu); Dr. Onin Lón (CENSA, Cu); Dr. Vróni Tolo (IIT, Cu) n Dr. Mrí C. Nápols (INCA, Cu). Th uthors r lso grtul to MS. Rgl M Cárns n MS. Elizth Cristo; n to th CITMA n MES ministris or prtilly uning th rsrh inlu in this work. 309 Biotnologí Apli 2010; Vol.27, No.4

EE1000 Project 4 Digital Volt Meter

EE1000 Project 4 Digital Volt Meter Ovrviw EE1000 Projt 4 Diitl Volt Mtr In this projt, w mk vi tht n msur volts in th rn o 0 to 4 Volts with on iit o ury. Th input is n nlo volt n th output is sinl 7-smnt iit tht tlls us wht tht input s