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Transcription
un$df, otlh. ptmhb
ROLE OF VIRULENCE FACTORS IN BREEDI'{G FESISTANCE TO /4 SCOCHWA.RABtEt A THESIS subrdn.d to UTTIIVERSITY OF THE h con?|.t. t tfl|r|cd PU JAB ot rh. ,.qttremlnr fo, th. d.sr.. o, DOCTOR OF PHILOSOPHY al .latonal C.nrr. otZAKIA LATIF Exco enco In Mot cut.r Biot6nu un$df, otlh. ptmhb t2000, *"d-"' DR. S. RIAZUDDIN c-u"Pli!*yrbl g1Tffi umr.rdry.of dt. tunhD l,*r* r"n* .. "tj8f I OEDICATE THIS HUMBLE TO MY SWEET MOIHEN tlAlE} AN l|EN WHO II{SPINED C TO HIGHEE IDEALJ FON L|FE AC|(NOWTEOGEMEI{' Ihank to !h6 sraciouE, rho grcrtesi Almighty Afl6h who btesEed m€ with tho couroge 3nd nade hy 6tforts froittut tor th. cohpt€rion o, rhi. pi.ce ot It i6 6 rar. privitago 6nd .ou,.e ot ptesru,e i. .&re$ing my profoond aod cordi.l grarirode ro my resp.ctod,t6.,n.d ano rev€rend Bupotui3o., Dr. S. Firzuddtn, Diroctot, N6tionat Contro or Ercellance io Motoclt.r Btotogy, Uhiv6,sity ol lhe Punj6b, L.hor6, tor th6 invsruabrg irutluciions, ebl6 guid€nce .nd t6€n inr.r$i tdkah by him in thl! asstgnmonr. I do not tind words ro 6xpr6ss my sincer$t 6nd tinittoss thdnkB and grrtitud. to Dr- Richad, N, St.ng€ tor gtving m6 th6 opparrunity to work tn his guidencg.t Univorsity Co .g€ London, U. K. I would like to rh.nk ths tacutry mehbers of CAMB ,o, th€h h6lp and sssist.nc6 ih tho conduct ot thi! wo.k | 6tso lhEnk M,. Muhammad trfdn tor torm.ring rhis th6sis. Spociat Eppr6ciations .r€ du6 to my p.renrs, ororhorB, lister. and husbdnd tor th6ir cooper.tive and accoftodattue bohd|our during rhe study, lher6by holped m6 to accohptish rhis study. coitTE Is PAOE NO. SUMMARY CHAPIES I NTNODUCTION ol CHAPTEF II L|TEnATUREFEV|EW._..........,_......_._.._................,.,.,.._.......08 CHAPTER III MATEn|ALS AirO MTTHODS .........................._.................... 21 Chiclpoa s€€d6 A scoc hyta /. biei isol6r6s... lsol'rion ol Ascochfta nbiei ttom dis€a36d pbnts........... ,. . Prap.rdtion ol aqueouB extract ot chickpea s..d............., .. Fungal growlh in liquid hedium ar d'll€r€nl conditions....,. ... Growth o1,4. .rr,Gl .nd bioactivity ol culrure liltr!t6.......,, .... 21 23 27 27 27 Cell BioaisEy...........,...... 28 Soed sto'ilization and sowtng...... . ........... 2a lsolbtlon ol chlctpea c.ll........... ,. .........., .. ... 2A Flou'€3cein discelalo 29 . . ' . Bio.ssays.........,.... Eoot growrh inhibition bioassay,.,................,,,.. Cdllus g'owth Inhibition bloss3ay........ .. ., ,, .. .... 30 30 3o D.r.ch6d l..v6r b'o!...y 31 Earr.Etion ol phytoroxlc dcndry bysolidphaa6€xtraciion................,,,,,........,..,..,.,,,.,..31 Pr€p6ration ol minicolumn _..._._.._..........._..........,...,..... 3t Cell bioa6s8y................._.._.._........................... 32 Thin l.yer ch.omdography.._....,...,...............,,,,,. 32 ' Specrophorom6i.icanstysis_.,..,...........,.,,_..._....,32 ' HPtCao!|ysi3,.,,..........,..,..,.,,,.....__,,.,,.,...._..,.,.32 Extraction ol phytotoxic activity by column chrom ogr.phy..._........_....,,........_..,..,.........,. 34 Prep6.6tionottl.shcotumn._....._.......,,..._.._..._,,.,35 . Flash chromatography ol ih€.amptes...._............. 35 Testing toxic f..ction! tor pu,ity.._....,............._.,. Thin l6ysr chrom.rography,.,._.............,,.._. HPLC an.lysi3....._.._...,,.,,......_.._.._.,..,._.... Ouanthative an.lFb ot phyrobxin in tiv6 cultivaool chiclp6. ....,..,,,,.,......._....,..._.._.._......_.. Toxin p.oduction by nins bot6t6s 36 30 36 36 3? 37 HPLC andlWis lsolation, poritication End tdBnttttcr(oh oI another unknown toxic compound trom tungus rree culruro lilhrt€ or /. .r6,bl iso|droCAMBt0....,.,,,....,..,,,,_...,...,..,.,,.,.........,,.......37 kinetacs ot phyrotoxln. producrton durtng tr vii.o growrh ot vartou. isot.t6s ot Parhogeni.rty ol tung6t isotlt6s. .. ..... ., . ...... .. Purilcarion ot the phytotoxin. Iroh 39 infecredchackp..ptant.,....,..,.,.,......._.,...__...._......_.....40 Genedc djversaty ot nin6 isolrt$ of A. nbiei varyi^g in vk!16nc6,.._.......,.,..,......_..,.,,..._....,tO DNA isolrtion..._....,_..........,..,.,......._...,...._.._..,., 40 - Rest ictio. ot ONA, .t.cr,opho,..is and blo(ing.-....,........,.,......_.......,._...._.....,...._..,t1 Lab€lling ot oligonlctootid€.....,..,.,...........,._....... 4t - Hybridi2arlon .od tmmunotogic.l dotecrion.....,.,,.,.-.,.,..,......,.,..,..,...,....,.,.._.._...., - Stippingm€lnbr.n€for.epo(ng,,......,.,.,._.._.....,4242 RESUTTS Opt'dum condifions for th6 gtowrh ol A. rcbhi ..-.. ...... 45 Producrio. ol phytoroxic activity.,,.,,........,........._........ Kineric6 ol secrotion ot phvroroxic .crtvtty..., ....... . .. Resolulion ol phyrorotic.cttutr'6.........,,.,........., . 57 Thin l.ye. chrom.tographtc lnatysi€.. ........ .... 57 Specfi ophotom6tric 6n.ty!i. Column chromatography .., Production ol solanapyron.s in tiv. cultlv.nofchictp.!.,,.....,.,,.,,.,.,,..,.....,.,,.,,.,,..,,.,,..,,.64 Growth, pH, toxlc lctlvily ond sotanapyron.. conc.nl.don of tungu! t 6. cuttlr......._..............._...... 64 Growth ol niE lsl.t . of ,. db,ir't l cullur. and toxicliy ot c!|tur. titrrt 3.....,.,,..,,.._.....,..,. Bz Solan6pyron€ cont.ot oI cuhur. liltr6r.. ol A. rsbl.l,....--.-.,.,..,,.,..._.......,.,,,,,,.,..........,. 76 Th. r.lltion ot.ol.n!pwon. cont nt of culruro lilrr.r.. b th6ir roxictty......,,..,_.._.._........,..,.. 77 Tt'! idontilicrtion of cytoch.l..in O in c'ilt r! tillrst€s ol CAMB lO........................,.............. 77 Tho lotlclty ol cytoch!16lin D ro chtctF..,.,,..,.,,_.........81 Ac.umul.lion ot myc.li.l m.!. ad tu.gol to,{ctty.,..,,_..8t Ch.ng6. in th. conr. of phytoroxtc compounds dudng lungal gro|!/lh................................. 92 Parhogenlclty of dllf.f.nt 160l.r.. ol A. tubttt..,..,..,,..,_.,90 G6n.llc dlvorsiry .mong diltor.nr isol.l.3o1,480,bi.,,.,,.......,,..,....,,.,_...........,..,..,..,...,.99 CHAPTEi V O|acussloil ......-......................................................_....... t07 cllaPlCn vl AEFERENCES 120 I-EGANDS Of fIOURES PAGE ITO. Lesion lorm.rion on stems.nd l66vss of chickpea. Fi9. 2. SpotasolAscochytarcbidi. 3. Growihor,4..rb,ir1onp|aie..............................-..-...-...,,.,...,25 .....,....,..,..,.,...,..,24 Intocted chickpe6 seeds lor multiple sporu|arion,,..,......,.,,.,.....20 Fi9. Fi9 .................04 5. Fig. 6. EIIocr of ditterent carbon sourcss on the induction ol synlhesis ol UV absoibing compounds doting growih cells/proroplasls biolssay tor th6 presenco ol phyiotoxiccompoonds.,,.,..,,..,.,..............,,.,,.,,.,,.,,,.,,.,,.,...,.50 Etlecl ol tunous lr6s cultute llltrote on callus r.rh'ii6n t.6; nod6s 6nd ihl€rf,6des. ...................-..-...,...,...,53 Fig. L Ertecr ol lungus lre6 cullure lilk.l6 on detdch€d ch'clp6a leavos .s evidenl by typical diso.s6 svmDtoms kioht t€atment with cultur6 tilt.6l6 0f ,a. .;rte,. rnJl€lt culture m.dium ss conr'ol'... .. . 521 EII€ci of culture liltrdl€ ol /4. rrbtei lenglh 6lter 72 hours incubation in d€p€ndent mann€r . .. .. 55 Toxin Ectiviry {a {) pH changos (o 4 5nd dry w6ight t,'--.o) ol lungal my66li'rm during incubslion on Cz.p6k Oox m6dium supplemont€d wilh chickpea s66d €xtact..................-.,,..,................-,..,,.,, 56 UV 3o€ct.E ol bol.ted phyrotoxlc compound3 0, & 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Fig. 12. Chromoscan ol sol.napvron63 A oo Fig. 13. Chenlcdl structu.6 ol solanapyrones A and C..............., . ., 1 r 1 lil 6nd C lii). Conftmaiion ol tho plrity ol lho soltnspyrono a by ih6 sup€rimposlng ihe sp.ctrum o4 tho chromat4srani. .....................................-..-........ ., .. 61 .62 FiO. 15. Conli.mrrion ot lh€ purity ot th6 sol6n6pyron6 C by the superimposing th6 specr'h ol lhe chromrtogt.m. ....,..,,.,..,,.,63 Fig. 16. Elroct ol the addirion ol dillersnl energy sourc63 on roxin ptodlctio.. ........ ..............................................,,--68 fig. 17. ol 12 day old culturgliltralos ol eight isolat.6 oI Arcoclryr, .rtidl as assayed The roxicjty by cell viability using lluor€scain diac6tat6 as a vilal slain. la) lGolrtes grown on Cz.p6t Dox nutrienrs suppremenled with exrr.cl or chhtp63, cv. cypr€5sa dnd tesred with cells trom cv. Cvor6.s6. Ibl lsolales grown on Cz.p€k Dox nutri.nts supplemenled with 6itr.cl ol chlcl$6., cv- cypr€ss5 and r€sr€d wirh cells l.om cv. 6153, {cl lsol.t€s grown on C2.p6t Dox nutrients sopplemented with oxtr.cr ol chickpas, cv_ 6153 and tesled wnh celis liom cv. Cypra.sa, ldl lsoldtes grown on Cz.pok Dox nutri€nls suppl€mented with exrrrcr of chickpoa, cv. 6153 and r€lted with corls i.om cv. 4153, oltl lro tor three replicat€s a SD. .. ............,,.,,.,,..,71 Flg. 18. Toxicily ot lungus lr€6 cultur6llltr6t6 ol Asco.hyta rcbiei i.olare CA,IB 1O asrinst isolated chickp€ac€ffs6v,6153...........................................,,,,..,,.,72 rrg, 19, lhe loxiciry ol 16d6y ord curtur€ trtr6t$ ol isorate CAMB 10 of Iscoc/'yta .abiei * 6ss6yed by cell viability using fluorescoin di6cot.le as a vil6r srain, (.1 Grown on czdpat Dox nut.ienrs supplem6nt€d wirh extract ol chickpea, cv. Cvoress. and tested wirh cells trom cvCypressa, {bl Grcwn on C2ap.k Dox nul aents suppl6m6nt€d wilh extracl of chickpoa, cv. cypr.ssa and rcsted wnh c€lls lrom cv, 6153 {cl Giown on czspek Doi nutaont3 suDpl6mnr6d with 6xrr.ct or chictoea, cv. 6153 and t4tod with cetts trom cv, 6153. Dat6 ar€ tor three replic.i6s!SD. .....-.........-....,.,,..73 Flg. 20. Comparison or lwo isol.tos ol A. nbieilot rha prodriction of solanapyron€ and toxicity......................_....._....74 rig. 2 r. Solanapy'one A rnd sol.n.pyron. a r C (onc€nr'arioo of l2 d.v old culturolilt.t.. ol nlno ilol.rot ol Ascochvtd nbiei lal SolanaFyion€ A concentr.tions lurvl) of isolales grcwn on Crap6k Dox nul €nts supplemehtod wilh extr6ct ol.hickp.., cv. cypr6.s5, lbl Solanapyron€ C conc€nir.tion luMl ol asolat€3 grown on c.6pek Dox nul.l€nls sopplemnt6d wirh ext,act or chickpea, cv. cyprc$a {cl Sol6n.py'one A concenlrations ot i6ol.rB groM on Cz.pel Oox nulraents supplo@nt€d with 6xk.ct ot chickp6a, cv. 6t53 {d) Solandpvron. C conc.olrations ot isolat.s grown oh czapek Dor nutri.nts luppl.monred wirh exr.act of chickD€d, cv_ 6153, OEta .rc lorrhre6r6plic6t€sasD..._.............,_........_........_........,.,,.,,.,76 Fig. 22. Nlclear m6gneric reson6nce (NMR) spectrum or lhknown compound. ...,,,. ..,,...,......,................._...........,..,,, 76 Fig.23. Distortionless enhanc€monl by Fig.24. P.oton/c.rbonspecttum..-. Fig.25. Protoh/protoncoupli.g,...,..,,.......-..............-......-.................43 Fig.26. UV spectrom oI cytoch.le.ln D. Fig,27, Conlirmatlon of rh6 puriry ol rhs cyiochalasih D by lho sup€impoding th6 spectrum ol tho chrom6togmm. -.......,.,,,....86 Fis.28. Chamical struclurc ol cyloch8lssin D.....-...........-...........-...-..- 87 -roxicny ot cytochdl$in O to chlctp.d cuttins. Epin6.ty of a chickp.. cuxlno a. holdiog buller conbining cytocholasi. D llooug/mll compa.6d wilh conliol in holding bufls !lon6. Note d6sicc6tion, consr.iction 6nd br€akdge of p.rioles..,..,,.,,.,88 Fig. 30. Chdng6s in lungal myceri6l d.y w€ight durinq /h u,lro q'owrfi ol nin6 isol5t63 ol 4. /rblel........ ... ................89 FiO.31. Toxicity oI lungus lr.e culiur€ tlltrsl6s dorihg 25 day srowrh of nine isolaros ol A. @biei.............................91 Fis- 32- Produciion of solanrpyron6 A d\ni^g in vito growrh ol v.rious isolato3 ol A*ochvtd 6biei. .......................93 Fiq 33. Producrion of solan.pyronE I & C dotinq 25 ddy growth ol nine isofar6. ol A. t6biei...-..................-..-...94 Fig.34, Production ol cytochalo8ln D duti^g in vn.o growlh of nine isolaios o1,4. 95 Fiq.35. olioonucfeotide lingerprintin g ol Ascochya rcbiei isoldr€3 l,hn d'g€.t.d genomlc ONA ot nine isol.tes ol A t.biei Figs. A, C, E, G, I md J prob€d with {GATA}4, {GACAI4, {cAA)5, ICA}8, lGlG)S snd lGGATl4 rgspectively; Taq.l dig66tsd, Figs. B, D, I tnd H ptobed wilh lGATAla, lGACAl4, {CAA)s .. . ..... .. . 10O.nd lcals respectivelv lc ropr6sonrs lor cAMBl..................... I02 FiO.36. ONA lingor prlnting .ndlysi8 ot th€ nine isolates ol /4. .rDlel puitied DNA wrs dis€ltod wilh fcoFr. El€ctrophorsed lloug/lane) and in blot hybridked to lh6 a) IGATA)4, b) {GACA14 6nd c) {GTGI5 non r.dlo6ctive labelled p|ob€ {dolocol6r woishl ma.k6rs ar€ 9lv€n in tilobas63 and c rspresents lor cAMB).................. 104 polori2ationlransfer{DEPTI......-...................,.....,,.,,.,,.,.,,..,,.80 .....-..................82 ,..,.,,..................-..,..,.....,..,..85 rariei TEGAIIDS OF IA8f,ES PAGE IiIO. TEbl6 1: Oill6r6nt isotat . ot /4scocryr, .sb&i coll.ctad from difl.r6nr loc.liti.. ..d coltivarsotchictp6...,,-..-..............-....................,....._.._..22 2: T€ble 3: T6ble Solvenrs combination perc6nr!96.,..,..,.,,..,.,,,,..,,......._....,,.,33 Growrh o1.4. rrr,bl in dilleront culture modia,,.,,.,,.,,................,,....... ..._..._....._...........,.,,45 4: Ellect ol temperature, lishr & 0H on th. srowth ot /s.o.ryr, /abi.i ...-..-.--.....-..................,.,.,,48 5: T.ble 6: Tronslormation ol pe.centages to prohits.....,.....,.........,..,.,,51 Tdble 7: pH, gros.th, toxic acrivity and solsnopyrones concontration of lungus free .ulture lilfiat.s....... ., ..... .................... ............ ......€6 T.bb a: Dry wgiqhr ol nin€ isolat€s ol Ascochyta tabiei gow^ o Ct69tlDox nulraents suppl€m6nred with se.d 6xtsc€ ol.hickp€a cultivars Cypr66sa and €153....,..,,.,,..,.............09 Tabl€ 9: 125,8 MH2 13C NMn ol chytochalasin D in CDC13..,..,,.,,,.,..........,,.,,.,,,,,..,,.............._......_....,,.,.79 Trble 10: Tsble Table Table l l Production ot solanapyrones in fiv6 cullivars ot chictpos-........ ....,........... -...............-...-..-...,.85 Nucl6ar overh.us.r slfsct INOEI €hnancementsofcyrochalsinD,.,..,......................_...........,84 Amounk ol 6ol.n.pyron. A, Bol.n.pvroo63 B & C r.d.vrochrlasin D exfisct6d from two chiclpoe cullivdrs inl6cred with vgrious koldt8 ol r. Ebt tdill6ring in vnubnce.....-.....-..-......-....,.,97 SUII ANY R.66arch work perorm6d during thb .tudy w.3 aim.d to .tuctd.rs rhe role or vnofance factor in breeding rcsista^ca to Ascochyt tthiei, During this .tudy nine i..leL. ol A. t.bi.], blisht ot chickp€!, w€r€ .cr..n6d ror b E causarav€ .O€nl ot th. pr.s.n.. ol phyrobxlc .crivity. which Opllmum condirions lik6 m.dium. pH, t.mp.r6rure 6tc- we.e iN€!0gat6d for rh6 gtowth ol A. rcbiei. Biotoxlclty ol tonous lree colturs lilria& ol r. .d0l6l was conlarmed by cells, nod6./lnt€rnod6s, d6rached lo6v.s and root growth inhibition bio63says. Funh€r invesrigations indicated rho pro.€nc6 ol phylotoxic compounds a$oclal6d with lungal pdthog.n€si6 in chlckpea blight, lt was obsetved th.r the synth€.b ol phylotoxlc fter.bolirolsl was indoced in ih€ presence aqu6ous extr6.t ol oI untnown ondogenous lactors presont in th. chickp6. 366ds. In ord6r ro d6r6cr rh6 phyloroxic compolndls' during growth ot /4. ..biei in Cz6p6k Dox m.diom suppl€montsd with chickpea 366d extsct, ths roxins w.re isolatsd from th6 cuhurb filrrar€ hy orOarlc .xlEctlon, solld ph6e exlraclion 6nd HPLC and id€nrlllsd as sol.naovron.s A and C. eslabli€h a prtt€rn ol production of rh4€ phytotoxinlsl in 12-day old fun0u6 Funh.r srudi6s w.r€ cari.d out io lr€6 culture liltr.l6s ot nin6 dillor6nt lung.l isol6r66. Thoy w€r6 grown ifl still culture oa Czapet Dox liquid modium supplem6nted with Equeous extrack ot rh6 chickp6d leeds oI one ol two cultivaG, 6 d€si 161531 or 6 kab(ll {Cypr€ssal, Toxiclly ol th6 cutlurs tiltrdtes from bolh n6dla wer6 .ssdy6d with rha cells bol.lad from rho 16d63 conc6ntr6tions ol both culriv.6 end ol th6 phyrotoxln., sol.napyron€ A and roldn.pyron€ C, in th6 llltratos w€f. determinod by high-podormanco llqoid chromrtography {HPLCl. For oioht of th. lungal i.ol.t.s, loxicily and solan.pvrono conc6nt6raon dccording ro w.ro cor€l.tod. th. vrlu6 $6 cuhiv6r ot. v.ri.s trom 0.383 ro O_a59 us6d In th6 m.dlum 6nd ....ys. Bur In rh6 cuttur. liltrato ot one lsol6te, thero waB no dotoctabt. tsv6t of sot5napyrcn$ but was 3howing l5 to 20 tim6s mor€ roticiry 6s compared to olhor isotal63. Th6 compou.d w63 lsolar.d End tolnd ro b€ .ytoch6t.san D by ma.s specrrohotry and NMF. sol.napyron. c 6nd cyrochatasin D was 6ko detect.d lrom th6 naroratty iht€cted chickf'€. ptanrs. To tind oul the 1016 ot t5otared phytoroxtc compounds in btighi ot ol phybroxic compound! in lniiici.ly int€cr€d chictp6r pl.nts with dlllsr6nt fungdl isolar€s oI a. .a6,id has b€€n chickp€a. rhe conr€nr d.t.rmined. Th6 6ov.rity ol dl6eas. w.s record.d osiog . 3cat6 ra.Oing trom 1 to 9 {6Gw.l, l9Al, havihs tiv. doftned crt.gori6. ot scv€rity.it6r 12 d6ys. Inl€ctod pl6nt mat€rials w€r€ atso procosa€d through mtntcotumn for lh6 ol ,. qu.nrilication of phytoroxic compounde by HPLC du,ing Th. srudior Indic.t€ th.t con.id...br. vrrt.bltry .xiBb whh isotat.. rrbr?i lor plthoggnlcity and virut€nco. Ih. tow.st g6ds ol dis€s.. sevorlly on 1-9 w6s 3 lnd th. highost 9. Thu6 no cuttiv.r showod complor€r resist.nc6. M.ximlh phytotoric compounds wer6 dorected on lhe t2th day ol jntociion, Toxih production vari€d in bolh cuttiva.s of chickp.. infect6d wlrh ditloront tsotato! ot ,. 6rlr/ varying In virutonc.. Cytoch.lasin D w.s r.covorod in high t6v.ts tolowod by $t.napyrones B + C and soldnapyrone A. th. tita.s ot solan.pyrone! A, B + C .nd cyioch6lalin D synth$iz€d by th. v.rioug fungal isolates upon plant int6ctioo 6nd lhe 36v€rity of th€ dis€as. a3 det6rmlned by th6 siz€ ol l.al .nd srom t6lton wB c6lcut6r€d as O.O8t, Howover. rh6 cotretation b6rw€€n O.r44 rnd 0.718 r*pecrlv6ty on dis.r36 susc.pnbh chtct(pea cv. Ot53 whil€ ir w.s 0.065. 0.25 a.d 0.122 r6lp.crtvety o. tuns@ rolt.tEnt chlckpe. cv, C44. The .esutls .€6m ro n.garo phytotoxlc cohpounds in lhe ons6t of fungat . crusativ€ rot. of isotlted btight in chickp6€. For rt thr dor.rmi.arion ot hruric! ot phytorox,n. producrion du.ino o groMh oi varioo. isorales of 4_ /r'el ,, vdrying in virutsnc6, th6 cuhuro liltrltas w€r€ proc$!6d lpro 24 d.ys throqh hrnrcorumn wath tho inl6,v.l ol24 hours ro r6sotvo rh! cont.nl3 o, vrrioob phyroroxE compound.. H.tf ot lts voluhe w6s used to. th6 qs.ntificrtjon o, phytoroxic comDound! bv HPLC bnd th6 orh.r h6[ w.B 6vapor.td to residue to chock rh. roricirv bv solanapyronos, A, s + c ,nd .ytoch.tr.in D production v.ried dmohglhr nin6 isotat6! ot,4. ,rr.iei, whue.s fung6, Srowrh as evidgncd bv lungll dry woight and spo.rrtation a! oblorv.d uoder oicroscope proc€€dod norha y. tosignilic.ntty tow tov.k ot phytotoxrc cornpounds cootd b. det.cr.d in lh. cuttu,6 fihr.t. o, isot.r.. CAMB 7 .nd s, In CAMB B, ontv sola.Epy'on6 In I r C w6re d6r6ctabr6 at 2.3 ug/ng ot rhe tungus. ofier isot.t6s production ot the varyi.g tov6b or phytoloric w8s obsorved during l, Ms..r€ compoond3 ,iaro orowth 6nd cytochrtrs,n D w'B obsery.d ontv h isolareE CAMA 5 and CAMB tO ft.se r€lults indic6!6 CAMB 7 to b, . Tox isotato at th6 pres6n! d€tlction tihtr3 wh6re.5 CAMB a produc.s ootv Boldn.pyrone I r C bd not sotdndpyron6 A Anatysis of gen€ric div€rstty by one ot $6 noasuldbto objoctivs of rrrlirl wore charrcre red by DNA ^. ,rD,Gt isot.t6s w.s .adiorativ€ lab.llod prob* compllmonrlry to simpt€ r6p€titiv6 s6qu6nco. n6m.ry,|GATA)4, {GACAI4, (GTGIS, {CAAI5, ICA)8, (GGArt4, G6nom€ ol /rr,br w6r€ srudied 6nd conrirm.d various isot.r.! or A. ,abi.i lpt€'rtoustr cnssorized sccording ro th6 dis.6se soventy 6nd ioxin productionl. Atl /4. requsnco motils w€r€ tound to b! Frasehr in th€ ,4. 6birt senome dlfi.r ln or9rnr26rion and .bundanc.. Tho ext.nr ot potyhorDhism obsetued and infomarivity obtEined ,re srrongty depond€nt on lhe prob6 as we[ a3 on rh. reslriclion enzymg us.d_ Irqt .nd tirfl arc found to bo hosr suit6bt. tor origonlcl€otide lingerpnnting ot /4. /rDiet gdnomo ds comp...d to 6coRt. amons six prob€si (GAIA)4 w.s rho moBt intorma(ve o..urcd t€s. tequontt .od .pp€ar6d to b. concenr.r.d 6r one or rwo predominant toct, In conr'ast. rh6 mo s tGACAt4, tGTGtS {CAA)S, iCA,8 ,nd {ccAD4 ting.rprihrs wer6 more comptdx and p6tt6rn cr6.t6d by TGACA)4 and {GlG,s probgs w6rc distincr bur monomorphic. Sev6o rsorarer, oul ot hin6. were 3imibr with one anothgl but dith.snr t om rhe r6sr of the isot.rss wh€n hybridi26d wath {CAl8 6nd lccAlt4. EHf,PfrEE ! NraoDucnoil Chickpea lcic6, a.ietinum rhe Leguminousa6 tahily. t l, !n indig.nous qrdm logumB, b.longs to lt b th€ $nd mosr impon.nr pulse crop in th€ th. M.ditorrn66n ..d A6i6n s6mi.rid .reas IFAO, 19941. Th. m.ior .re. ot prodocnon b In th. Indl.n .ubconraoont {B.nsl.d.sh, world rnd fiGt in Buma, Indi. and Pakistanl wh€r€ Em6ll, 6nsular, d6lk sooded chiclp€ds, known as desa typo6, rr€ predomin6nt. Paki6t6n as s6cond moFr important chickp6! ptoducing couniry lh lh6 world with a sh6.6 ol 9,96% in sG! and 7,37% in produclion {FAO, 1978}, Th6 iotsl cultivated area ol chaclge. ia lhe world l. 6bout 10.4 million hocl6ros and ,nnu.l production is about 6.8 million tones IFAO, 1978,. In P.kistan. th6 .v6rsgo yield per h6ct.re is 6stimar6d lo be only 5OO Kg lH.q, .! .t., lgal) as compared to Ks/h€ctdr€ in India {singh 6nd Auckland, 19751. In 1975 av66g€, Asia prcduced 5'0.OOO_OOO tg/ha, led by India, which producld 900 kglt.; MI, yi.lds 4-5.OOO.OOO Alraca ptoduc€d 250-3€4,000 MT, wllh 70O !o 1994, on th. rangins lrom 57c700 Ml, r.noins from 5Oo- yi.Hs..nging trom 600, 660 kg/ha. North and Cental Amsrica prcducod 180-260,000 Nll. whil€ av6.aging 1,600 t(g/h5. Europo ptoduc€d 5O,t 1A.OO0 MT, while .veraglng 75O ko/hd {Fdo, 1976, 1994} Ih€ .omDdriBon of amino acid cont€nt ol vadous di€lary proteins r6vool6d that except fot acids, chickpea is comp€rable to bool or ils quantilatlv€ impo anco is Chickpoa is valued lbh (FAO R6ports, 1970,. Atthouqh qu{l€ 3igniticanr nuiridnt3 in rh€ di.Gry requircmonk ot sulllr conlaining amino r gr.6t in supplementins tood majority ot the poputation. lor its nutilivg s.o& with high protein cont6nt, 25,3, 28.9% alt6l dehullins lHulso, 1991). li b on6 of rho maior sources ol diotary protein for tho poor .nd av.r.!6 incom. f.frilies, not onry in P.kl6r.n bot also in neighborlng countri6!. As human lood lt b .!tsn as lr*h gr..n v6ger.bles, p.rch.d, nied, rostod, rnd boilodi .3 snacks tood, tlou can b. used as 6oup sweer aad condimonts; !6€ds 8rc ground and th6 , dhal, ond to make breEd , prepared with p€pp6r, salt 6nd l€mon it is sery€d as 6 3ld6 dlsh (Sax6n., l99O). |t3 dry 3talks.nd husks conrbhino smdrl brok€o ploces ol grain du.i.g milling, ar. t6d !o animals, Chiclper is known by orh6r n6me3 such as B6ng.l gram llndirn,, G,dm, Egyptian pea, Ch$lnut bean (.ll Enslishl, Pob chichd lFGnchl, Chrn. {Hinda), Homm6s, Ham6z {A6bic,, Garb€nzo or Garavsnco lspanish), G.'banzo (talin Amaric.|, Nohud, Chickp66 t.bl.bi {tud<oyl, Shimbta {Elhiopla). is grcwn all ovu P.kistan bul its cultivarion is conc€nrrargd in s6l6ct.d r6gions, Ecologically, Pdkistan cEn b6 divid6d into ihree chickpo. It hrs high 6inf.ll, .nd 3urf6ce idgalion tacilitio6 allo m.inly g.own und€r r.lnf.d condhlom. c.nttd R.slon: ll Produciion h63 highly lortile soll6 but th6 6lim.t6 b rcnly !id6d by sorfsc. tno.lion b mo3tly s€mi'arld. bul chickp66 ia tl6o grown undlr r.int6d condiliom. lhis region can b6 divided into two disrinct ltact3, dju€ring in rainlall, soll 16 lllly, and s6ophy6ical fe.tur!. a3 follows: (6) w6st6,n lincludes Sargodhr, M.inwali, p.ns or attock .nd Jh€luml, 6'84 mm/month sulffi€r rcinfsll, irsct, lbl 313 it i. s low rslnfsll lod Sialkorl, 26 374 |m/mnth mm/month in winter; E6.r€rn (includ.s RlwalDlndl 6!mm6r rainbll, 23-65 mm/monrh in wlni6r, High Gintlll lract. South.h R6gon: Frinl.ll is scanty .nd agdculture is lotally d6p6nd nriglrod condition- How.vor, chiclpo. is grown on r.6idu.l moistot6 on .ft.r iic€ wilhout .ny 3urtac. inig.tion- Th€ lmpodant dls66s€s ot chickFr arc witr lFusaium orysporum lPddmickf Snyd. & Han!.), dry rcot rcl lRhizoctni. brrrrbol, lMlobll Borl.tt MtcrcphomioE phaseotina tMaubtl A3hby,, bt.ct root ror tFus.rtuD so/a,t lM.rrl App.r .nd Wr.l, !r.y motd tBotlytis ch.rca ?68. .x F..1. Phytophlhora rooi ror fPryrophthon magasp'tma Dtachs.l, pythium ror see.t rcr lPythrun utidn lrowl, srunt lp., t6at lnd ro vtus), lnd Fi torh, Almost .ll th6 chickp66 grosn io rho coohtry ar6 susceptibt6 to . p.!i. .nd dE66$s, especially rh6 pod borcr, wtti rnd btight. Among.t rh6 p.si., rhe hort damaqing oneB lro pod bot.t tHelothis snige6l,.ur woth (Asrotis sp.) and seed w.6vll6 tqtuchus sp.t. Tho climat€ ol Pakistan is v6ry suftabte to. tho growrh of chtckp.r. Howevor, lh€ rverage yi6ld of chickpoa jB very tow In p.list.h. Mdny trclo.s .16 r.sponsiblo tor rh. bw yt€td!, whtch Inctude orc6..tv. veg€lalive growth and poor h.Nest indd lshakoor and Ahsrnot Haq, t98ol , but th6 most import8nl limiring facror, in 6xptoiiing th. yiatd por.nti.t ot pr€5ent dry cllrivrrs in P6listsn, is th€ occurr€nce ot b ght dtseas. caus6d by tho tungus A. .abiei / sco.ryr, w$ ti6t ru6ri.,, a phyropsthog6nac deur.tomycere p..!!1t6, nam.d Zyrr,b 6O,bi by p.sse ni tn 1867 on the b.!is of onic6lluldr.nd hyatine pycnidio8porc6 A. /abiei anack. rll 3.riat p.r$ ot rh€ pt.nt. Circut.r tesions wtth conc€nrric ngs or pycoidi...e prcduc.d on t66ves..d po& ss a r€sltt ot 4. rab,id int€ction wheraa., th€ tesionr rrs stongale on psliotes .nd st6ms {Fls.1l. Ih€ nedoric t€sions prodlcod oo aeriat p.rrs or chickp6. Fig, 1. stems and leaves of chickpea 3ugs63i thtt t loxln nlshl bc i.volv.d whlch cd& d8ma n|.n!br.n. dy.lunclion .nd con..qu6nc. lo.. ol tliCor. Anoth.r .yrptom wfilch cln d6o br l.r..pr.t!d in $b lo,' ot rurgor In c!ll! suirounding tha valculrr tilru€r w€.k6n rh! !t.m. lh. inloct6d drntt lhow .pin..ly, rollow.d by drylng .nd bl.ct.nlng ot ..n.1 plrt! l..dln! to dmt llght b lodgi.g riE6 tho h.! b..n 6 v.ry ..rioB lnd dlv.slltlng dl.6!e to 6t !1.,t9941,.pp..r. dmst r.gul!.ly in.t.mtns Chickpo. blighl lh6 rcgion {K6k6r, 6pid6mlca in both th. r.lny and upto lOO% lols ol lh6 crop 19921. /4. roD&t lrig.t.d ,ru.! or Punirb rnd crn r€luh ls.x.nr lrd i! p'rticulnv dwlst'tlng In Singh. 1984, Sinsh ,nd S.tcnr, In arc6! wh.r. lD.iog t. coot .nd lhu. th. tonglJs c.n Inlllct 3.nous d.mlgo l. coot .nd w.r *.!th.r, p.dculE ly, Et on3.r ot tlow6ri.9 .nd truiltng. A.cochyra blight whtch huoid. c6u..d sorious yi6ld lo!s!, during .rdy .ighd.s topto 7O%l hrv. b..h . r$s.r probroh du ng th. l!6t 3-4 y..r! b.c!us. ot dry w..th.r. Thc opllmqm tomp6r.t!,. lo. conldlll g..mln.tlon .nd growth l6io.! occur on l6av6! wlthh 7 days lz6cho. .t Th6 lunsus rt l! 2G26oC .nd 13-t6oc rnd 5 dryr rt l&25oc !1., 1903,. Int.cr.d .66d t. !n tmporbnr lourc. ot inocu|lm. i! crd.d both on .nd wiihtn th6 !..d, .nd lho int.cd pod! crh show 366d inr.rbtion of 5G58% s6od tomarion, .t2., g€minrtbn 3.6dllns srowth !r. 6dv6r4h .ft6cr.d lHltton,Motrt, t97q ldhra.t rd C., 1935; M.d6n 6t.1., 1975; Saiirr, 1933t. h $. r.gton 3..mr to b. in ! vlciolr cycl6, wh.n th. .6!eo t. dry, rh.6 i! l{. alc@hyt bleht but rh. crcp yi.ld. !16 poor. Whrn rh. r.rlon ts w.r, rh.ro t. pot nti.t tor good yl6ld. bur rh. qop l! d..troy.d by blghr. Tht. di...tou! .ttu.don t. A.cochyr. bllqht lnd chictpo! productio. th. 3c.rcity or corl9loro .b&nc. ol g6mtic ,..i.t.o6 ln ih! .v.il.bl. comm.rcad cultiv.6. Ihu! in o.d.r to incro..! and cl.billz! oo accounl ol chickp6a production in Patlrtsn l!v.r!lm!.rur.r h.v. boo. .motovod. Th. brcoding of plant3 wilh dur.bl6 rusi.r5nco from wild rcl6tivs3 h.vo boon tho pl..t d13..... in dovoloDino tn|3 t.dition.l mthod do.. nol orovo to b. most .ommon.si rppro.ch t6r rh. conttol ol countri*. unlortunar.ly, durrbl.. rh€r6 havo b... a complimonr.ry .ppro.ch lo d6ttn. th. m.ch.nbh3 oi rBist.nc6 ot ih! Dl.nt .nd th. m.chrnisms ot virul..c. ot tha p.mdt€. Onc. th.ss !16 lnown! roltltrnco fuctoc In th. ho!| mry b. 3.l.crsd whi.h mly counl.r rh. p.r.!it . h.r by dn6ct inhibi on of t$ growlh, or lhdt.crly, by...dding lr! vlrul.nc. .nibuto. h.ttocrtw. Th6 p..sl study wa! d..lgnod to tlnd our t.cror ln brooding r.si.tmc. to Ascochyt r.blct- To Itfi th. rot6 ot vnobrE. mdin obietiv.r !.. r. co.lirm thr biotoxlclty ot lungu! 1... cuttu,. tttrr.to ot A..ochyi. 2, lsol.don, puri{ic.tion .nd ld.nrliic.tton ot phytoroxic comDoundl.), 3. Corr6l6tioh colffici.nt b6tw!.n rh6 snFunt ot toxin{3| and tortcfty .mong nino bolar6! ol A.rlbtot .g.tn.r chicrp.. c6I! ot two cutttulr!. Comp.rblon ol phytotordc compound. producrton in tiv. dtfi.r.nr P.lhogonicity of nin. bolrtc! ol A.r.bi.l. L bolrtlon .nd id.ntilicrtion ol phyiotoric compoundtst t om lrrifici. v hter.d .lticrp.. 9Lrt' 7. a..aLl D.bminatid ot tbdc. ol p@ro,fiLl g.owrh ol 8. bv vrloo. Lobbr of p.odrrcdon duitg ir vlto A.r-lC vrtng h vhJ.oco D A 6ns€.p.irtlno ot nh. boht . ol A...!|.l vtryhq h \rl!ta6 to d6t6min6 tlto q€n tlc dlv.raty. EHf,PTEE !! LITEMIUFE FEVIEW Plants are continuoully chall.nO6d wiih . b.ord spgctrum ol potsnlially pathogenic micro organisms, yei only a small frsclion ot rhs dlllerent sp6ci6s or slrainG with whl.h the pl6nt com63 in conract a,6 c6p.bl€ ol .clu6lly inf€cll.O 6 glv.n pl.nr speci.s- Th. plEnf3 lkat lin. ol delense mo5! certEinly inclodls physlcrl bade6, such 6s rh€ cutictg t.y6r on lhe surfEc€ ol most ti6sues, as w6ll as tho c6ll watt jrs6tf. In mlny casos, how6vor, th€ physic6l bari€rs ihdmlolv.s do not accou.r for rh. in.bility ot 6 pdrhogen to propagrr. Int.r or intrlcelutsny. Th. fung.l hyphao pen6lral6 pl.nr c6ll wall .nd spfead b6ne.th rhe optd€rmat tEy€r bolore this coll.pse process cell ol ti3!u6 occur!. lt has been a$umed th.t during this w.ll degrsding en2yhes ar6 b6ing p.odlced by rh. penetadnO palhogen {Hohl 6t al., 1990). Pecrir dogrEding enzymes se6m! to be wadety involved in rhis procoss 53 hss b.an shown fo. soveral ptant pathogsn inl6raclions lcollm€r and Koon, 19861. On6 ol rh. maior .rolh.nrs .g.t.rt the Involv.m€nt or cotn,3€s to tongrl pen€talion into ptsnts hss bosn ths low lev6t! ot cutin636 rlsoci6t6d wilh some pathogenic tungEl spor6s, which is obviousty too tow to elloctively pgnetrEte rh. culicutar brrior {WotGhuk and Kot.ttutudy, 1986). Howover, r6cenr studies h.v6 d6monsir.l€d rh6t th. inittrt tow l6v6l3 of rh6 onzyh€ ar€ used to produce unaqu. ot curin, dghydroxy C16 .cid and tihydroxy-Cl8 dcid, which ih.tuce th! !y.th6b ot cutrnase n.€dod ror p.nglrrlion ot rh6 funqu! into pt6nr {M6iri and xofartlkudy, 1979t. Sporos of the lungus Fusa un sotdni f.Bp. p,irl us6 production ol inducer ltrol€cut€s lo s6nsr . contlct with rhe ptant. With CDNA probes, curina& monomors gen. tbnscrip$ cln b6 d.t.ct6d within l5 minotos all€r addition ol lh6 inducer hol6cul$ into lungat culurss iD v/to (Woloshuk 6nd Kobtutudy, 19861. €t al.,ll997l ch.rect.rir6d .nd cton.d cutin.s€ tbm As@chyta ,abiel which seqei6 . curin.s6 l..t€rasat in tho artturo fitoai6 wh€n ir i. inducrd by curin or hydroxyt.r.d irrty .ct&. rh. .nzymo wrs purilied and lound mot€cutar w€tsht of 22 KD in SDS-pAGE and d..vedesler bohds of 3H-tabett6d curin or p-nit.ophenytbutyr.t, wirh maximal activiri6s .rouod pH 8_ As s.rin. osteras6, cutina6o i. stronot Tenhakon, inhibited by otganophosphorcft compou.d! .n.l lh6 msr afiocrivs inhibiro. + {MAT 2,3,5-tdchrorcpyidin€-O-(O m€thyto,n-butytl-phGphateestsr+ + 95641 shows d (i v.lu6 ot 0.s nM. Th6 cutinaso s€ne was ctoned lrcm a senomic cosmid library by screening wiih two otigonuct€otid.s direcred rgainst cutin3s. cons€nsG p€prid.6. Th. g.n€ was subcton6d 10 . 1.7 (b Sall/Hindl I 1 in.6rr rod sequencod. Tho cutinase g€ne codgs to, a 223 6miho acid prorein wth srrongo homotogy to orh€r tungat cutinls sequenc€s. Tho purili6d curinase t.6ncod6d by a sinst. copy g.no. in ptant. t6 . comptex phEhomonon r.qukino ot g6nes The .!pr.E.ion of q€nes invotv6d in lh€ Db6a36 ,e€ist'nc€ conlrol oi hundr6ds &cumulation ol loxic secondary mlrrbotit6s (phylo6toxinsl, inhibiroF of psthogen 6nzymos {proro.sa and p€ctinso inhibiro.s}, 6nzyma. to d.or.d. pdhogen srtuctural components (chttlna!€ .nd endogtuc6na!.1 .nd ptant wdrr compononb, is dependent on 6lth6r dtrocl recoonition ot palhog€n constitu€nts or .crion ot these constiru€nls olaboration ol pllnt i3 dofsnses pr@lded on ptdnt tis6!es. by Thus, pathogon doiection. Subs€qu6nt srodi€s wirh a wound p.rhog.,n, Mvcosphaa.e , sp. showid lhar rh€ l6v6r ol 3p.cittc curinase enzym6. c.h deterhine rh. c6!.cirv of rh6 lungus to pen6irgr€ th. fio6t cuticl€ 6nd cau6e dis6.!e {pego .nd young, 1982), Ihe fnvolv.m€nr ol culins!€ in lungat intoctton with Fus.tbn ebni t,sp. p,si ha! boen tudhdr imptacar6d by utrrasr.uctur. srudies wilh t.ririn cohjus.r.d 6ntibodi€s pr.p.rod againot th€ curin.s6 tshoykh Et 61,, 1977, $d proveniion ot lungrl Infoction by spocttae Inhibirton of rh6 €nzym€ (M.iti and KolsttukudY, 1979'. Chickp€. btiohr western F w.. tnot obsotuod in 19r 1 (Burt6., ontior Provinca ot roporred trom the lottowinq 26 l91al in rh€ Nonh India. Chickp€. btighr diseaso hrs b66n cou.tri6!. Atge ., Aus&.Ii., B.nglad€sh, Burgrri6 {Atanb5ott ahd (ov.chevski, 1929); Craads {rvtotral and lvrcKonzie, 19741j Cypru!, Ethiopi.. F.rnc6 {t!brou$e, t93l}, crcec6 {Sar.j6nni, 1929,; lodia, h6n {Z5tpoor, !983); n.q, tsraet lHatfon Meni, ,970); Jod.n, L6b3non, r\4orocco {Labrousse, 1930}; pakistsn, Bomanis tsivut63cu, 19321; SFin, Syria. Tanz.ni. lwalac6, 1948) and Tu.isir, Turroy.nd rho 1935t. In 6 t5rrer pubtic.ton, tt is,epon6d !h.r rh. di.e.s. w33 more kdquontry ob3€ryBd in Ats6 6, Bdtgaia, Cypflrs, cr€6c€, k.q, USSF lPavlov., lsrE€|, Jodan, Sy a, Tuni.i. lMoral .nd McKen.io, 19741, Tu oy and USSR. The natur6 or the primary inocutum in d chickp6a fietd is lril or discussion. P.eviousty, pycnidiospor63 d.,iv6d rrom ini.cred . matrgr s..ds .nd dlEe8od d6bris h6v6 been considerod tho onty sourc€ or p(m.ry inocutum lNon6 and F6ddy, 1987t. How€v6r, th6 perdct arEge ot ,4. ,rr.bi Di.lyhela nbiei, was atio id€nrifiod on over wintonng chickpoa dobris in sov€r5lcounl.ies tKsis6r, 19921, whit6 trB inftu€nco on dF€ase epid.miotogy an.l pathogon dtversaty is stl|t unctear. Feceht .vio€nce sugg6st! rhet rhc tereohorph hight b€ mr6 wid6ty spr66d lhdn pr6viou3ty thouqht, and th6r 6scospor6s may slso 36Ne as. p marv inoculum (Trapeto_coses and KaF r' 1992). pisi t d Oth.r r€cen! stodies havo shown thli /4s'oc'vra "Diei' A A.hb.e vodtc. ioxin. whon grown ln 'h v'tto cultur€ und€r d6fin6d condilions. The toxin lrom /4, t.bte and A pisi ha3 beon isolst€d tnd that th' id€ntitiod 6s ascochitine. lt has b66n fu'thq demonsttal6d suoc.Diibilitv ol pea cutivar. is dn€cilv ..cochitln. .nd rh. vnubncB ol lungrl obilltv to svnrhesize lhe toxin a vil.o rolated lloltt" lo their s6GitivilY ls timiltnY r'br6d to th'k ln EimildnlY with these obs6rvation'' it msy b6 assumed that sentitlvftv ol chlctp6. culrivdrs is r€laiod lo 6ynih6siz€d duting ,h v,rro growrh ol A. vari€lv ol to /.bi.i. to{n' Sev€rd! fungi produc€d phytoloxic compounds Cvtoch.lasing and phalloidirc g.oups ot small, natutallv occuring oro.nic mol.cults ihtt bind ro ot' ' lwo 'clin tnd ah., its polvmerizalion. Thov hsv6 b.6n v',idolv t!6ed to studv tho '016 of scnn in biolosical process6 6nd .s dodols for acrin bindino ptot'ln! Cytochaldsins, a group ol lungtl m6l.bolit*, po.m€rte cell montb"nos md up (Schliw!' 1982 lnd 'ound Y.h6r6 6t al , l9a2), bocomo less stilf tcoop€r el .1 , 19871 6nd 6nucl6't' (Goodm6n an<t Mnands, 1974) ln 6ddlllon to bindins .ctin. cvtochalasrn' A c6u3€ cetls to stop ruttling and trsnsloctllng, and B also inhibit mono€achlride lttnlpott scross the plasmt m6mbnnr; howsver, cvtoch.lasins C, D, E, H, 6nd 2l' 22 dihvdrocvtochalalin B do€B al., lgaol. Cvtoch.l.sins bind to the btrbed lnd ot tctin filrm6nt., which inhibits both lhe Bloci.tion .nd dis'ociaton ol suhnitt !i p" tcdn rh6t 6nd. The stoichiomlry ol binding is .bout one cvlochtlssin nor tFamodl .t lilamonl {8rcwn and Spudich, 1981 Functlondllv, cvtoch.lt.int & Flanagan 6nd Lin' t636mbl6 c.pplng proteins which block 1980}' tn 'nd ol bcrin tilamont!. hucle.t. polym6rizsiion, and 6hort lilah.nts No known acrio binding prol€in stabilizos dctin lilsmlnts ds ph.lloidin does, but such proteins may havo been missod. Oth€r sp6ci* ol ?4s.@rvta also produc€ vrrl6lv or toxltu. Ascolursnon. snd $cochlo.in havo bean isoltt.d liom ,4.v,.i," 6nd d.cumbin fion A.npen.h. A.ch.vstnthomi s\n\n.16. €ooxvdon 6nd its monoacotEte as w6ll a! chrys6nlhone. CYroch.l.sin A and B have abo been isolatod lioh A. het@romotpho 3s welr cytoch.lasin, .scochalasln, A severo toliar blaqht ol $ 6 n6w OlgandEt {l/rtr,rn ol6.nde. L.l .atsed hy Ascochvta hatarom,pha lsch. 6t s.cc.) curzi, characlerized by oxt€nsive brown leal zon.tions, w63 oborved in 6 nuBery n€.r B6ri doring April 1985. lnvdarigdtion on the toxin motEbolllos produced by the palhogen led to rh6 pt.ilic.tion of a n6w cytochalasin ntmcd a3cochalbsin (capasso 6t al,, 198a), tog€rher with larg€ amounr ot cytochalasin B 6nd small6. .mounis ol cytochglasin A lcapasso ol El., 19871 and d6oxaohomin lEtinder 6nd T!mm, 1973) obtained lrom th€ lilftotes ol thr66.w.€ts old siill cultures incub!!€d 61 21oc certai. lungal planr p.thogens produco ertr.c.lllla. toxins thEt lro to bo causrlly involved in dboase. Some of rh... .r€ roqun.d fo, parhos€nbny wh6.eas othors contribut. to vnulenco of ihe or96nlBm thar p.oduce th6m. Toxin irc€n8itiv6 plants crn bo cflici€ntly seletod dir.clly or regenarsr€d lrom insens av. cells t vtuo. lt the toxin is. p.thog€niciry fsctor, 6 high l6vol ot resblance is lit6ly to b6 obtllned, iI th6 lodn vlrol6nce laclor. an interm6di6te level Eith€r l€vel can b6 oconomically r€slstancg lo ol resistdnco i. lik€ly to b. obtainsd. valurbl.. Genetic modilication of pl.nts lor dls.36e, whoth€r by convontional r€chnolosy, roqlires th.t 13 a or goneiic ongln.orang rosistanl andividoars could be idontili.d among l.rqe population oa susc€ptibte pl.nt.. pldnrs wath lhe prrhog.n irsott, . ThiB iB usu. y done by inocutaring proc6dur6 thar is cumbofsom and inoliicienr. Sasral aothors h.v. .dvocrrod $. uso ot toxins producd by pathos.ns lor more €fficiont id6nritic.tion ot d,s!.s6 r6ssbnr panis or planl parts such.s lassues. ce[6 and proropt.db {E6,t., 19871. Planrs hav6 evotv6d . corr|pt€x .ystom of d6t6nse mochani€ms acling at.ialler6nr tevels in ord€r ro r€pct 6n ,ll.ct by p6!hog6ns. phyroatexin! ar€ d6rln6d 6s anrimicrobirt compolnds whtch Ere synthesiz€d .te novo upon inloction or eticiror reatmBnt {Dixon, 1986t. th€y a shr.e the characrE,istics ot b6ing roxi. to lhe tnvlding paihoO€. by rh€ .ccumut.rion ot 6ntibioric cohpounds in r6pons6 ro intocrton ond oth6r stresses. necsnt studies havo demonstratod thrt 6€rirt psrts, cel susp€nsion cullure€ lc€nsenbach ol .t., 19771, 6nd cotytodons {ca,tson, 1973) of runOus resisranr crttivars cao b6 induc.d to sccumut.r6 sev6r6r totd hish6, ol medicarpin and maachicin wh.n comprrod wnh tungus susceptibte cultivars. Furthe., evidehce with rcgsd to th6 rot€ ot pterocarpans l6vel3 and jsoflavone m€t.botism isolatlng .) in chickp6a lungst rosierance was obrain€d by .! d6id2.in, tormononorin, cdtycosin 6.. lhought ro b6 Inrermodirt€E in phyroahxin ph6notic compound3 6uch 6nd psoudobapitigehir, which bi@ynlh€sis, b) sp6citic inducibt€ enzym4 such sB NADPH; isoflavone oxrdoreducl.se, midolom.t isoft.von. 2' .nd 3, hydroxyrases ahd mordnyrost€r8se which 6re a prcr6quisite tor $6 con 3hown rhat srrains of a ,. rabi.l pathogenic to chictp., c.n .fiicisnlty d6grade medicrrpan ro ov6rcoh€ the ptant detonse moch.nism. A con.titurivgty etpressing oxido.€ductase enzyre h66 boon plrrir y purfi€d nom c,uo6 prorqn exrGcr ot th6 lungus .nd shown to ct6.v6 tho bonzytphonY| €rher bond of th. phytoalexltu (Van All6n & McMillan, 1942 rnd coodman .nd Mnrnda, 19781. 8612 and Cowo 16 hav€ recgnrly shown that alrer infdction wirh . vnubnt slrdn ol Ascochyte nbiei, 6 fungu. r$i€r.nt chickp€a lcicet ..i.tin@l colrly.t .ccumul.!.d 4-5 lotd high.r t6v.t! ot phyio.tartn. medicarpin and maacki.in, when compared culrivar (Mli6lmon with a tungus Busceptibto et 61., 1975). Phytoatetitu hdv6 been shown ro bo pioduced by over 10O pl.nt sp6cies, 6nd the av.it.bto informario. ha3 bo6n .dequat.ly .ovi6wed (B€m.s.oni rnd Pat6r, 1986 & Ebot, 19861, Th6 prodoction ot phytoal€xin5, appedr5 to b€ uoder rhe rranscription.t contiol ol a s6t or gonos indocgd in responlo to tungst intecrion, Th. procssses or gen6 .ctiv.don .nd adhe,onr synthesas of spociltc mRNA mot.cutos of en:yme prot6ins involvod in phylo6l6xln biosynthesis hlv€ .nd r6p€at6dty b6.n repon.d lBaru er al., 1990; Eb€tot.t., 1984.nd Rydd or 6t., 19871. Howev6r, th€ impodrnca ol ihec€ g€n6s to diseas€ .6sistanca, is cohplicated by th6 .biliti.s oI th66 g6n66 to b6 .t.o indoc.d by oth.r factors, such a Uv risht {Amin et dt., 1988) o. wounding lDslvil and Thus lh6r6 app6.r to b€ rwo ind6p6ndent mech.nbms invotvGd in rho onser ol Ascochyta btight and chiclp€. ptant resi.tsnce. Ths mod€t b.sod on ddta lrom Brrz and coworkqrs a6sum€s s princtprt rote tor phytolt€xin6 wh6reas lh6 other studl€! !!6igh a mdjor ,ote lo tungat torins. At this sl.oo, the physjologic.l signiticance ol rh. rwo m6chanisms .nd rh6ir intordction, It ony, jn derormining chtckpo0 tungus resistance is unct6ar, Th€ toregoing distincrion smong ryp63 1016 d€termanins rh6 qoality ot rasasr.ncd ol toxin has vatue in $.r mishr prcdacting it3 b. obtained. tt rhc toxin ha8 no c.us.l 1016 in dii6a!6, th.n ol.nG 8.l.ct!d for r!3istlnc! to it will r..i.t.nt lo dl$.l.. rl th6 toxin i. . Ddthog.nicity f.ctor, pl.nt. hiohly ro.l.r6ni to tt. toin will b. corpl.r.ly rdbrlnr to died.. rt p,ob.bly not bo lho toxio i6 . vnul..c. f&lor. plant! [email protected] to th6 loxin will b6 p.dirny ..sisranr to dis6.s.. Prrti.l r6.i.t.nc. !6l6cr6d wirh . vtul.ncs tlctor cln 6. lully ..rbi.cto.y ror d1..... conrrol ll rh. oortlo. ol th. dr!.... ..u..d by lh. torln b lh. .conomlcr{y lnpon!.t. M.ny pl.nt spei.. ao6 .s hGts tor r padcular parhogon can d6v.lop . sp.clnc r.!i.t.oc. to th.t p.thog6n. A ho.tp.r8it6 p.i i. . comr,l6x ol two org.nism6, rhd.lo.., .0sc.ttibitiry o. whlch normally do r*bronco of th6 host lnd vkul€nc. or p.ihog€nicity ot pathog.r on th6 olhlr. aro and6pcnd.ndy. lt iB mor6 rel.rion .. hosCp.r.lit pdhoson.y.i.h w.3 tlr.r rocogniz.d by Ftor ttg7tl duing hb 6xt6nsiv. siodi.3 ol lhs lt.x rulr g€n6 . .ccor.t. ro d.rign.t6 th. 3r.t. ot lv.turt€d comp.llbl. or incomp.tiblo. Th. q.n6ric ..t.tionlhip ondortying cultivEr 3p.ciric hosl .rch bo if,rordcp.nd.nt conc.pts, wlych cannoi lungus Uelrrloson th.t govsrm r.6i.r.nc. h th6 ho.r, rh€r. b, ,iri For coro.oondtng o.n. in ih. paihog€n thrt govorffi a virul.nc.. In iht! co.r6xt, . vnubnco i! rh. ol a rdc6 ol . host,aD.cific Ollhogon spocios to i.t.cl p.rricd.r cultivrr ot its host. Reirt.nco in th. ho3i b u3u.tty .tohtnant, .! is . inEbility vlrul€mo in th. palhog6., Thi. rotationlhip. gov€rning th6 int r&tion ol m6ny whicn b ih6 g.n.tic brlts pl6nr3 6nd rh6ir plrhoo.ns t. c.[.d rh. g3..Jor'96n6 Glarionlhip lswahlon ot.t., 1988). Tho g...tic ralorlonlhip b!tr66n ho.rs .nd plthog6ns i. provoc.tivo it! .impliclty rnd it! mol.cular ..mific.tiom. E6ch g6n. tor dt.6d$ rBktanc. in a plrnt b prn.d wlih E compt.m.niary 96.6 to. rvnubnc. i. In borh th6 @thogen. Only a ilnglo p.ir ol con6sponding grn6s amoog maoy i6 roqunod to triggsr th6 plant's ddlenslv€ m.chani3mg. Tho g6n6rallly of rh€ ''Gen€-lor Gen6" r6larionship i. thoughr to 6xrond trom vnuses .nd b..t6rid to include tonq6l pathogeN, n6m6!od*.nd insoct p6st3 ol plants, Ono molgcurr interpr6rarion of thls prolound gonetic relolionship is rh6l ptanrs produco .eceplors (diseas€ r.sirbnce gono productsl thar bind p.thoqen mol.culca ldi.6ct or indn..t producrs ot .vlrul.nc. s.n6' rnd Inlil.i6 th. suit ot biochemical r6spon663 obsoNed .s r6sistancs lH66th, 1980). Althoush no ad6quar6 mol€cula. .xplanatjon ot g6n6 tor gene interactions yet 6xisls, lt is wld6ly thought th6t the g6ns product ot th6 host resist.nc6 g6n. inl6racts wllh rhe prcducr of rh. p.thogen o.n. tor . vnlbnce. This inter6ction mry be eirhor direct, rE in rhe int.rlction of specillc cell surfoce rec6pror pror€jns on borh hosr rnd pllhoqen, or m.y b€ mediatod ihdn€cily through mrcroholecul.. whos€ producrion r6utl3 trom tho .ction ot 6 sp.cilic grn6, such .r lipopolysacharides or comptex catohYdfales (DEly, 19841. Vnurence ch.nges ar. prob.bly .ri.ing conrinu.lly in popurrtions of plant pathoggns, but in rho fi€ld msny wlll not be dotoctod on .ccoont ot th. gonotype an which thoy occur. lr b propG€d rhar 5 ch6ng6 trom rvtul.nce to vnulonce on r pdrticul6r ho6i is on6 involving t€w g6ncs, w6 can a$ume lrom lho work on th6 inhorttanc€ ol growth rate io oth€r organl.hs, that aggressivsn.ss iE und€r polygenic contol. Th6 rcsr dtam6tic popularion chang€! thorcloro aro likely to tollow wh€n a variant havinO a particold g6n€ tor virulonce 6ris€3 .lther sexurlly or rsexoslly in an .ggfo.sive w€ll ld.pted g.notypo. IhB ge.€nc 6nd holecular basis ot chickp€5 blisht r6.isranc. is v.ry poorly undorslood, Sources ol r€Eistanc€ occlr in wi|d typ6 ,ol6liv€6 and r6po( chickp6a blighl rosi6tanc€ i3 conrrolsd by . 3ingl6 dominant sen€ (Vt at.l., 1975)- Th6r. h.s beon no 6rudi.E d.lining rh€ mechanisms ol reslsl.nce ol th6 pllnr or ot vnul€nc6 of lhr p.r.3h6. 6ccordino to on6 Henc., blight .$l5rdnr v.rieties hav6 b6on r6le6sed, but b6.am€ .uBc€ptiblo rlr€r only lew growlno 666sons du6 ro rh6 6pp€6renc6 ol viruloni drdins ot th€ p6thogen (Jamil,F.F. €tal., 1993; S.x6ns 6hd singh, 19871. Singh 6nd Reddy {19961 imp.oved chickpe. yisld by incorporatino re6blr.c6 to in m6in se8on. Th. vi.ld assessmenl bog.n with F7 line3, ft.t .r ICARDA sit€s 6nd l6t.r Ascochyra btghr. C,osses wero m.d6 internrriondlly. A tot8l ol 1584 &cochyt. blight resistance w6r6 devolop.d with the raoge chicle.. ol matu ty, pl3nt height, and s.€d lih6s siz€ not to growers in lha blight endamic 6roa3 if, lfie r6gion. th*6 includ.<l 92 lin.s r.sist.nr b 3ix r6ca3 ol tha previolsly .vail6bl6 M.dite(anean Ascochy$ parhog€n, 6nd 15 lrrc6.so.dod .nd 28 ea y maturiry lino!. N6w crirtivars produc€d 33% more seed yield lhan th€ origindl r€sbtEnt sourc6- Fecentlt an inl6gr6r6d di36a6€ control prooram was impliment6d (k.is6r or al.,t994l to us6 dillerool culrural pr6cticos to m.nage intect6d crcp dobris that remainod in ths ti6ld aft6r tho bliOht-inlected chickpss crop w.s hatuested . d6v6lopmenr Tr.p€ro or al., {19961 studtod ottoct of datter6or tr,ngicides oo of th€ r€lomorph d6bri!. Thl.b€ndazol d€vElopmont sttdies, rreared chickp€. ss6d3 w6r6 ettocrlv. In prgventing ol lh€ A. .rrlel on naturally inloct€d chickpoa d6bris. ioconl .s prrt of prosrdm 6t ICARDA ol ol Ascochyt. rarlbt on anl6cr6d chickpea ths int€matlonrl Ini6grated chickp6, blight conkol h'v! dis€a3e, dolinlng contribur6d to .h und.Btdndins ol rh. proorossion of temp6ratur. .nd .6l5riv6 hunidity on th6 r0 d.volopmnt ot chictp.. blioht In !.l6ct r..l.t d lungo. on .u.copribt. {d nr cultiv.r! {ICARDA, t984t. ko9r6m3 fo, d1...$ cont.ot 6nd .o8i!ranc! b...dtng in chiclp.a d.plnd on rh6 .sli.blo ld6milic.rion rnd ch!rucrod2.rion ol fungll ttthoiyp$ ..d popul6iion!. Ascochltt r,btatt nor'iour lor vdi.bitirv In morphology lx.b.., 19731, plrhog.ntcny lPort pugtir t9921 .nd phvrotorln producrron lalm .t !r., 1989; Hoht 6r.t., 1991 rnd tarit 3r at., t9931. Variabiliry in th. p.rhog.nlcity ot ?4. .rOiri h6s b..n .oponod froh Indi., Syri., t.banon, lunasl., lraly and Prki.ru lN.n., tggrt; Oor.shi lnd Atrm. 1984; F6ddy 6nd K.bb.b.h, t98E; cow6n 1991; 6r.t,t989; Hu.$in Jrhll €t El., t993.nd Mod.o..r.t., t985l. pathoryping t6chniqu!6, ustng K.bb.boh, 1986t . Th€ and M.tik. cta$icrt biotoltcll .6r ot difiorant ho.r g.notyp6! {R6ddy lnd i. t.boftou., rrho conlumins 6nd r.quiro! 3t,tci sltnd.rdiz.tlon ol t8t condlton. b6c.6. trs roprodoctb iry b otran poor. Th6rolo.., rny idontilicrrion bls€d on morphotosic.t, ch6nic.r .nd/or palhoganic ch.ructorbtlcs h.s blon ditltcutt ,nd !u .F trcll s.varal Th. .n.lFis ol $n€tic v.rt.rion in ptrnr parhog.n poput.tion! i! .n import.nt protoqublt. for und.r3r.nding co-.votutio. In ptd p.rho!y.r.m lMlcDon.ld or .1,, 1989.). intlulhco. B€ddy th. th. h@r poput€rton in 96n6ric div.rstry .od . ptrhr pErhog.n poputation .lrucrur. lssr.lrtulty .q.ln!r r ..r ol ho.r cutrtu.r.. r.c.. m of p.thoq.nr. .nd K.bbrb6h, 1985 .nd w€ilins .r .t_, t99lc glou!.d o1,4. .rOri.l colbctld in Syd! aM tjbrnon Inro 6 .y!t EO t.ot.io. occording to th.ir Tht. co.c.pt ot btotootc.l .olt.r. lroh .ov.r.t di.!dv.nt.g$: rh6 mothod i! {h.coGumrng, rh. oxpon6o i3 .hormos, r.producibatity i! poor, .nd p.thotyping inr6.n.tionrl 6xch6ng6 or d!r6 b re.rrlctcd th.ough quar6ntlne reoul.tions. Sinc€ mors inrormationr about population generica, geogEphical disiribution ol pa(lcular clonos, rsc6s and pathotypo! ol th€ lungu. 6.. of considerablo lnt€r4t lor chickpoa planters, .lt.rnariv€ 6nd genelic variability .onceprs ot pdrhotyping 6re cbany oaed€d. ln rocent y66r8, potymorphi.m incre.singly used lo at tho DNA t6vot complom€nr tradtiton.t mdrkers h.v. b€.n in the snlty3b ol 0on6ric id6ntity, va abitary, .nd retarodn€s5 in tungi. Two merhod! r€coived host attentlon (Macl6.n et moltilocu5 probes .t., h.v. 1993), DNA ting€rprinting wilh th.t d.tect num.ous r*r,iction t.agment tengih porymorphl6ms sihulraneousty lP6na 6i at., 1993) .nd pCR tinqo.prtnting with arbitr.ry p mors lhat 6mptaty dnonymoG regioft of thB Oenom. {walllams 6t .1., 1990}. Both t€chniqu€s hrve ahosdy yi6td vdtuabtB imishts inlo popula(ion genotics, palhorype divo..ily, m6tin! systoms .nd phytog.ny or. wld6 v.n€ty or runsat 6p6ct6s lH.m6, et.t., r9a9; L6vy €r 6r,, i99ti MlcDonald and rvls inoz, 1991, coodwin and Annb l99t .nd Schrtrd and Wostemey€r 1992). Th6s6 r6chniques .r€ atso idgaly suited lor srudyinq clonsr popirlEtiom {Schar6r lod St€v6ne, 1987j Boromgo ot,t., 1990 and t6vy ot al., 1991; Turnq 6t 6t., 1990, by m.rchins ONA hybrir,iz.rion pallorns on southoh btoG. Rec6ntly, th€ dev€lopmsnt of motocutar m.ny area3 ot pt6nt br.€ding and r6vo6l€d msrk.6 h6v€ revotultonized baotogy. DNA potymorphasms th.t by RFLP.ndtysis tL6vy er.t., 199!i Chiu 6r.t.,199S), ar6 DNA ling6ninting (Descgnzo and Hanington, 19941 6nd pCn-b6s6d lachniquos {W6lBh and Mcclell.nd, 1990; taroch6.r,t.,19951 ,r€ now bems.ppri.d to lh6 mark€r.sgisr6d breeding ot crops, th€ devetopmeni ot tink6g6 maps lTanksley er 61., 19891, tho isol.tion ot r..isi{ne6 gen6. lYoung, t99O', .nd to th6 structural anrlysis ot plant populatiom {Sch.at €t Et., 1991). Most recenlly, polymorphic DNA m6*€ts hlvo atco b€5n Introducod to rha ot tungat O.nofts_ Hybridiz.iion of r.rt ictiondiqosred genomic ONA to endog.noB 3in9t., .nd/or mutiitocus prob63 mol€cular char.cr.nz.don .rrowed to ditl€r6nri6ton ot ctones, torma6 spociats, r.c€s 6nd Darhotypes vafietv ol lungal species tsch€r6r snd Sr.v6nB, t9g8i H6n6. et 6t., in. 1989; Levy et al,, 1991r &own et st., t99O; McDon.td 6nd M6.!tn6z, Kisller et al., 1991). Ih6 t99t; olhq h6thod, r.cantty d6v.top.d bv s6v6,.l r6bo.atories, is callod RAPD anatysis trlndom .mptiriad potymorphic DNA; Willisms et al-, t99O; Fish€r cr at,,l99El, Ap_pCF (.rblrrrity prim€d polym6r6s6 ch6in rc.crion; W6kh .nd Mccta .nd, 1990) or OAF {DNA amplilic6rion ling€rp.inting; Caetano-Ano €s €l at,, lggtsl. tl b bas€d on th€ PcF m6diar6d 96n€r.tion of random or semi rcndom g.nomic oNA rr.gmenls lhal may o. may nor b. potymorphic depondioq on rh6 tongth 6nd 3.qu.nce{s} of lh. short, ccjich 9rim6(sl osod tor amp fication, Th€ resurting polymo,phi3ms sre.arr6d FApDs {Witti.ms el rt.. l99O) or (amplili€d fr.gmenl lengrh potymo.phisms; Caetano Ano 66 .i AFt_ps at., 19916. EHflPfrEE !!! MATEBNLS AND METHOD! All inorganic compounds, .olv€nb and chromalogrrphy m6leri.ls w..6 obtlln.d lrom E. M.rck, o6roo o.rmd.dr, F.F. Grrm.ny; rll org.nic compounds wero purchased ftom Sisma Chsmicalr, Si. toub, Missouri, USA. All Orowih m.dia insradionr. w€r6 lroh Oirco Labotatono., USA; Mrc6r.s6 R1O, C.llulysin-Rlo 5nd oth6r enlym@ wers lrom C.lblochem, USA, Synth6lic oligonuctoorid€ probos wero oblain.d iiom Unavertity ot Cincinnatii Labelinq and Dol6ction Kits were lrom Eoeh.ingd M6nnhotm, corm6ny. wlostly, Incuballons wsro c.iri.d ot,t 22a2oc. All 6&erlm.nts w.r6 dono in r.lplic3t.. lcic.. orietinun t t s6eds, cultivars 6 53 lsulcopraue to ,4' mbieil1nd C44 {tasi.tant to ,. .ab,b,), w.re obt ined rrom rhs Nuclatr Chickpea 1 Institute lor Agriculture and Biologv lNlABl Ftisalabad' Pakist.n throush tho courlosy ol Dr. M, Ahsanol Hsq and cv Cvpfossa from t local supermtrtst ot London. Diseasod chickpea plants wsro collecl.d lrom chickpea ll€l& ot th6 Ndtional Agriculrur. Fosearch C6ntro INARC), lsl'mabad, Ptkisr through rh6 courlosy ol Df M. Ba3hn Malik Atcochn. 6blel bolrr.. A. rsbiol i3olat.s CAMB 2, 3, 5, 6, 7,8, 10 and 1l w€to bolatod liom intected chickpe6 plsnts 6t Nallon.l Centre ol Excellanco in Mol€cultr th. Puni.b. tahorc. The inlocted chickpsa cultiv6rs w.re corr.cted lrod dill.r.nt loctliti.6 {T!bl6 11. l6olat. 6( w8 obt6ln'd hom the Biology O6panFonl, Univ6r$itv Collego london Flngtl spofos Biolosy, University of DFTEAETT ISOIAIEA OF ascocttwa ,uatf, colucrEo DFFEAE T LOCATTTIES AND CULNVANS OF cHtct(?EA CAMB 2 NARC PK 51792 NARC CAMA 5 304 CAMB 6 KC 1285 CAMB 7 CAMB 8 -C 44 6K CAMB IO CAMB I1 lcc 115!4 Fio[ w6'6 sror€d 6s a suspencion llOT 3por.3/nr' in lo% gryc6rol .r '7OoC lot turlhor uBa in experiment'. |'olarlon oI Asco.hyr. r.bi.l lrom Di!.r..d Pl.ntr lnl€ci.d l..v6s, 6r.h. and pods w6r. w.6hcd tho.ouqhly wlth tap w.ior and sreilized by imn€Blon ln l5% Naoc! lor 5-8 minut€s. Ait€. Mshlng threo times in 6todli2.d distill.d wdt6f, cov3.6d wlth sicrillzod dirtlll.d w5t.r .nd disch6rg. th.ir spor$. inoculat€ 2% wsier th. lh. pl6nl msterlel w.s pYcnidl. w.re .llow6d ro Th6 r63ulunq spoie susponsion (Flg. 2l was used to .9.r pldl€Ej pl6t6s 4 dry3. Colonios drislng lrom sln!,16 w.i€ incubal.d at 2212oC lor 3 to spor€. w.re u!€d to inoculat€ pl6r63 contsining 9..m 36ed 6xtr.ct IGSEI rcdium lchickpe. 8.€ds gluco.e 2%, ag6r 1.5%, pH 22i2oc lor 6 to I daF 5.6-5.8l Th6e nl.les lFaO, 31. 6xtret 6osm/l wer6 incubaied at Coloni.s lrom GSE medium w.rs u$d lor rhe production ot inocutum on storile chickp6. s66ds {Altm €i order to produc6 copioB numb.r al.' 19871. In ol spore. lor inoculom, chickP6t seod6 w€r. sollenod by boilins lor 30 minut€s in walet ,nd drain6d. Aboul 25_30 s€€ds w6re autoclsved in a looml ttask lor 30 minutds. A looptul mvcolium tiom GSE m6dium pltr€ was ldd€d in tho tl6sl and dlsrlbulion ol th€ inoculum. Altof lncubstlon 61 wd sh'kon to onsurs 22l2oc lor 6 a d'vB, ihe (Fiq.4l w€re dgllal€d with 10% stvc6rcl solullon and .diusled lo q've a r63uhlnq 6por€ 3usp6nsion ol l07 3por€sht lh€ infqcted se€ds susp.nsion wss th€n divld6d into ampoules (lml spo.e 3u5p.nsion in Eachl lor storag6 6t -7OoC. sporcs ol Ascochyta abiei. 25 Flg,3. Growrh ol4. rar@- on Plat€, Inlect€d chickpea seeds lor nulriple sporuBtron- Pr.p.rdlon oa Aqu.o6 Errt.cr ot Cniclp.i S€€d! Sixry srams ol chickpa. lcicet a.ietinun t.l es6ds w€r6 boitod in distill.d w.r6. lor 30 minutos. B!to.6 cooling, 3otid m.l.ri.t6 wo.. sep.r.t6d by p5$in9 the solutlon rhroogh tiltor p.por. cooling ol th. axtr.ct 5OOml in tho prasence of chlckp6a B66ds w.B €voidod ro provent furthor €xt.action Fung.l Orowlh In Uqold M.dlum .t Dir.r.nr CondliDn! Art6r cooling, the clo.r chlckpa, 6!!ds 6)<tract was .ddod in Czap.l Dor liquid m6dium lsaccharcs6 3O.q NaNO3 3.0; K2 HPo4 i.O; lvtsso4. 7H2O O,5O; KCI 0.50; FoSO4- 7H2O O,O!t. Th€ pH ot the m.dilh was adjustld ro 4.8, 5.2, 5.6, €.0, 6.4 & 6.8.nd distiibutgd in 3omtltiqu.res in 25oml Erlenm.yof fldsks- Ihe flaBks w..6 ptugg€d wtth coton woot ond coverod with duminum foil b.lor6 autocl6ving. On6 ml spo.6 susp.n€lon dliqoot, .tor6d was mullipli€d on sterili2€d chlckp.! s..ds at providc ftcsh inoculum. Each w.3 Inocul.ted wlrh in d.rk .r w.rl .. Growlh of A. 2OOul llOhr, 26h .por. in 10% grycorot .r ,7OoC, 22t2oc tor 0.8 d6ys ro fl!3k containing 3omt grcwrh m€dium Busp€nstoh (1O7 sporo€/mr) .t dtit!...r t€mt.r.rurs. .nd Incob.t.d vrz., ta, 22, 26 .nd 3OoC r.bl.l .nd llo.ctlvity ol Culrur. f,r.rr. the rungus w6s srown In 25omt E onm€yer ttrlk$ contdining 3omt ot c2ap.k Dox riquid medium lpH 5.6) lupplomht€d wnh chickp.a..od ert.ct ot cv. 6153. E.ch flr6k wa. iocub6r6d wirh 20out 5por6 luspemion at slallonary condlilons tor 12 dsys at 22l2oc in conrinuoo6 dilfuse tight, 2a Aft6r thdt p6iod, rh. culruro littr.t€ wd! hrrv$t€d by passino the incub.r.d mixture through eith6t Whlth.n cF/A or No. I fitt€r lihrat. wa. olrher p.oc6$€d imm6dt6t6ty or ir.ezed u!e. st 2OoC tor to.rh.r liltrrt. w.6 divldod and used tor rh6 to owing difieront ...'ys io conllrm rh6 pr6s€nc6 ot bio-toxicity in tungus t,.6 Cultur. bioactivlry lrl p.per, Th! cutrur. C6ll Bioassry Inr€.nod€s {ii) Rool srowrh Inhtbirion Biosesay Bio.$.y (ivl O.r!ch6d t.!v.. S..d St rlliz.do. S€ods ol 6 tiiil Nod€s and Sio."sav_ and Sowtng locally 6dapGd, blight .usc6pribto lin6 6153 wo.o lhoroughly wa3hed with tap wat6r rnd surlaco steriti2.d lor tO minot$ wi(h 15% comm€rcill sodiom hypochloila tN.OCt). After washing with sterat. dlsnlbd w.te., the s6e& w.re blott6d d.y undBr as€pric condattons .nd 6llow6d to go.min.te by plociog them on solid 6gar basst medium ot Ganboe et al. {1968) dovoid ol amino.cids, vir.hins and hormon6s. Tho tubes wsr€ incubared.t 25oC ot dn inciddnt light p6riod ot ot 16 h. Filtoon d.y8 old s.6dling! w6r. u€€d IOOO .r tur wirh 6 tiqhr sourc. mat.rial fo. diff€rcnt bioEs3ays to contum ih3 pres6nc6 ol toxic compounds in lungus l.olallon ot chlclp.r Coll. lsolared cells tiom chicteoa le.llets w6r6 obrained by proc.doros d€scrib6d by Shoh€t .nd St.anqe lt989l. L€afl€tB wer€ €xcis€d rrom chickp6. planl3 (12 15 d6y3 old) grown in rhe growth.oom. Th6 leatlol6 29 w€re v6coum inliltrat6d wlth lO 2OOmg; Mac€rozyme R .n enzymo solution conli8rinq ot Cellulase R lO 3ohg.nd Bovin6 66rr,m albumin IBSA) smq in toml ot holdlos butt.r lcirric 6cid monohydr.t. 1o.5gm; C.C|2.2H2O 5mM, KH2PO4 1mM; MgSO4,7H2O 1mM; Glucos€ loogm and N6OH 6,2qm in I lit.r dlstill€d H2O lnd.dju.r.d to pH 5.a1. Th6 susp.noion ot lorllots in thl dagesrion rolutlon w6B .tlr.d on . mlgnotic sfird .t .bour 100 120 rym until they bsgan to disint€gr6to 115 to 20 min). Afi6l tittlation lhrough tour lay6.s ot hualln clofi, rhe c.lls w6r€ w.Eh6d threo holdlng bultor by 6usp6tulon .nd conrrrug.d .t IOO X s. Th.y w.,. tan.tv susp€nded in tho same bulf6| 6t 6 donslty ol 0.1A unttg Stock Bolurion ol tims wirh st = 820nm lc. fluo,oscein di6c6r6te wss prep6r6d monrhtv in Ecotone tsmg/mll and sror6d .i 4oc. I was dilur.d tl:sol wilh hotding bullot wh6n r.qun€d and rdded 50ul ro €ach wett. Tho isotarod cetts in the w6ll ot miciot€31 pldte w6r. viewed und6, an Otympo6 invorr€d mtcroscop€ (Modcl lMIl squipped wirh .pitlour€3.€nce optics. ExcitEtion tluorochrome was achi6v6d by llghr 6n.xcit€r lilt.r tom r qusnz 1F49O End.€noct€d Inro rh. ol rho hatogon lamp, titt6.6d by microto6t pt.to dichroic mnrc, IDM'soO) containins s builr. in o 515 by. btu6 bari.r rilor. lr wa. rl3l€r to visuallze rhe d€rd c€llB wh.n th€ ptar6 waB ittuminated rddt{onaly by low intgnslty lighr provid.d by . rongst€n filamsnt tamp equipp.d with a r.d barior lilt .. Fitty cells w6r. counrod in 6.ch well snd sco'ed tiv6 it rh6y lluo.escsd yellowish greon whil. d6ad c6lls Eppearod as r6d bodies, 30 The c6ll bioass.y was portormod 6ssenti.lly as pr6viousv dsscnbod {Shohet and StranOe,lg8gl. Frco:e d 6d sampl6s filt.r.s ! wero dissolvod in holding buller to qive ot lungls fro6 concontration culroro t.n timos hjgh6r that ot lhe onghdl cuku.e likr.te. Dupllc6te serisl two lold dituriohs w€r€ made in holdiog buttgr End add6d to tha w6lb of s micrctsst pt6i6 {souuweD rolow€d by the c€ll suspension {soul/welll. Aftef inoub.tion fot rt 25oC. lluor*c.nr di.c.i.te di*olv€d in holdins burrer ttoous,|mtl wB add6d l5oul/w.ll; Wldholh, 1972j Strange et .t., 1982t. Th. 3h porcontage d6.th ol csll a5 d6t6min€d by faitur6 lo ttuorsGco yolow gr€.n w.r corecrod lor control vEtues and protted as probits .gainsr the to92 ot th. ditution tacro.. On€ unit ot sctivlry was delined .s th6 do.6 which cous6d 50% c€ll d6arh. Th€ diturion und€r 6n inv6rt6d lluoroscenco hlcroscop6 tacto. co .spo.ding to this LD5O value was absrr6cted troh th. graph by 20 ro 9iv6 th€ numbo. ot unir3/ mt lar 50ut, ot prep6r.tion w.. us6d lh 6ach woll; Srango 6t.1., 1982)multipli€d liil Roor OroMh lnhlbltlon lO toxtn Blo.!s.v: chickpo, seods lcicet minul6s wilh and . .thun t.) w.r. N.OCI - Th.y wor. w*h€d surtsco st.ririzid rhr€€ lor 10 tih.3 with lt6rill2od distillod watd rnd g6minalod in st6rlllr6 peiriplares on a pioce of tittgf pap.r moisl6n.d with 12 day6 old culior6 liltr.t.. Alter 72 hour. {ncubstion at 26wC the lonqth ol66ch radic.l wrE m..sur6d. {fill C.llo. Growifi Inhlulion Abr.s.y Ch€mically st6nlz6d chickpea 6..d3 were g.own .t 26OC In mdifi.d BE m.dlum lcdmborg 6t a1,,1968). Nod6s and internodss seedling w6re coltur€d in c.llos troh 15 day! otd induclng medium supptomgnted with suc.os. 2%, NAA O-25%, 8AP O.O5mg/|, agar 0.8%, pH 5.6 conr.inino .n appropra.io ahoun! ol tho tesi solurion. callus orowth w55 mniror.d durlng th€ 20 d6ys incub.tloh. livl D6l.ch.dL.av..8lo.!.av Eighl l6afl€ts ra2or ol 15 d.ys old seodliDgs were dotschod wirh a shrrp bl.d6.nd lloatod in 6 p6tript.t€ cont.tning lomt ot th€ test lotu oh. Treated lssvgs were aocub.tod Et 26OC and inducrion ot dis6as€ symptoms w.ro monilored during th. noir 4a hour.. Ext6cdon or Phyroroxlc Actlviry By Sottd Ph!s. Eftacdon Preprrudon of Minlcobmn Bluo Gilson pipotto rip wEs ptuggod wfth qt6$ woot tor th6 prep6ratron ot ninjcoluhn. Atter .ddinq 4OOm9 of ocrodocytsityt IODSI sfitc. {10'4oum: Technopr€p 25140; HPIC Tschnotosy, M.cct€srietd, second pluq was tampod on rho top mrnicolumn by elltion w(h 10 mt or of OOS sitica lnd Un, a condirionod rhg h.rh.not tM6oH) 6nd water s.p!r.t ty. Altor p.$fng the culrur6 tilrr.t€s $,A)2,16' 20 dry3 otd separdtety In separat. minacolumns), toric compouod (sl was 6tur€d in acetonit ts .nd was os6d for c€ll bior63.y as w.tt as tor TtC and HptC .n6tysis jGr to coolirm lh6 prBsenc€ ol toxlc compound 16l in tungus tr€s cutruro tithat6, 12 ll C.{ 8io,s8.t: For colt btoresay, acetonarrflo w.sh us by using sahpto conc6ntr.tor and tho r@idu. w.6 dk&lv6d in holdano butior ro giv6 . concenrrarioo t€n tims hioh.r ovaporrlod that ol th€ original culrur. tittrat€ and bio.ssay w€B portorn.d u.tng th6 m6rhod oI shoh€r and sirang. (19891. ll' Thln t.y.r Elul€d acoton(rito w.s Chromatoghphy: chrcn.tographed with vrdous sotvsnt3 combinalions {r6bts 21. Th6 sampl& w.re spofted on M6rck p..coared pt6r.s tM.rck; TfC prrt6, sallcd q.l 60 F254; O.25mm' on, lh€ chromatogrdms conr.antng . floor€scenr indic.ror. w.ro d€v.toped in. !.tor sotvont swt.h contdining €ither h6x.ne: 6thyt .c€tat6:6c€ric acid {50:SO:0,1%, or h€xan€: sthyl .cotare: merhsnotr 6c€lic acid (SO:45:4:t). Th6 ptElos wor6 drl6d in an snd viGwod und6r 3!Onm wavot€ngth to mark da.k f,l Sp.ctrophoiorctric A..tyC!: D.rk spots appoa.ing on th€ abov6 TLC plrres, w€re sepEr6tety .crapp.d to dassotvo in hothrhot. All6t .emoval ol the undi6sotv6d mrtorist by c€ntitogarion, rh€ ctear soluiioh w.s scanned in 2mm qua.tz c6 tor UV .od visibt€ .b.orb.nc6 in a Hardchi Specrophoro@rer U 2Om lvl HPIC Amty.is: inj.cr.d onto Samptos of rh€ aceronitrato etuatg l2out) w€i6 . PhitiF Hptc conlbting ot a qudr€rn.ry pump, wh{ch co'Jld b. proofahned ro dotivor vsrytng mixto,o of up to four 3olv6nis, a colomo (250 x 4_6mll i.d.t or OOS silicd (1oum) .nd a diod. a..!y detector which .outd b6 s6t ro monitor 6ithd lblorplion in th6 UV ll9c3gonm) or th. vi.ibto t3gcsgonh) f.ns.. Th. h||2: tnEE{ 4Vqr!@rrcqlt Ito tE ry ol 1, L t6 6 ot 60 !o o1 I I ,t6 36 15 0,i 60 !, 33 ol L 6 11 66 ot ol lo a 2n 1tr l5 .o 12, ol 20 60 i{ to .(t 5 30 la 2t 1t e6 2a 17, 16 ll, lo0 1t. ro 20, b 21. .6 !0 N 80 stationary phes6 was r w.r.6 tw.ttod, u() {4um particle si2.: 3-9mm gua.d coluhn ol t50mm), which was protact6d by oi rhe samo matoriat, .nd rho mobitG ph.s€ .cetonitial6: wrter Ilow rat6 x Nov.-pak c18 cotunh ll:lt ! w.e which was pumped throoqh th6 sysiom at . 1.5 ml/min. Samptes wer. ini.cr.d m.nu.fly and p6ak poraiy and identity w6. .stabtbhed by compsring th6i, r.tsntion .nd char.cterisia. UV spoctra taten from th6 upBtopo tim. .p.x ,nd downslop ol rel€v.nr pcak. .nd matching rhos6 by iup€rimposttjon on those ot rurh€nric s.mpt6s. Fo, quantitativo !nrty.i6 cnromatogr.ms w6re abdracted nom lhe thr€€ dim3nsion.l =322nm.nd compuratod usihs .urh.nlic sampt.. ot rhe soran.pyronas as exrcrnal st.ndards. thia m.fiod p..mth6d s.ps.6tion ol th€ toxins. In rddhio., the programmod to m66sur€ lh. concontataoh comput6r coutd bo ol any cohpound wnh 6 UV or visiblo wrvolongth chrono6ph6r6, Exrr.crion ol Phytotoxlc Acdvlty by Column Ch.om.toor.phy Alter removal ol lungal mycelium, 12 d.y old cultur€ tlllr6t* w6r6 adiGted to pH 3.O wilh lN HCI dnd pa^itionod thre6 tim.s agaanst.thyl ac€r5t6. almosl all uv dbsoding m.t6irl3 w.r€ 6)dracr6d in rh6 organic phaso. The combin6d olhyl ac€tltg lrdctions w€r6 dded ove, anhydrous sodium sulrat. (Na2SO4l and atrer filtr.tion, lho 6lhyl .c6tal6 w6s conc.nrr.hd by tllm ev.porrtion at <35oC to a 3mall volum6, Th€ concantr.ted extr.ct wad ul6d to 36pardt6 rh6 compound ls) by column 35 Pr.p.i.lion ol tl.rh Column Ihe column was pr.p.red by lillinq th€ column wnh silic. q.l i23O,4OO mesh, E. 60 M.rck N 9385: Still et 61,, 19741. Th. tolal column longih w.s 45cfr and disn.ter 2.5cm.nd its 15cm Ms lifled wnh Ellic, g.l 60. Belor. filing the column wirh silica gel 60 a sm6ll plug ol gr.$ wool was placed in lh. tub6 connecting th6 sropcock to the column bodv, and 6 ol 6and add6d to cover rh. boltom ol the column A laYet ot B.nd i3mml was car€lully pl.ced on th€ Ial top ol th6 drv gel. Th€ column w56 layer carolutly lillod with th. solv.nt 3.lect6d lor chtom.tosr.phY 5o .! not to distud the ad$orb6nt bsd and oponsd the stoFcock Th. llow controllgr w.s fitrod lighrlY to the column with strong rubb6r b.hd!- The main En fin' valvo lording ro the llow cootrolld. was openod slightlY. This caused lho pr6ssur€ 6bove th€ .b3orb€nt bed to climb bpldlv snd cohpressod 6lllc' 96l !s solv6nl w.s.apidly lotcod through th€ column- lt was impottant lo txp'l all the 6ir Jtom the column 6nd not to dbw rhs low6r pa't ol tha column lo ropacking become hoated as oth6twi.e th€ cotumn lrdgmentod tnd r€quired {srilr er r/ 19781. Fl.!h chtomatogt.phv ol tho S.mPl. Alter wa3hing th6 column rhoroughlv with m€rh6nol (3oomll' ih' column wss.quilibr.tBd wilh 3OOml of hexan€: €thvl ac€$t' {!:1) conr.inlnq 0.1% acotac acid conc6ntat.d 'thvl Ec6tat6 samplo ptrtiiio'€d liom th€ origintl culture filtr.le wss loadod on tho ad3orb'nl b6d with ' (2ornll warr pasture pipotto 6nd dllowod to run into rh6 column Fractions cut off.lter lh..pplac.lion ot 6ootnlol th6 'am' solvsnt conr'ining O l% r.6{ng Toxlc F.rctiom lor fth Puriry Lry.r Chrcm.toraptry Thin layer chrom6tography w.6 G.d to monitor rhe prdenco of tortc compoundlsl in tactions colloct€d by column chrom.rogr.phy of concenrrar.d 6ihyi 3ce€tt. Ihe lr.ctions w6ro .pofi6d on M.rck silics gel platos {0.25mft1 cont.ininq doveloped . fluoroscent indjcalor and in rho .olvont in which th.y wer6 elured during column chrohrrogrgphy lh€xrn€: ethylac€tat€: dcetlc acid 50:50:0,1%1, rhoy wsr6 vi6w€d under short UV lradlatlon. Spots w6r6 llso r€vool€d by o(poslng lho plat6s lo lodlno vapor3 or 3p'ayins lh6m wirh [email protected] acld (1O% lh €thanoll .nd he.tinq 6t gOoC in 6n ov6n for 10'15 minutes. it, Hish p€dormaoco liquid chromalogrEphy wa8 dono as doscibed 6bove ro chock the purity of th6 lrrcloG collect6d by colutnn ooalltlrivr An.ly!|. ol Ptylotorlnt.) h F|i. Cuhiv.B ot-chictpea Tho ronsG wa! grown fot suoDbm6nted wilh the oxtrtcG 12 d.y3 on czap.k Dox liqllld modlum ol liv6 cttliv..s ol chictp€a (d€d cvs 6153, CM 72 and C 44; labull cv.. czapok Dor.lon6 w...160 used I ltc 2Oo End Cvp.e5.6l 6€p.r.t.lv. control. cultur€ tili6l63 {10rnl} w.t6 .lbiected to s6lld Dh.$ extr&tton. The phvtotoiic compound{el sre elutod ln 2ml .colonitrll€ lnd quanrnariv. .n.lysis wt6 donc bv HPLC udng authonlicated srandard s.mpld. 3? torln Prodoctlon by In. l.ol.r.! ol A. t.bl6l Ascochyta ftbiei isolates CAMB 2, 3, 5, 6, 7, 8, 10, l1 .nd €K wad grown 3aparar€ly ln c2ap.k Dox liquld mcdlum tupplem.nt.d with .xtt.ct ot chickped soeds cvs, 6153 ond CYpress6 lor l2 davs 33 d63cnb.d betore. Alt6r rhat period, the rlngus lree cultur6 lilti.r6s w€i€ divid6d 6nd h.lf was used lor c6ll bioaEsay 6nd th€ other hall HP[C. Cutllr€ littr.r* tor lor sol.ntpv.on€3 eslimarbn by c6ll blo.56.v w6i€ lreoze dri6d and thar lor phyiot6tin{s} analy.is w.3 p$sed through . lolid ph.e Bxt'ction lhimbro Toxicity ol lyophili26d cultu.e tallrates sgalnst isollted chickpoa c6ll. cvs.6l53 dnd Cypr*sa w'3 perlorm€d bv the 66m€ method .3 doldibed b€lore ln c.ll blo66say t6ction. Sampl* lor phvtotoxic compoundlsl 6.alY.is wer6 p.6p.ted bY solid phase 6xtracrion and proc6s5.d tor HPIC 6F Totlc lsol.don, Pudldtion .nd ld€nlificadon ol another Unknom 6bi6l ltoltt' Compound lrom Fungu. 116. Cul$.. fitraL ol A.cochvit CAMB lor ascochytD Bbi.l llolat6 caMB 10 wat grown on cztp'k oox a3 nurrienG supplom6ntod with sxtrtct ol chickp€s s6€& {cv cvpt636'l dtvs' d*crib.d abovc .tcept th.l tho incubttion tlme wd' exrendod to 16 pH Aft.r the .omoval ot lungus, ih6 cultur' lill'6r6' w're adiu't'd to 3-O 33 .nd p.rrjrion.d thr.. timB. .g.ln3l €thyl !c6r.1., Toxblrv ol .rhvl ...t 1. traciion and .qleous phrs€ was tscorded .gainst chickpe6 c€lls ol 6ithr cultiv6r {cv- 6153 or CYp.oss6). th€ combinod .thYl ac6rar€ fr.ctions w6.o dned ov.r sohydious sodiom sulf.to (N.2So4l End, 6cetato was rcmov6d by tft.t lihration, lho .thvl roi.ry 6vapo.6tor. Th. r6sidu6 w.s dilsolved in chlorolorm 6nd psrlitioned twice againsi w6ter. -rhe chlorotorm lracrion w6s 6vspor6led to dryness 6nd th6 product crYsi.liz€d lfom rethanol Samplos ol iho c.Ftals wo.6 6xamin.d bv st6ndsrd mass 3pecltomtttY 'nd nucl6.i msgnetic re3onsnco {NMFI tochnique6 Klnetica ol Phylotoxtns Ptoductlon Dudnq Nin€ isolat€s ol,4 .ra@ rt vrto Gtowth ol Vttlout l3olttot wsro grown 3€parat6lv in 250 ml EdonmY6l Ilasks containing 30 ml c26pek Dox medfum suppl6m€ntod wilh chickpt' ot cvo153. Each ltdsk was inoculated with 2OOul One fl'sk ol sporo susponsion llo6sporos/ml) .nd incubated al 2212oCmvcollal mar8 66ch lsolate was taten 6ftor 6very 24 hours lnd th6 tlngal soed €xtract {6ostn/!) drlsd 3t was sop.rated by filtration through Whahan GF/A ol.3s liber 'nd 6ooc ov6rnight io consisni procossed lhrough w€ight. Each aliquoi ol ih6 cultur€ lllt'ats {lomll w63 to qua itat! th6 minicolomn as d€scib€d b6lorc lor bioactivitv 6ssav 6nd oc'bnnr 6 contonG ot vdriou. phvtotoric cohpound{sl On€ halt ol th6 bv w.sh (1mll was $6d lor the qu.ntllic.tion ot phvtoioxic codpound3 HPLC while the * oihd h.lf llmll was €vaporaiod to r6'ldue lor cell bioassEY d$crlbed b6toro In coll bioa!36v socrion 39 The E.mples ol tho rc€lo.itrilo ebare w€r€ inj.ct€d onro HPIC consisling ot 6 doubl€ pomp. Tho .nalysis was . B.clm.n c.tri.d our on r.var.. phas6 C 18 column and lhe mobil. ph.so w6s.caronnrib: wau l5O:5O), llow rate 1.5m1/min. The phytoroxic compoundr w6rc rccoqni2od by thoir relenlion tim. snd quantllied Paihogrnlohy of Funsal by r.l€rence lo .xt€rnal sttnd.rd6 or l.olrt.t Tw.lve 3sedt or .6ch chicte..lch..t t.ieti.um Lt .tltivar 6163 tnd C44 wer6 grcwn io pois t6r 20 d.F und€r conlrol lompor6lute {25_2aoc} p.r pot ro grow lor 45 days Funoal inoctilum w.5 multipllod oh chictpea s.e& (Abm.r.l., 19371 Elght d€v old cult!.. ol and then thinn€d to 6ight each isolate w6s rskon lrom rh€ infocted .€sds .nd agitated in 3t6ral. distilled w6t6r io obtdin 5 spor6 susp6nsion (104 spores/ml). Pot3 wi6 with 12ml ol ihe rosult.nr spore susp€nsion ol each spmyod in duplicato isotate on boih culrivars sepaiatsty Th6 inoculal6d plants wsro t€pl !t 80_ th. lirsl 4d,F, ihon io noimar environm€ntsl conditions {25 28oC) fof oPlimal 90% humidity aad 22!:2oC l6mporatlre durinq rranslored growrh. The !6veriiy ot disoase was rscorded using 1 9 scals lcrcwsl' 1981) havinq rive detin6d catego.ios or s.v€rnv alt.r 12davs lsr'd6 1 repr.s.nred no visible ldion on .ny plrni lhighlv .65ist.nil; gr.d€ 3 w'3 bdsed on a l6w minulBlesions on thd stsm 6nd/or upto 5 lolidgo inlsction k.sistant); grado 5 was bas€d on 6tem l€sions commonlv 2 to 6mm long an{,/or 5 lo 25% loliage infection lmodorstelY su5coplibl€l; grtde 7 was assign.d lo 6 Eituation where l6Blons w6.e very common' largsr lhan 6mrn and/o. 25 ro 75% lolitg€ intoction l.usc€pliblel and Orade 9 showod exrensivo l€6lon3 on 6ll younq shoots End/o. le.vo. kllled lhlghlY Purific.llon ot lh. PhYtotoxin! lion Inf6cl.d Chlcl9.! Pl.nt' t66ves (2.5gml w.ro dot.ch.d lrom aniticitlY inl&tsd ts w'll as h6llthy chickp6. planr3 .nd v.cuum intiltr.tod in soml mftsml Att'r tilicr papot 24 hou.s, mothdnol soaksd toav.3 were bl6nded, filt6red ihrough fho liltlals was centdlugod to obtain a cl6tr p"s€d through supornsr.nr th.t w.! conc.ntr.t d bv lllm .vdpotatlon tnd 6 minlcolumn ol octodocvlsilvl loos) slll.6 pre condniooed wlth loml and wdshed with methanot. €luted wiih meth.nol. Alter washing wilh smlwalsr, ths phvloto)(ins wer€ descnb6d b'loi' 2ml .cetonitrlle and quanrlfied bv HPIC analwls 65 c.notlc Olv.r.ny ot Nln. lsollt€. of /4 G.nodi. t"ttl VttYing In Virulenca l'obl6 ol /4 t'Otti wtt 'rtr'ct€d eco'dinq ro mdllicailons' {w6i6ing 6t al, 19911 wlth tht lollowing ONA ol nin€ a CTAB mEthod chickpoa s66ds w€r' Stock crituros (10',6 glYcorc[ grown on 3i6tiliz6d incubtled on 6 rotafv inocutatd inlo Czapok Dor nutri'ni medium snd we'6 h!N6!tsd bv filtr'rion shakor ror 6 8 da\. ar 22A2oC- Mvc€tia maloial 12 sgml wts through muslin cloth and lvophilizod LvoPhiliz6d t morlar and 3 p63tl! 6nd ground to a fine powde. in liquid nlt'o96n with bull6t lTn6 Cl IOOmM; disp€rsed In 6oml ol prehot (6OoCl CTAA txtrdction o 2% 1.4mM; EDTA 2ornMi cTrdB 2% pH a o; B-h'rcapb'rh'nol NsCl ot 96niv swirling nucltic tcidt iust balore osett. Aitd 30 min srhaction with an w6ro isolated lrom tho re3uhing c?ll lvstte bv 'qutl rdd.d volume I6Oml) ol chrorolorm: isoamylalcohol {24:11 and phaBor w€r6 sepa.ated by centriluo6taon ar soooxg lor 30 mlnul€s. Nucl€ic rclds wer6 th6n p.ecipitated lrom the aqooous phaso with 0,6 vol of isopropanol .nd collecr6d by centiilugation (sooorphl lor 30 min. Pollel3 wor€ wr6h6d with and di$olv€d In 2.5m1 TE bulln llornM Tris HCl, thM EDTA, pH 8.0), snd treatod wirh lO unlt8 of DNAse lr€s ENAse A 70% 6thanol lBoehringe.l lo. 3 hou.6. phonol/chlorotom RNA6€ was .6mov€d bY .n .ddrtron.l .xl..cllon .tep. ONA w.3 elhanol pt.cbn.t€d, cenl lug6d and dissolved in ao apprcpdtto volumo ol TE buliet. Re.rrlcrion ol oNA, Electrophor..i. .nd Bolling: Genomlc DNA ol 6sch i6olat. w.s dig.3l.d wirh thr6e .ndonucl6a36 r33lriction €nryrus (E oRl, Irql .nd /ihtl) 3.ptt.telv according to th6 supplior's insiruction3. Diglsted sampl63 w6r€ load.d onto 1 4% aga.o6. bullot {4OrnM Ttis HCl, 2OmM sodium dcetate, lmM EDTA pH a.o) and run.t l.sv/cm lor 16'18 houre al room tomp.raturs 128 32oC)' Afler €tsining wi$ erhidium brcmid6 and Phologr.phins, th€ q€ls wors qers in TAE depurinsted, den.iut6d, n6ott.ll2€d 6nd blolled the DNA lrom th6 capitla.y trsn.ler ro the nvlon dembrano lHvbond N, g'r bY AmEhtml ov'6i9ht alr6r blotting, m.mbran66 w6r€ n!6d with sxssc bult.t lor 1 min 6r toom r€mp€rdture to romove q€l debris lnd psrticul6l6 'onreminonG that w6r6 Inroduced dunng th€ ir.nsl6r proc€ss Air dried th€ m6mbr6nos 6nd DNA w$ immobiliz€d by h66l tteatmont 12 houG al aooc)- tabeling ol Ollgonucl.otido. Oligonucl€otidos w.ro lab€l€d wlrh digoxig€nin ddUTP 6ccording to rhe m.nuldcturer's lnsi.uctions lDlG ollgonucl€otid. l€b6ling kit, 199t' Ao.hrinsd Mannheim, Gorm.ny) lor nonrrdioacriv€ tnalysis. Hybrldiz.rlon.nd lmmunologlcal der.crion Prehybridizaraon, hybridizdtion 6nd ws.hins stops wer6 p€rlormed 3s by Ali 61 6r. 11966) and schaller 6t al, {1988). In short, nembranes werc prehybidiz6d in rr6hly preparod hvbtidjz.tion bult.r descdbod I(5XSSC, blockinq rdas6nt 1% (w/vl ,Nloufovlssrco6ine O 1% O.O2% lwv)l at Hybridizdtion OSoC lor lw/v) SDS tl l6.st 1'2 hours in rollq botde ov€n' ol blots w6s portohod lor ov6rniqhl in hvbridiz.tion bulre' inctuding th6 prob6 {2opm6l./ml}- Annealing rempeiaturo {Tml ol tho (19911, oligonucl€otides werc calculat€d .ccording to Eppten and Zischlar llm=4tc+Cl+2tA+T)l Hvbridizdrion was c.died out ar rm'looc i6 to. IGATAI4 and lCAAl5; 3AoC ror IGACA)4, lcAla and {GGAT)4 tnd 4ooc ror {GTG)5. Washcd tho nFmbrtnes lwic€, 5 min p€i wash' in 2x 3OoC {zxssc SDS O.l% w/v) ) rollowed wirh O5x wash bulrer t€spectiv' {O.sxssc, sos o 1% w/v) twlc6' 5 min p€r wdsh 6t the wash solution temporaturo ol the p.obe detormlned empiric6rlY lmmunological dstedion .nd color devoloPment rosctions weto p6rform.d according to th6 procodure rocommond'd bv th6 mlnuftcr'rrer G€rmsnYt Nuclqic Acid D€locllon kit, 1991, Boohtingor Mtnnh6im' tDlG Slripplng M6mb6n€! lor n ttoblng cotor lha Prlor to rehYbrldiz.tion with OIG ddulP ltb€ll6d oligonucl'otid6s' th'm ln N N_ wa3 removod rrom rh. nvlon membran6s bv incubatinq until tho blus color dim6lhvllotmamide lDlvtFl at 50 6OoC in a glass trav precipiiat.s wor€ remov6d trom th6 membran* DMF solltion w's ch6'ged a! h.ruc 0. {..d ol d.o.b.E dlo .d ha.. o.r ttd {. ||rr|trn fio.lqf y h i,td.rnd dfrd ,.F. fft. !.!!r w.r 7.no{.d w r,!.tlrm wi,| tm{ o.2rd/t, 806 o.t* {dvl !t cToc for 30 mln. ftnid mn!tf.t!.. tr 2t€ac ltrd .fr.r dD& .br.d .t a9€ for notd lr.qt.|tdy ro hvbddr.tlon. EHf,WET EV FESULTS Ascochyta Ebiei coloni66 wore s6par6led lrom dise6sed chickp€a pfants, as describ6d in th6 mdte.ialo and methods. was €stablished by -lh. idantitv ol A. tsbiei a) morphologicsl ohdrdcterisrics as obs6rved und6r th6 microscope, bl rh6 ability ol th6 lungus to planr3 und€r sr€rilg gnvnonment! and cause blight disease in chacle66 {cl DNA lingorprintinq with svnthotac oligonucleotide prob$ complim6ntary to simple .epetitive sequences. Th6 said isolatos w€re arbirraily dosionatod.s cAMa 2, Ihe tungus w.3 grown r.padly on chickpea 3,5, 6, 7, 8, 10, 11 soeds lor th6 pioduction of multiple inoculum upto 7-10 day6 al 22ll2oc lFis.4).When 7 1o days old intectod s6€d6 w6ro lgitol6d with si6.iliusd distirr.d w.t€r, a h6.vv spore susp6ncion, d€void ol myc€lium conraminarion w65 obtained. oprlmum Condirlotu lor th. Clowrh ol A. 6bLl Sevor6t medi. and vatious oth.r €xperim6.kl conditions were employed for tho maximum grcwth o1,4. raAlit such as l€mperatur€ lighl or dark, pH 6lc. ll was found that Czapol Dor medium suppl€m6nt6d with 6rtr.ct ot €iihet clltivat lhowod v€tv good growth ol the fungus {Trble 3,. Msximum dry w6i9h ol mvcdllum i,e,11 13m9/ml 8nd a-36 mg/ml wss observsd in mediun .upplorenred with chicrpe. s66ds chickpea s6€ds 6xt6ct ol both cuhiv.F Cyprossa and 6153 respectivolv aft6r 12 dtvs of lhcubrtion period 61 22oC t6mp66r!.e Ftom th6 r€tults lTabl' 4l h is cl6a' thst lhero w.s signlticdnl oltoct ol light on the orowrh oI llngG maximum growlh 15.9omo/ml and 13.o0mg/ml obs6rved in lnd 6153 resp€crively in continuols light at the cvs 6nd Cvpt€ssa abov€ concluded a5 Table 3: Gnowttl oF r. t{rE II OlF EnErr CULIUnE IEDIA. 7.to cDM+SE {CM72} a.a6 cDM+SE 16l53t 9.83 cDM +SE (C44t 8.OO CDI$ + SE lcypressal 9.76 3.1 sE (61531 0.8 1.33 s&K 6.77 cDM+6t53> CDM+Cypr6.$ CDM+CM72 COM}C44 MF> S&l(> CDM> MF ' ' sE GSE S&K - CDM Uoditi.d F i€l m.diurn Cz.Dat Dox M.dium seed Ertr*t Gl@o* S€6d Eft&t oEdlum S.mina and Kh.n m.dlum GSE>SE a6 Table 4: Eff€ct ot lempe.rtor. Atcochyrt nbld Ory Growth condlfont , Light, Oaft.nd pH on the Crowlh of w.lght of riycellum lmgrml).tiar 12 dryr of h|c0tadon 0t53 TENPERAIURE ('C) t8 8.54 6.66 22 . t3 8.36 8.53 30 5.30 3.39 LIGHI 15.90 13.00 DARK r2_70 8.43 4.8 6_50 5.,11 5.2 8.20 7.51 5.6 10.20 9.76 8.0 4.70 6.19 8.4 7.43 5.9'l 6.8 5.83 1.79 pH temp6reiuro i.e.,22oc tlrble 41, under ditiorent pH conditions, mdximum dry werghl tlO.2O and 9.76ms/mll w.6 obs.rv€d .l pH 5.6 wnh both cullivar ol chickp€d Cypr€ss6 End 6153 r€spebtivgly (T.blo 4). From th$o siudi.s. it i3 concluded thel czapek Dor modium supplem6ntod with 6hher chickp.. s.€ds 6xtract in lighr 6t pH 5.6 lnd 22oC l.mp€r.turo suppott. good growrh ol4- /aDi€i Dilleront carbon sourcos such a3 lructose, glucose Yeasl chickp66 seeds otlract of llngls r6Bi.t.nt t(irtct, culiavtr cM72 and gxrrtct ol lungud 6usc6ptibl€ culiiv..6153 were used as suppr.m€nr in C2ap6k Dox liquid m.dium on the Induction ot 6Yniha3b ol UV tbsorbing comPounds du ng growih ol A- .rbiei M.imum UV .bsorbing compounds 6t 33onm w6r€ monnored in Czap.l Dox liquid dadiom sopplem'nled with chi'kp" tbitrv 3.ed. €xrracl of ellh€. cultiv.r' Both chictp€a cuhiv'r' diller In th6n lo synthesi2e UV suppl6m6nt€d wer€ dbGo.bing compounds Oth" carbon sourcEs iGt en€rgv sourc.s .nd havo no inducing elt6ct lor lh' synth.si. ol UV absorbing compound6 {Fig 5l AdllnY: bollt6 ot /4 r'bla CAMB 5' purltled trom di6ea3ed chickpaa plant w.s srown in clapsk Dox rEdium ol 6 rungu3 suppl.mont6d wirh an .qu6ous €xtract ol chickp66 seods Prcductld ol Phytotodc Phvtotoxic aciivity ol 12_ susc€plible cultivar, a3 dsscrjbod in th6 merhod3 bv Btudving its eff6ct on day old lungus free culturo liltralo w.5 inv$tigtled ot cell wslt ol ptniallY disostod chicl$ea c€lls/p'otopltti3; dunno Itowlh ol bl crtlua Inducrlon oi nod€3in!6rnod6; c) root Brongarion sYmptoms on chickpet se.dllngs and d) appoat6nct ol ivpictl diseasg a) rcg€nsi.tion d6tdch€d chiclPea r€Eves. tiE of incuD.tion lhyr) p.,;i$"gl:: ',;el*u:r***isiffi ffi tQ, when chickpea mesophyll cells r€tsining psriisllv dis.st6d planr c6ll w6ll w.re ftosted with .n .pptopri.l. dilutaon ol rungal lree culturo liltrat€. it completelv inhibited c€ll wall r€g6n.r.tion and th6 tre6tsd colls .Ppear6d as red d65d bodies when vi.wed altet staining wirh fluor*cein diacelato under . ph.se contrasl microscope {Fiq 6) Under th€ Oiven 6xp€rim.nt,l conditions, non tr66ted cells compterelY svnlhesiz€d tho pa.tiallv digostod . m.ss ol stowing ctlls which appo.rod .s yellowish gr.en bodiss (Fig. 6) Filtv colls from 63ch w6ll wore examin.d cell wall ro regenerato into 5nd th6 nudber ol stdinod l.live) lnd unstainod ldeadl c6ll3 were conv€rr6d to 6 corect€d p6rcent c€tt dealh using totmul. ol shoh€r 6nd sttanqe (19891. Cor.cted po.c6ntage call d€drh= Td Cd x 10O/Cr' wherB Td is ih€ estimdted mean number ol detd cells in last well3 Cd is rhe esrimatod maan number ot dead colls in conlrol wells and Clis tho oi live colls in control wells Po'ceolage coll d€trh w.s conv6,l6d lo probit vslu€s {Table 5l and plolted sgainst loglo oI th€ €stimated hean numbor dilution lactot to obtain tho tOsO Unils ot loxicitv por ml wore c6lculat6d by mutriplying fte dilulion lscror coit€sponding to th€ lDsO bv 20, bocause rhe volumo ol culture lihmro in th6 wells w.s soul Th. 'ssay ttom Figot' sensitive, r€liabte and reproduciblo As is clear is 'xtemolv 6' lh6 d6td 6nd bodi66 can b6 qusntitativolv distinguishod from th6 regsnerating c€lls thr€lore the msthod .6n bo usod qu6ntitativelv M6an c6ll viabililv tit6r period isol.tion wE 9Oa5% which d€clin.d noglisiblY over tho tsstY ol rhe fungus fr€o cultu.e lillrat6 wdl iitrh6r ils iohibilion Bllecls on rhe induction ol callG troh nature 50 Fig. 6, bioass€y tor ths Pros€nce or Tabl6 5: Tnmlom.tion of Porc.ntcgos to Probtb3 0123456789 2.67 2.95 3.12 3.25 3.36 3.45 3.52 3.5S 0 3.77 3.82 3 87 3.92 3.66 405 4.O8 4.12 4.33 4.36 4.39 4.42 4.45 4.61 4.U 4.47 4.69 4.72 3.36 4.O1 10 3.72 20 4.16 4.19 4.23 4.26 4.29 30 4 48 4 50 4.53 4.56 4.59 40 4.75 4.77 480 4A2 4A5 4.87 4.90 4.92 4.95 4.97 50 5.00 5 03 5.05 5.08 5.10 5.23 80 5.25 5.28 5.31 5 33 5.36 5.39 5 41 5 70 5.52 5.55 5.58 5.61 5.64 s.67 5.7't 574 577 5.81 80 5.84 5.88 5.92 5.97 5.99 6.04 608 6.13 6.18 6.23 90 6.28 6.U 6.41 6.48 6.55 64 6 75 6.88 7.05 7 33 5.13 6 5.15 5.18 5.20 44 5.47 5.50 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 99 7.33 7.37 7 41 7 46 7.51 7.58 0.9 7.65 7.75 7.88 7.m ol 2 wook old chickPes s€€dting, or induction ol dis6as6 lik€ symproms on dot6ch€d chiclpe. l6.v*. R€sults in Figuro 7 d6monst.t.d th.t pr.B€nc€ ol phvtotorlc &livltY l..dt ro 'tiol'tlon ol nodos and intsrnodes. wher66s, r65ult3 in Figure 8 3how6d lhat lungal nodal/intetnodal portions cuhur€ lilrr.te induces th€ .ppoar.nc. of diso!.€ like tvmptoms in d'i6chod The phytororic nalur. ol lungus fr" coltur' Iiliraio was turth€t its abilitv to inhibit rool elongation in E dos€ d€psnd6nt papor mobton'd wirh mdnn6. when chickpea 3.od. w€r. gtown on s lilr'r to lungus lre6 cuhure tilttalo lFig 9)- This olf6r3 tnothot qutnrnariv' t'36v established by of th6 iungal culture filtrate On rhg of nodes/internodes (Fig 7) and 'ppeaiog ol dise$€ monitor th6 phylototic activitv contra.y, etiolslion symploms on det.ched loev.. (Fig St c5n nol b' procitelY qu'nritl6d' howev€r, the 6lJ€cl was visudllv do!6 dep€od6nr' Kin€rl.! of Sectetlon ol Phvloloric Aclivltv gnwlh m€dlum wag Secrsion of phytotoxic activily inlo tho lungtl 4th dsv during a 16 daY ss6.y.d with ,4. rrbiei isollt. CAMB 5 ater 'verv rh6 dtta ot th€ ongoing incubation period lFig- lol tt appears ltom of phvtororic tctivitv i3 ind€pend6nl ot the w63 conlinuouslv tungal mycotisl gtow$, Growlh ot lungtl nvc6lid oxperiment that p.oduclion increasing lhroughout th€ 16 davs p€riod, whor6* secretion ol phvtotoxic €nd continuod to lhs activity started llom about the 4th dtv ol incubdiion tesult€d in ihe lo$ ol 12rh d.Y Growlh ol $o lungG boYond !2 ddvs' rhat tecretion ot blo'cllvitY bioaciivity. These lesulrs lerr 10 th€ suggestion gtowth phtse whor'e' pH into $6 medium mav b€ a tunction ol a cenain 5l E tecr ol lungus fte€ curilre nodos and inte.nodes. tartr6te on calluE formalion lrom 54 Fiq. 8. culturo lihrato on dorach€d chickp€a is wiOinr Uv tvoical diseato svmptomc {risnr_re'tm€m rcoiei ena bft curtu'6 medrum a3 iJtruie li't,atd oi Errecr -r.i".l "ii''r, ot lunqus lr€s a C.l- drad. O.r. .!tdr a g ta C.t. r1ll O.a. .th.l t ot t..at. i... cllroE 'l|"'l' ('rt F's. 9. ffi ffi1.l''#iffi''tud'ti' 'dffi ta s B i 12 I t0 €0 t 60 t l0 t a12 tl-ol d--{ I tH di!n!'|hcDtbo - roidn.ctlvcr '" rc, 00r 'ri-'!Y !| ar'*"r'€{hl dt&d d; ol iunoj ntddn t .. erle rtr cl'&rrc Fh. n dun eflhnlrid oI cultur€ lilrr.i. .onrlnloGly incre$6d but gradually upto rhe 18th d.y ot Rmolurion ol Phyloloric Ac(Mtl.3 Thln Layor Chromrtographic An.lwlr: Whsn lhe organic odr.ct of lungus tr6. culru.. lihraro was rosolved by rhin layer chromrrogrsphy dnd the chromatogram was dev.lopgd in a solv.nt 3y8tem contdining h€x!n6r €thyl 5c6t.re and acotic acid {50:50:0.1%), two UV ab6oding compoun& with Fr vatuss 0.20 and 0.29 wer. s€p.r.r6d. The two spots, bivrrily desiOnated .s compounds I and ll r*p€cliv€ly, were separ.t6ly f.movod and dissolvod in moih8nol for specl.ophotomotric anrly6i5 ol rh6 isolated componenrs. In addirion to the two compoundE, 6norh.r componont with e lifil€ or low abiliry in the existing solv€nr sylt6m wss detect6d loc.ti2od 6t lhe origin. Thb compoo.nt w.s r6chrom.to0r.phed in 5 !.cond sotvcnt sysrem containing hora.o: .thyl rcet.t.: m.tha.ol: acotic .cid {50:45:4:l). In the s€cond rolvenr system, compound lll hsd an It vdlu€ ol O. I 4. All rhree compononls wsre s€parately invosiigar.d lor UV absorption spectrum and biotoxicity studi6s. Compound I Egainst chickp66 w6 lhowing thre€ times mo,. toxlchy c.lls than compound ll. Sp.cbophotomtlc An.lFi.: All thre. componants whach wc,. loc.liz€d on lhin silica 96l pl.les gave a dislinctly diff€r€nl UV sp6ct.um lFig. 1l) suggesting lheir eoporale idsntity as fung8l mBtabolites- Cdum Chrom.logrEphy: Before rpplyinq th6 mixturo ol 6olv6nls on coluhn chrom.logr.phy, it was usod tor lho soparation ol toric conpound {s) Irom €rhyl acotat€ traction on TLC plst€s. lt was toond rhrt hox.n6: €thyl acerare: acetic acid 150:50: O.l%l gavo good Goparalion of spots F!, l l. w sp.€!! ot ttoht { thtEiotlc c'mtlrndt tl'll e nl' wh6n TtC pldt6 was vi.w€d und.r UV lighl- Fr6ctions (2oml) we.6 coll6cled lrom th6 column wiih th6 .bove dentionod solv€nt. Pur. compound I was r€cordod in fr.crion3 4.7 6nd the socond in rractions 12 17- Mixtut€ ol lhe two compoonds wEs louod in irdction 8-11.F.ction4- 7 and 12-l7 on TLC plal6 were giv.n lh€ 0.23 6nd 0.14 nf values. Whon lr€ctions .lut.d trom th. column w..6 run on HPIC in a solv.nt system, .c6tonnrile: waid l!:1), as mobilo ph.se, both compoond! had dirrerent tetention rim€. Th6 thre6 dimensional chtomo6can on HPLC revealed lhal each compound h6d a UV spoctrum (Fig. 12). TLC, HPLC 6nd spectrophotomet.ic studiss rev6rled rh6t rhose compounds wer6 id.nticrl wirh solsnsphyron€s A dnd C {FiS. 13, which wer6 pr.vio!.ly repo(6d by Alam et dl., {19891, using kdbuli chickps! cullivar ltc A. rabiel. fhese id.nrili.d as phytotoxins compound3 have 6lr66dy b6en sssocidt€d wirh earry brighl 200 in vltro gowrh ol ol potdto€6 .nd romaras c6u56d by /t.rrrrt so/ari (lchiha,a et a1.,19831. Purity ol lh6 cohpounds was conftmod by superimposang the sp€ctra oI the chromatogram Iroh th€ up slope, rhe 6pox .nd rh€ down slope, (Fi$, 14 and r5). (.uss.sred rh.r th€ compou.d .nd ll w6r6 98% poro. Recently Hamid 6nd Strdns. ll99&) rcpo od !n 1 easy and speedy moihod lor isolating the 6olan.pyro.e loxins lrom th6 .rbir by using dichloromelhrne, cyclohoxsn6 and .thyl .cetale insr€sd ol dcetonar.ilo: warer {l:ll- The etur& w€r€ culture lillrE.€s ol ,4icocryld collected 6s 25ml f.actions and quantiligd according to rh€n UV spect.a End extinclion coollicients. Ih. lechnique wa3 Ep€6dy (10-13 Becond lor 6.ch fracrioh) and yielded pur6 prepardtions HPIC .nd $6n UV sp.crra. ol th6 compounds rF d6r6hin€d by 6o mlnruN dou ?oarh|N ttg. tt. 61rroi$.!i al.bn?tona te llal.lafl drL t A l! ad F.csn C 00 Soln.pyronc A Sdalpyrcr F S. 13. C Ch3tridl .t uctur. ot sobnrpyrond A and C. !. lrd AO12, C5{ \i 11. F{rslRrlrr r:i i sn.2,5r -.3: ir!.1 -.xe: - lrec -\-:: \ RC.15, Conffriiatlon ot ilt! DuttY ol ih€ loL gvrono C by lh€ s'4.rlm9oilng dlo |p*tum ol tn. ch|wlEro0'3m' \i'':. Producllon ot Solrhapyron.s tn Fiv. Cultlv.r. of Chtctp.. S€€d extract of chickpea cutriv.r3, 6153, CM72, C44. ILC2OO and Cypr€.s. werc u3.d s.paraiely .s . inducibte supptemenr liquid fredium to quantify tevot ot thr in Czrp€k Dor sotanapyrones producrjon. tr was rh.t 6qu.ous extracr ot runsur susc.pribte cuhiv,.6tS3 induc.d th..6 lold hlOh.. .ynth.!t! ot phyroroxtc compound3 wh6n comp.i.d wlth discovered the inducibl€ €llocts of similar tov€t5 oI Equ€ous gxtract ot tungus resistanr cultivar C44 lTable 61. No sohn.pyrono w.6 darecred in Czrp6t Dox tiquid m.dium wh6n chickp.r 666ds extrcr w6s otiminared trom th6 mdium. tt is concluded l.om th€6. srudies rhai djlt€r€nl chiclpea abilary culivrr. diffsr in rh.ir to induco soldnapyron6s p.odlclion and specia y lungus .U3cepribte chickpea s66d3 .ontain qu6ntitativ6ty hi9h6r tov6ts of unknown €ndogohous r,ctors responsiblo for rh. indlction ot phyroroxic cohpounds. Growrh, pH, Toxlc Acdrtry and Sotln.pyro.$ Conc6ntratton ot Fungos AscochvtE rcbiei gr.w tapidty on Cz.pet Oox m€dium supptBm.ni6d wirh chickpga se6d oxiracl as compa.6 to Czlpet Oox tiquid m€dium atonE, Th6 medium 6nd culrur6 liltrares wore oot toxic when chickp6. .{rrct was omitted f.om th. modaum llrbte 7). pH ot rho cutture tihr6r6 3uppt6m.nr.d with chickpe! sood €xtrsct cv, Cyprasla was decreasod atr6r 8 days incubation wh€roas in case ol cv. 6153 ir decr€ae€d rttor 12 davs (rrbt. 71. Ioxicily ot the culture fittrsr6s containgd chickper €xrrscr ot cv. Cypr€ssa and 6153 d€creas6d att.r 12 aod t6 day3 r€sp.ctivoty when ch6cked again5i colls ot eith€. cuhivrr5 lTlbto 7). Maximum sot.n.pyrones produclion w.6 r.cordod.Irer t2 days.nd rh6n gradoa y decr6a6.d rtt€r T.ble 6: PR{tDUcnOx or €OtAllApyEOtlE8 tt{ F|VE CULTNAna OF cHtc(pEA. Sol.n Drom. lhM) N.D N,D cDM+6153 o.647 0.104 CDM+CM 72 o.062 o.273 cDM C44 o,064 o.163 ttc 200 0.085 0.346 0.344 o.tt8 + cDI\4 + CDlt Crapok Dox M.dlum t.bt.7: Chbrpea pH, Grdth, Toic Fun90. Fr.. Cult!re lnc|lbatl pH Ac(wltv Flltr.i.!. tnd sot.nrpvronc! concentrttion ol Dry welght loxicl|y (unlt! / mr c.F) lil) po.lod (lngrnl (daF) B+C 6t53 c.F) CDM alone cDM +6153 CDM + CDM N.D. C F 5.60 0.00 0.00 0.00 0.00 0.00 6.58 1.27 0.00 0.00 N.D. N.D. I 6.69 2.08 0.00 0.00 N.D. N.D. 12 7.U 2.53 7.50 4.60 N.D. N.D. 16 6.30 2.68 0-00 0.00 N.D. N.D, 0 5.60 0.00 0.00 0.00 0.00 1 8.30 8.00 0.00 't3.92 0.00 0.00 0.00 8 8.86 8.90 34.27 11.71 0.046 0.064 4.32 13.00 181.00 107.63 o.444 o.120 16 7.90 13.80 131.23 .57 o.229 0.091 0 5.60 0.00 0.00 0.00 0.00 0.00 8.00 7.00 5.46 0.00 0.044 0.00 I 9.26 10.28 20.60 46.85 0.078 0.046 12 7.44 15.90 74.79 187.40 0.304 o.112 16 7.50 16.50 59.70 r53.02 0.133 0-0zl 0 Czapek Dox Medium. Not Delectod. Cullufo Flllrale. th.r p.riod. When Czapck Dox liqold fr.dium w.r suppl€m.nt6d wrth gluco36, lructose, yeasl exlracl, yeast nilrogon base, casamino acjd in dddltlon ro chickpea se6ds oxrrsct p.odu.non ol ol culriv.rs C44 and 6153 lor solanapyron6e, m.xinum toxins wero d6r6ct6d suppl€montod wnh chickpca 36.d3 errGcr the in modiuo ol either cultivar, wherdas all othor supploments seNed as engrgy sourc66 lFig. 161. Ono crit.rion of the importance ol loxins in the das6as6 syndfome c.used by a pathogen is that roigoniciry shoold b6 r€ldtod to pdrhog€nicity or virul6nc6, In the initial invostigstion3, th6 Eolsnspyrones A dnd C wore obrained lrom only one isoreto ol A. nbiei- ln ord6r to 96in lu hor knowledg. ol th€ pot6nrial importanc€ ol solanapyrones in th. blishr di6€a.e c6o6.d bV ,s.rdryta rrrlei nin€ isol6ro8 wB.e used lor totlcaty, solan6pyron€s prodoctaon and palhogo.icity or vnul.nce, Groeth or lm lsolar6 ot A. 6bl.l In Cultur. and Ioriclty ol Cultlr. lvlost isolates ot,4- /aOtei€xc€pr CAlVlE 7 and CAMB batw66n l0 and l4ms/ml dry weight ol mycelaum 6116r 1l accumutat€d incub.rion for 12 d6y€ on C2ap6k Oox nurrignt. supplom.nt€d with .qu6ous €xkrct or chickp.a s66ds ol either cv. Cypr66sa or cv.0153 lTrble 8). CAliS 7 .nd l0 s'own on cv. 6153. Tho ol CAMB 7 and CAMB lt . when srown io m.dium CAMB 11 p.oduced less mycelium 6s did CAMB weighl ot mycerium contElnlng 6xtracr ol cultivars Cypr€8s..nd 0153 were & 6.53i1 o.llms/ml dnd 7.3o ! O.38ms/ml respectively. Th6 high€st vdlue i.e., 15.91 mq/ml w!6 grown on cv. Cyprossa (Tdble 8). 9.16l l-22m9lml & 5.44_l o.3omg/ml w$ obtsin.d wh6n €( 5a sol.c (ug/mg dry wt,) 2,6 2 I CDEFA Dllt€rcnl onErgy aoutres Fb.l€. Eft cr ot th. lddltlon ol dltLrairt efid (Bl. Pn roy soutd on ioxln Oox rdluh tA, wrt .utpLm !d Yvlti glucos 216 lnFtore 2t6 {Cl. c.dnolcd 296, (O) v...t qt!.t o.b96,tEl ntuoo.n 8.s 0.5* lR cBclo.6 ...d d|ncr ol ca/., (Gt, chrcto.ri @d .xr..d oa 4153 lHl sol.n pFone lonennrtion luo/mo dry e',.lstrt ot dD tuncutl a ..rimr.d in 12 &y old lungus tlo€ cunlr. rBlie- ORY WEIOHT OF trII{E ISOLATEA OE ASCOCH||A RAdE GROwl{ Ot{ CZAP€|( OOX t{UTitEt{TS SUpptElEt{tED w|rfi SECD EXTNACIS OF CHICI(PI4 CUINVARS C\?IEAAA AXO 6t63. CAME 6153 lO.9OlO 37 lO.4OlO o5 6153 12.17 12.77 or53 10 6t 53 10 CAMA CAMB CAMA tl 4OlO 50 8610 a1 6153 12 96aO.64 12 o8to.27 to 6r 53 tl 20.i0 9 16!1.22 7 3O.!O.34 ot53 CAMB 2O.iO 30 6t53 6K CAMB lO a3 lO 44 6t53 15 91lO.4t 9910,33 to 74tO.26 9 46a0. | 3 5310.1I 4410.30 Th. r6cord.d roxiciry ol lh. cultora liltr.t.6 ol all isol.r€s v{i.d according ol lh6 lo the cutiv6r used in ths modium in the bjo6$dy and rhe isolaro lonsus- Th€ colls ol cv.6153 w€re s6n6r6lly mor6 s6nsniv€ rhan those or Cypress. ond the hiohosr Elso cont.ined extrdct tit€rs w.re recoid.d whon th. m€dium oI cv.6l53 (Fig.l7l, The lowesr titters w6r6 obtained when the medium cont6in6d the extacr w€rs 6ss6yed with c6lr3 ot cv. ol cv.6153 Cypr6$.lFis. and rh6 culturo liltrates 17). By lar lh€ qr€at€st vsriarion in th6 toxicity re6ord6d wa. caused by the dille,ences in th€ isolates. Culture liltralos of CArvlB 6, 7 ,nd gene.ally the least toxic I wilh <10O onits ol activily/ml in all combindtions ol m.dium and 6asay cultiv6., ri3 onry werc louf .tcoplion b6ins CAMB 6 grown on cv. Cypr6ss. and 16st6d whh cells lrom th6 oamo cultivar lFlg. l7l. OJ rhe isolar€s Bhown in Fis. 17, 6K h6d rhe hish€st,vsras. dctiviry on media containinq th6 €xtract ol 6ithor culrivar and assay€d with cells of gilhsr cullivdr. Th€ aclivily ol cullu.o lilkat€! ot other i6ol6r€s wds more vdri.ble. For exampl6, CAlvlB 5, whan g.own on medium cont8ining extract lrom €ith6r cultiv{ had l€ss thrn 50 units ol aclivit/ml wh€n 63say€d with c6lls ol cv. Cyprossa. In contrr.r, wh6n as66yod wirh .6lk ol cv.6153, culturo filtates grown on media conroining ortracts |rom cv. Cypr.ssa.nd cv.8153 had 111.4 a 24.3 6nd 238.9 rBsp€clivery lFig. 17). The toxin litl€rs a 37.4 unil. ot 6ctivity/ml, of cullore lillral6 of CAMB 10 toachod at a maximum aiter 16 day3 jncub.tion (Fiq. 1al rdth€r th6n 12 days. Thay 6lso .xc..dod lhe titt€rs ol rhe other isolates by mor6 than .n ord6r ot m.sniludo lFig. l9l, mor€ ,4, Eb@ljsolare caMB 5 {Fig. 20). lh.n 30OO units/mlwhsn compsr€d with 17 o I - 17, Fio. .-q@@ Tha toricitv ot 12 dav old culture tihrales ot e{ht isolatss ol Ascochyts ttbia as assaved bv c€ll vbbililv using-llL'oresc€in dracetato as s vll.l stain lsl rsobtes grown on uzao€K uox nutrienrs suool€m€nred whh €ttract ol chickpea, cv cvpt*sa and rested lh cotls t,om cv cvpressa lbl lsolates grown o' az;oet Dox nlt enrs supplemsnr€d with ext.act of chickp€s, cv. bvoG$a and tosl6d with colls iiom cv 6153, 16l lsolat€s orowd on czaook oox nulrients suppleme.t€d with oxvad ot ;h'cko€a, cv. 61 53 and lestod with c6lls iion cv cvpr'ss6, {dl l3ola6s srown on Czap€k Oox nltrients suppl€m€nr6d with 6rmcr oi rhickoea cv. 6153 a.d teslsd wnh c€lls lrom cv sD' 6153. Dara are ior throe rsplicates i coo Toxlclly (Unll.hl) too aoo 200 too 12 10 lncubatlon perlod (Deys) Fig. 18. ot flngus tr€e isolaie CAMa 10 againsr Toxicity E Flg. 19. of l8.dsv-old culturs llltrttot of bolgt€ CAMa 10 ol- Aacociw '€blsl .. a..a\el bv G3ll vlab-llitv u{ng iii,oieiiiin itticctate ar i vlt6l .tiln, lr) Grcwn on czal€k Dox Tha loxlcltv nuvlgnt' srrool€m.nt€d wlth axiGct of chlckp!!, cv cylresss and t6st€d \ivlth c.l!! from cv. Cyprusaa' {bl ctown on czape( cv' Dor nutd€nt slJppl€mlnrad wllh .xtact -of chictp€a, on Grown 6163 f.om cv. {cl wlth c€llt cvorer!! lod t€liod ol chlckpea' wlth ci5p6-t'oo, nutacms euppl.m€n!.d lfJract ;.'6t54 rnd t€tt d wlih c.ll. ftom cv. 6153. Dat srs tor F/9,20, Conparlson of iwo lsolat€3 ol A. hblel rot the producrion of solangpyron€ and toxicily, 75 Sol.n.pyron. Cont.ni ot Cutrlr. Fttrur.r ol r, arn The solansplron€ contont ot th€ culture tittrdt. v6ri€d according to the currure medi6 but more panicut.rty eccording lo the isotatc oa rhe tunous, Tho variarion in sotanrpyron$ concentralion ot th€ culrora titr,lio3 trom nln6 isolor€s o1,4. .r6lbl is Bhown in Fig. 21. Nono oI rhe sot.napyrono w6s d€rectod in cuttur€ fitrratgs of CAMB tO atthough they w€r6 tho most roxac {.omp6r6 Fios. t7 & l9l_ Turning ro the orher ilotares,6K prodoc.d th€ high.6t conc6nr.aton. ot sotan6pyron$, wirh v.t!€s ot 3a4 solanapyrone A snd 118 I i tot uM 33 uM sot.nlpyrone C when grown on Cz.p6k Dox mediom conrrining oxtact ot cv. Cypressa. Wh.n rhis isotato was grown on medrum conrrining €xtacr ot cv. 6153, v.tu.. tor sot.n.py.on.. A and C w€r€ 547 jt 342 rnd 104 ! 53 utvt, respoctivety. Simit., Emount, ot soldnspyrone C {tO8 a 56 and |O a SS oM) wera pioduc€d by CAMB 2 on borh media, bur rh. .Dounrs ot sotan.pyrono A were ros5 rh.n rho3. obrainod liom 6K (301 153 6nd 223 uM on m6dia conrEining -t !112 exlract ot cv,6153 snd Cypr6ssa, ,.epectav6tyl, When Orown on $6 msdrum containing 6xtracr ot cv. 6153, CAMB 6, 7, Lnd tO did.ot produco derecrabl€ conc.nvations grown on m€diom containtng c ot lh. cohpounds. In conrr.sr, wh6n exrract ot cv. cypre$., sotanapwonos A .od w6ro detecied in cutlure tittmtes ol CAMB 7, the sam6 modium, solanapyrone C w.! but hor tO. Simitarty, on dstscled in cutrurc ti[6ts6 ot CAMB 6 and 7 but not CAMB 3 and 8. Oth.r dmounls ol the solan6pyron6s lFig. 21). I isorares producod inr.,m.di.t€ tr r-- I A,..bl.l l&1.16 Fig. 21 Solanapyrons A and solanapyrono B+C concentration ol 12. dav.old culture fihrares ol nin€ isolatss of /4scrcrvra .aO'e, {a} Soianapyron€ conc€ntrarions (ulvll ol isolales grown on Czaoek Oox nutients suppl€menred wilh exract ot chickpsa, cv. Cypressa, (b) Solanapyrone C concenlration luMl or i3ol6res orown on CzaDet( Oox nutienrs suDoleFenred wirh a -ol cypr6ss6 lc) solanapyrone a concsnvarions of isorates orown on czao€k Dor nutisnrs suoolemsnr€d with extlacr ol ch'ctDea, cv. 6t53 (d) Solampyron6 C conc€nvatioB ol isolales groon on Czapek Dox nuu'€nls suoDlgmented wnh oxllsct ol chickpea, cv. 6153, Oala a.o for thr6e ,oDlicatoslso, extract chrckpea, cv. Th. n hdon ot Sol...pyrom conr. ol C\aroE Fltn.t.. ro th.lr iorlctty In ord6r to ascortain th6 contribution ol rhe sotsna!'yrones to th6 roxicity ol culture liltr6tes of CAMa 2, 3, 5, 6, 7, 8, 11, .nd 6(, toicity w.3 plolt.d 48insl sol.napyrong concontration. The coii.l.tlon coollcl€nts vari6d accoding io rh€ cultiva, us6d in ih6 ftdium 6nd in rhe e.sry, Th. highesl co6llicient k=0.8591 wss obtained wh€n th6 cutturo titoat€ coniaining exbact ol cv.6153 was $6.ysd wirh c6lls nom Cypr6..! and tho low*r lr= o.3a3l Cypro5sa wd8 assay6d coolficienk when rh6 when rh6 cuftor! lirtrat. conraining €xrrrct of cv. with c€lls iiom 6!53. V.lud lor the srh. corfotauon culrivsr w!3 irs€d both tor ortrlct aod .E6ry cell., wera O.6lO 6nd O.635 tor cvs.6153 .nd cyp.6!sa resp.cttv6tv. Th. ld. mc!tun ol Cytoch.l!.ln D In Cuiturc Fihrlt.r ot CAirB Ihe n66dleliko c.ysr6b ol th6 36mpte oxhibtr€d .tO .n M+ .t h/z 507.2020 lcdlculated lor cao H37NOB=507.2€21). Promhont trasment ion3 ar m/z 43, 91 and 120 suggesrod tho pr€3once ol . ph6nytatanine .osidue and an ac€t6t€, Th$6 dalE, toggth€r with rh6 proron nucloar magnetic r€3onanc6 (NMB) sp.ctr6 lFis. 221, indjcst€d th6t lhe compound Fudhsr svuctural confirhation was soughr ftom rhs l3C. NMR spsctrum run 6t 125.8 MHz in COC|3. The carbon €hifis clo6Ely prrall€lod th...solts r6pon€d lor cytochllllin B {Gral et 61., 1974i L.ber & T.m, 1974; Ved€rds el al-, 1974) run in Ds-pyridioo, with somo 6li9ht dilt.runcgs in shitrs c6used by the dlll6r€nc€ in 6olv6nt lTrbl6 9). Obrortlonl@s €oh.nc€m.nr by polErization r.!n.ler {DEPTI expo.imenrs w.r. G.d ro simplily th6 speclral assignments (Flg. 23) in conjunction with 19 t25.a MH2 t3c irMF oE cYlocHALAa D { cocl3 ch.n{c€l Slift 3 174.1 53.7 5 49.4 32.6 l 6 7 a 9 lo ll 12 lol 147.5 69.8 46.9 63.4 45.1 13.0 t 14-5 130,6 37,7 42.3 t1 la 19 210.3 77.1 '127,6 20 21 22 77 -O 25 19.4 24.1 169.7 20.8 2'.6' t29.r 24 3"5' 137.r 128.9 127,1 .carbon ltoFs !r€ numbor.d .ccording lo Gr.l €t.1. ll974l; L.bet lnd Trmm t19741 dnd v.d6t.6.l .l ll974l proton/c6,bon cotreldtion spectG lFig.24). The rglariv€ stereochgmistry ol tho dol€cul€ was conftm€d by oxaminadon ot the prolon/proton coupling constants (Fig. 251 and by analysis ol iho nocloaf overhauser efloct (NoE) dilldgnce speclra lTable 1Ol. T.k.n togelher. lhe dab lrom both tho mass sp.crromolor .nd NMn .xDeriments, clearly delin6d rh. toxin r. cyroch.lasin D lFig- 26, 27 .n.t 2al. Th. Toxicity ol Cylochala.in D to Chlclp.. The LOSO concenrr6rlond lor cytochal6sin D in rhe cell assay lor cvs, Cypr.rlr rnd 6153 wsr6 64.a6!7,1ug/mr rnd 49,07!4,1gug/hl re6p3ctavsly. Wh€n cuttings ol cv. 6153 w€i€ plsced in solutions o, cYrochal.sin O in holdins bufl6r lloouo/mll, th6y b6cam epinrsdc and th6 periolE desiccared, coost,icred.nd btolo lFig. 29t. Accumul.lld ot Mycaf,.l M.!. .nd Fong.l Toriciay Nins tung.l isolates were grown at pH 5.6 in 30 ml Czapek Dox ldttB m6dium 3!ppl6m6nr€d with rqueoG 6rtrct ol chictp66 s.6ds of a fungo. susc6ptibl6 cuhivar 6153 and th6 mycelisl m6so wa6 6x6min€d 24 hours- Figu.e 30 pr63ont3 ch.ng8 in mycoliat dry w€ighl dudng day growrh peiiod. All isolalos oxhibil€d w.lghl lo th. , ne{ . 25 grrdual increase in myc6li.l dry 15th d.y of Incubrtlon. Thi. docroas6 during the !lt6.6v6ry w$ tollow.d try tiv€ d6ys, ltEbilizing atte. day 20. . 20,4096 ln CAMB 2, CAMB 7 6nd CAMB lO, how€v6r, tho obseryod deqease in myc6lial w€ight conlinued €ven beyond lhs 25 d6y incublrion period, The obs.iv6d decre$6 in the mycelium weight 6lt€r opllh.l qrowrh may be .ttribut.d to I{UCIEAN OVEBHAUSEF EFfECT TNOEI CIIHANCEME'{TS OF CYIOCHALASII{ D. 1l CH3 48,5%, 58-6%, 3_'1 1%, 138+ 10%,2/6 1% 23&CH3 lsHar 3%, 15H.q-3%, 168-5%, 7__1% 16&4%, 7,-1, 19 1l%, 2e2% 1sHeq 15H.x-19%, 16&2%, 7 -0%, 14H-A%, 21-'2% 4AH 5B-4%, 8B-2%, 10 2%, 7 --3%, 2 16''1%' 20'2%, 22c-cH3 21_-13% 258'CH3 ac-l 7H 19H l6&3%,23-1%,25-l% 21_H 20-9%,48'a% 13H 2O-4%, 20H 21 -11%, l3-3% 7 -8%, 156x'4% GrEl C.rbon 6ioms ere numb€tod .cco-tdin! to Lebel and fsm I19741' gaor rr i,'."1.|j:l*Hff 'J::i'."""*":i"i"Jvil; llt'il'1""133"""i?li'i"?iSlofftittinos ttra' equarorra (ear p'otone ftfltg! IrEltttcal RXIRX dltr frfl HlFlS.CSl o!. lrul 0.1200 0.ocn 0.0/O0 hvelg|gul Flo.2c. W .9ac$'n of cyto'ltabdn o' (ni 85 85 lOSIRlx lll,lta floo A-l.c8L FlP2OOS.Coo ab€: AU llD: soc r.o2oo r.0150 r.0100 r.0050 ).0000 ST, 03:52 9t,03:52 I is-3. soo ).5000 1 4o0o . 1. .2000 .1000 .oooo 0 Yalr?t .^^ "i:UBUE iu6:66i 27. . Contlflnatloo ol dre g|lrhv ot tla Gtochdldn lup..impoattg lho ap.crun ot d|. ct'odLio$dn. Dby$.' il"\ .H ./ \ o olc ovt/ Ctaochahd! D FiC. 28. Chmlcd .tuctur€ of cytoclbbln 0. 3a Fig 29, Toxcitv or cytochaasin D to chickpea cutting Epinastv ot a chickpea cltinq in holding bulfer containing cvtochaasin D l-old'.g buffet alone. tlooua/mr comoared Nore d;siccaron. consr(lon.nd brearaoe ol pel'oles 16 ^14 , >6 z, Ilouballon Perlod (Day6) Fls. 30. Cnanges in lungEl mycellal dry CAIVB2H CAMB 60F--o 6K IH CAMB ffilqht du ng tn 3 A--{ CAIVB 5 3iilBi,H3i$3t, rohgrl d6generalion and d€rrh. I have prevaoosly ob66rvod rh6t accumutation ot mycetial malE dopends on th6 chickpes cultivsr us6d 6s a slppt€menr ro the Czrp6k Dox nltrionl medium- The prgs€nt ddl6 d6monsrr.te thar m.ximum myc€tisl ma3s accumurares dlrang rh6 insr t5 d6y iocubation period. Howev.r, th.ro is 25-40% dillerencB in rho m6ximom mycotidt msss .ccumutar.d trom 4th lo 25th day oI incubstion by ditto.ont tunglt isotates grown und€, When loxicity ol lonqu! 116o cultur6 rittdr€ p€r lnit wotshr ot th6 lungal mycolial mass wrs monitor6d as o tuncrion of incubation rim€, it w6E obsorv6d th6t the maximum amouflt ot bioacravity v.ri€d signatacsnrty trom on. l3olar. ro the oth6r tFig. 3l). Maximlm amounr ot bioactiviry w.s obsdtuod in CAMB lO a1 496 uni6/mt att6r t4day incubstion. CAMB E o,(hibit6d maximum sctivitv at 239 unit6/mt ,trer isol.l€B oxhibited md6r6t.lovol. ot msxim6 15 12 day ancubation. Orh€. .r 30 75 units/mt b.rw.6n t2- dry incubation excopt isolates CAilS 7 .nd CAMA A which show€d tirl|6 or no roxic activity throughout rhe 25 dsy inclbarion p€riod. It wa€ interesting to noto that subG€quent incubation ot th€ fungug cullure l..d to 6 Srddual lo$ ol tho bioactivity. Thrs virruatty no 6.tivity w.s o&€rv.d rllei 2l day incubation in .tt the i5otates except CAMB 10 ln which the cont6nr ol phytotoric sctivity srabitized ar 45% ot rh6 toral aclivily. These data cl..rly show rhll all phytoloiac compounds und.rgo indctivalion durang prolong€d incubsrion 6nd the obs€rued inactiv6rion .pp€ars to lollow a similEr prolile 6s th6 lungat myc6liat mass. 91 600 400 300 200 100 tncubation Period{Days) Flg.3l. lotlclty of lungu3 n€€ culture fihratG during 25 dav growth ot nin€ isolaros ot /4, .rb,bt srMBsHElMii"ffiE^ME?,H Ch.ng.s in rhe Cont nt ot Phytoro{c CotrDomd. Durtn0 Fung.t c.owlh In ord6r to invostigab rhe quanrtr€rive tevets ot v.rious Eotanapyron€B .nd cytoch.l$in O, .r ditt.ront stag6. ot tungat growrh, tho cont.nt ot phytoloxic compounds ol static cuhuro3 w€r6 ,€sotv6d an Czdpek Oox h€dium supplemonrsd wirh aqoeous oxt.acr ot chickDe. s€.d6 oI a desi cullivdr {6153). Fungus cuttur6s were g.own tor 25 dsys .nd samptes of lungus lree culturo lilt6t6s (lomtt wer€ colected eve.y day, Fiqs. 32, 33 8nd 34 clearly €howed thar producrion ot the phytotoxic compounds varied consider.bly among rhe nine isotat.6 ond thar the maximum ahouor ot production w6s b.tween day 11 snd 19 whereas tun96t growlh .s 6videnc€d bV luns.l dry weighr (Fig, 3Ol .nd sporut.lion microBcope p.oce6ded normatty, .! ob.orv6d uod.r No sppr6ciabt6 amouhb ot phytotoxic compounds could be der€cted in the cuttu.6 tittates ol kotsi.6 CAtvtB 7 and 8, In CAMB 8, only 6obnrpy.o^€E B+C worB d.r.ctabt€ .r 2.3 ug/rq ot the fuogus. How.v.r, unde. simit!. condaraons, i5otat 6K produced 4G5O u9 ol phyrotoxic compounds psr mg dry weight ot tho lungus. Th€se resutrs Indicaio CAI4B 7 to b€ a Tox isotar6 Ei rhB pr€s.ht d6t6ction timns whersaa CAMB a producos only solanapyron6s B+C bur no sot.n.ptrone A_ CAMB 5 produces cytochalasan D in sdditio. lsolalion ot phytoloxic 16 rhe sotanapyronos A rnd B+C. compounds .nd ih6ir r€soturion by HptC rndysis were conlod our gxdcrly a3 r6porr6d in merhodotogy. Ouring rh6 sludios it w.s di€covered rhar sobn.pyrong A an.t sol.n.pyrone C h.v6 similar retention times !nd6. th6 expe,im€ntot conditions smounl ol sol.nopyrone C presrntod an .nd rh.reIor6, th6 ths on going Gtudie r.present3 . mirlurg ot rol6napyron65 B 6nd C. F6c6ntty, Shahid el .t., (1998) r6porred a soF€nr 6y3l6m con6isting ol Irlrahydroturan: h.rhanot: wdr6r 50 - 4A 30 20 10 hcubetlon Period(Days) Fig.32. Production ot solanapv.ono A during tsolal3s ol Ascodryta .abd, CAMB 2 CAME 6 OK siMBg CAMB tsL 10I--J A v/to growth of variorls 3iMB: CAMB 11 ffi L - 60 50 ; E 40 30 3 @ 20 10 o 10 15 '20 Incubation P€riod (Days) Fio,33. P.oduction ol solanaPyron€s .i.E isolates ol ,4. .abrei. CAMB 2 CAMB 6 H H 6K a+ar B&Cduting25 CAMB 5 CAMB 3 CAMB 7 ,A,--lA CAMB 8 CAMB 11 CANIB 10 t--{ H] H 95 a 80 70 ; E 60 50 40 ! 30 20 10 Incubatlon Poriod(Daysl Fig. 34. Productaon cArvrB CAMB 6K 2 ol cytochalssin D duridg cr--{| CAM83l!--l-\ CArvrE 7 A---{ a---{ CAMB 10r-----.l 6 oF---O CAMB 5 CAME 8 CAMB 11 H H 121-6%:20.6%:57.8%l ror rh. puriticltion o, mixtur6 ot sotrnrpyronas 6 and C. Fudher, inducraon soranapyrones ol Eynthosis ol sol.nopyrono A preceded that of B dnd C. Th6 amount5 ol .ll rhe phyiotoxins decreased during prolonged incubstlon sradustty ol lhs lungus. Thus alter the 2oth day of incubation, no sol.napyron6 A w6s obsoNed in 6ny ot rhe lungus rree cuhure lilt6r6s (Fi9.32), A iimjtar pa 6h ol 0r6doat disoppearance of solandpyronos B and C lFiq. 33) wos obsewed. Howevor, the t€vet of rhe phyioroxic mat6ri6l was .ot ro z6io 6v€n .trer 25 dDy6 ot incubation. Thus rh€ production ol solanapyrono A r€rched a maxima between 12th and 15th dsy incubstion whoroas p€ak producrion ot solandpyron6 a and C w& obGinod b.two6n th€ l6rh and 2orh d.y ot It w.s r€duced observed rh6r isol.t.s CAMB cytochalasin D {Fiq. 34). lt 5 rnd CAMB 10 produced w.s obs€N€d rh6r producrion ot was not ddpondenl on the addirion ol .qu.ous cyrochatasin D to the Cz.pek Dox mediuh- N6v€rtholoss, in simitarity wth the toss oI chickpea eiftact solanapyron6s during subsequent incubEtion, th6 amounl oI cyrochatasin D also decrea.od but did hot cohpl.t€ty disapp..r P.rhogonfcfry ot Dlfi€r.nt l.ol.r.. ol A. .s was th. toxicity. ..bLl All isofat6s ol A. nbiel wsts studied tor lheir pathogenicity cultivars C44 and 6153. on The r66ult. dsmonstratod th6t both culivars developed dis€dso symptoms lT6bl6 1t), Howev6r, it tungst p.thogentciry was ral6d on the ba3as ol th6 siz6 ol l$lons produced on th6 ptant, ir ditl6r6d signiticantly among rhe nino isolat6s 6nd wds dlso dependent on pfant susc€ptibility Ascochyta rcblei isolaros CAMA 6. 7, I, tl and 6K 97 T.bLll: AMOU TS OF AOLAI{APyiOllE A, SOtAttApyRO|lEA B & Cd..nit.dn ot phytotodc [email protected] ugrn|g pl$t Sol.8&C Cyr.D aolA I o153 CAMB 5 CAMB N,D. N.D. N,D. 28oar rO rr c14 5 N.D. N.D. 344a53 83.ir r 3817 405.i59 31a5 14!2 63a14 N.O. N.O, 2O2}.23 3 N.D. N.O. I 6r 53 3 N.D. N,D. N.D. c44 3 N,D. N,D, N.D, N.D. I417 26O.t92 5 8_i18 t12 c44 3 N.O. 189a4 480180 6153 5 51al3 143!21 690a14 c44 3 2ti5 4153 9 1Oa2 c44 7 I 6153 3 cvt 8oa17 N.D- 6153 10 N.D. N,D. 6153 6K N.O. 5 c41 CAMB I 6153 0r53 CAME C CYTOCHALASIIT D EXINACTEO FFOi' TWO CHICT?EA CULTIVAHT I{FECIEO VvlrH VAnOUS tSOrArlS OF ,4SCOCrtrr AttE OtFFERItc ||t| vtrulE cE. 2OOas3 36a5 3621!4r7 29!2 8ztot83 N.O. 2819 9o9tl80 N.O, 3al7 t jO.5 3a4l3t o 98 produc.d only gr6d6 3 lt€ri.t.ntl on chlckp.. cuhiv6. C4i1. On rh. oth.t hand, all lungal isolates.rcopr CAMB 7.nd CAMB 10 p.odoc€d gEd6 6 (modarately susceptiblel on cultiv6r 6163. Borh culrivais 6153 & C44 dov.lop.d lesions subs.qoent ro intoction by tuns.t isotate CAMB 10.nd the dis6ds. severiry was rar6d ai 9 {md.rarety susceptibt.) End 7 IsuscBptiblel rospecrively. Thus on a 1-9 sc.b of paihogenicity, the nin6 i$orat.s were car€gorized a3 pathog.nic troln gr6de 3 ro 9. 4|16r a, 10, 12 6nd 14rh day3 ot Int.crion, samde w€re an6tyz€d to, roxin produciion. Maximum phytotoxic compoundB w6.e d6t6cted on th6 12th day of inlection. Toxin pfoducrion vad6d in the lwo cuttivars ol chlckp66 inrectcd wirh dirfer€nt i6ot.r€3 ot T6bl€ 1l /. .r6ie,. presonts th6 amounts ot ditfor6nr phytoioric compoundE isol6t.d trom chiclpea ptanr. iotoclad wirh diltorent tunsat isotar.s .nd subsequentry incubatsd tor 12 days und.r oprihat tunq.tgrowth conditions, It w.s surprising thar all lungdl isotaios induc6d synth€sis of cytoch.tasin D and CAMB 7, which was cstesorizod as Tox', atso induc€d trac63 of toxin! includinq cytochalasin O, In 6rttt cuttu.e, onty two isotat@, CAMB 6 and CAMB lO, produced cytoch.ldsin D tFrg. 34t. Fu her, CAMB 2. CAMB 3 ahd CAMB 6 induc€d no syntho€l5 of Eol.napyron€s white a[ other isot.t.6 inducod synthosis ol solanapvronos .l det€ct.bt€ t6v6ts, It w.s surprising to nol. tho r€cov€ry ot sotanapyron6s tioh borh chickp€a cultivars subsequ6d ro intecrion by CAMB rO rhar induced no th. 6till coltur!3 {Figs. 32, 33 .nd 341. On th. oth6r hand, CAMB 5 .nd 6( th.t Induc.d Bigniticant tev6t! of th. synlhesi5 of solanapyrones in sol6n.pyron6 during ,h ,n@ growth abo induc6d signiiicaor tovets ot rh€ phytotoxic compoundG in lungus iotoct€d plsots bul 6xhibit6d 6 mod€rat€ ol di6or3€ 3€v6rity (Tablo 11). Th€ discropshcy In th6 lev.ls or production of ioxltu during i4 vit@ 6nd in vivo siruatioN sugg4rs rhsi degree production ot phytoloric compoun& in slill cr,lrures has no be..i.g on the disoase severaty or longd vnubnco. Th6 cotr.ldtion co€llicienr beiw66n disease srad. ,nd amount oI solanapyronoE A, B End C and cytochalssin D varied lrom 0,055 !o 0.718 dccordins to tho curtivats used in rh6 €xp€rimenr {rabts ll). Tho costficienr betwoon dasea56 grado aod cont€nt ot sotan.py.on& A, B & C .nd cyroch6r.6in D w.s calcllrr.d ro b6 0.o81, o.144 and 0.71a, respectivoly on dis€a!6 susceptibl€ chickpe6 cv.6153, wh€r€a5 corctation was 0.055, 0.26 and 0.122, rosp€ctlv€ly on lungus .6sistant chickpea cv.C44. Ths 3ignilicanc6 ot coftol6lion co€llici€nr is not ctoar al ftt! stsqe. Gen.dc Dly.rlity ADons Ult.rcnr lELr.! oll4, Er,@- To inv€stigato th6 prosence and polymorphic behaviof ol differenr simple repetitiv6 sequences in th6 Ascoch|t. rcbiei genom6, EcoRl, faql End Hi,Il diq€l6d ONA of nin6 asol6tos or /. rar,bi varyins in roxin production and vnubncg were consoculivsly in 96l hyb.idizod to six dillercnt 'rho r6s!lt. .howod th6t rll te3t.d motil6 are obvio!6ly prss.nt in rh6 g6nom6 ol alf Ebundance (Fig, A. rcbiei asoldla. bul rh€y dill€r in o.g6ni2ation and 351, lt was obs;ryed thrt 6mong sx prob€s, tGArAt4 track occur less lr6qu6nlly and 6pp..r.d ro b6 conc6nt.r.d 5t ono or two p.edominant loci {Figs. 35A and 3581. Among th6 probes tesr€d, IGAIA)4 was the host inlormarlvd in two rospocls (1) clear cur bandihg plrr€hs l9,.----r Jo83033:l ouAoooooo 9,4 43 05 :9 " o u,r6oourruu :3a:tS5:: ':rY91969.1 f .j r GTC q o. l'.ge'P r'no ol ol 4 ,rD,e f'!. a c E G so .-..- *, o.. D-NA o 1 ;p(GALAI4 "res (CAAr5 lCA'8 rc-Grs ald an"a.: orooeo *'rtr IGAIA)d, 7"q1oge-ed I'gs B D r 'd1r pobed {GGAl,a e,p"-r,e/ respecnv. O.'qo1r',-u wirh rGATAj4. rcAcA)4 {cAA)5 and 1cA)8 v lacking any background Em6ar w6rc produced, 6nd {2} polymorphic brnding p6tt€ms mad6 it possibl6 ro dill6.ontiate betwc.n isolrros. as compar€d ro iGATA)4, th€ hybridizallon patt€rn producod by .ll the oth6r probos llGAcAl4, lcTG,5, {cAA}5, {CAls and lccAll4i are mo.e complox and in some casos hnds wero usu.lly 3up6rimpo36d on 6 3m€6r. In order to opriFize DNA linO€rprinting pattohs, ihr€€ ditlorenr r€Brricrion enzymos wor. resr€d: E oRl, t ql and Hinll. Monomorphic (OATAl4-pa(erns were produc€d bi.llonzymos {Fiq3. 35A, 358 and 36). The six bp ctrtror fcoFl was le.st infomalivo and lragmonts were ctusrorod in th€ hlsh mol..ular woishr .Enqe 6nd did nor a ow 6r3y evatuarion in the vicinity or th. IGATA)4 rep6.ts (Fig. 36dt. lhis probs produced 36n€ lingorprlnting pattorh with lour bp-cut6r onzymo5 I€qt and l/tlt in ths eiz€ rdnge 1i,6, botwe€n O-5 6nd sKb, which are €s3ity dnatyzabte. The r6sr,tt. pr$snt€d in Fig. 36b show6d th.t {cATAt4 wrs us€d ro hybridiz6d onty wilh hish hol€cular welghl lragm€nrs produced by ti'tttFig. 3ga) and tow moleculfi weioht t'aqm6nts prodoced by raqt {Fig_ 358). thu5 Irgfw6r€ lound mo6! suitablo tor otigonuct.orido rinqo t/rfl and ry.inting ot A. rcbiei. Th6 resulb obt.ined by DNA ting6rprinting with th6 morits (GACA)4, lcAA)S, lCAlS snd cr€atod by lcGAIl4 E. mo,6 comptex tFigs. 35C-J)_ patl€ns {GTGI5 End tcACA)4 p.obes monomorphic. l6ol6t* CAMB 2.od CAMB 3 w6,. disrincr but app€ared showGd simitdrity in b.ndinq p.ttorn bot were dilferBn! lrom tho r6sr ol rhe i6otates when hybridized with {c4,8 tFiss. 35G .nd 35H) and lccAT)4 tFiq. 35Jt. Di6ti.ct difl€renco was ob6erv€d in hybridizstion with two probes, (CAA)E .nd {CA)8. Ih.r6 ,re tully di3lribured ove. th. whot. g€oome wh€n dio6sr6d with Irql dnd l/,tfi 6nzym6s s.p.r6t!ty (Fig!. 35E, F, c ,nd Ht. probe (GGATI4 36quenc.6 ,1.t,!,i.!.!dJl lo4 l.l GATA)4 ii::i 3:i r (GrG)s rIiSis!i s ;ACA)4 DNA ridler p,inring 6.€tysB of th6 njns i.olaros ot /4, 6Dlbl, Purified DNA wrs dig€sted wirh 6.oRt. AecfioOhor6s€d {1ouo oe. |3_n6r and n b|ot hybridirsd ro rh€ ai (GATAI4, b; {GTG)5 6id c; (GACA)4 oonradioacrivs tabetted p.ob6. Mot€cutaa mioh, markers arc grv€n in t'tobases tKb,. In band,no r|€n€hs- l .epr€senrs p.osent 6nd o repr€senls abse.i, c rcp.6san1i.s tor caMB produced vsry 3impl€ pattern wnh tirll dig.st6d DNA .nd rhi. 36qu6nce was onry pr66nl in small frasment3 1i,., b6tw€€o 0.5 ro 2 Kbl. Hinll restriction iiaomonls aro v€ry specific in A, rrbiet Oenomo in 3uch 6 m.nner .ll the {GGAII4 sequenc* concrntt6tod onty in 3 or 4 tr6gm6nts {Fiq_ 35J). Tho bsullo obbin.d with th6 combinaraon or f.oFl .nzym6 6nd rhar (6,4T414, {GTGIS 6nd lGACAl4 probos were not discriminarory as th€ molits w6.6 highly lbundanl and Eppe.r€d ln th€ rorm oI smear lFig. 36b and 30c1. Thus th6 conveniont discrimiortion ol polymo.phism obseN€d and inlormatiotu obt.in€d bv DNA ling.rprinting ..e srrongty dop.ndont on the prob!{3l E w.rr .. on th6 restricrioo onzymo ls) !sed. Th€r.loro Isqt add Ahll wer6 lound no5t suitabl. r6stricrion en2ym6s tor otigonucteorido linserprinring ol /..abletDNA. Poutson or at.,t1993) snd tvlorjon€ st Et., {1994) afso found rsql and Hinll 6s rh. mos! lrs€tut enzymos ror ONA lingerpriniing ol Anssica odp\s L €nd ,4. .rbtet respocrivety. Our oligonucl.otid6 probos resi6d in rhis study, IGATA)4 dnd prodocod ho€ dislinct moehologic DNA linqerprinting 6nalysl6 ot th.t !. tccA-fl! ot six panoms bsnds, rh6r wer6 €a6aty d€r€crabte in frbldt colsrtinsor 6r st., (1996) ropod.d oligohuclootldo llng6rprinting d€r€ct! conside.abte goneta. dlv€rsiry betwssn Ascochyr! isol.tes counkywis6 .r rhe motocutar t€v6t .nd rhis divsrsity increasds with lh6 inercase ot rogional distahcs. lt rcv6.l6d rhar all simple repothiv6 6equ€nces testod by DNA tinq€Drinting a.e presenr some degreB in Ell the isolat€5 inspeciion geographic ol lh8 ol A. tsbiei. Ihis c6h b€ lesn by vislal g6n€ral6d prclile. 6r6a. t!'leye. et al b of tho st..ins isolstod Irom lh6 ,119931 Edm€ .lso repofted that rher. w8s mofe similarit b.tweon lhe sr..ins isolar.d lrom clos.r qeogr.phical tr€a. From ting€tprantinO th6 l. forgoing discusston, v€ry 66td lt can b€ conctud.d rhat rh. r€chniqu. lor anatyztns g€n€rlc divor.ity of lungal isolEl6s in contfaal ro conv6ntlon6t m6lhods. Und3r th6 orss€nt .rp6rihonrs, all lsld.. p.ttom in ling6.prindrg g6nomic slroclur€ How€ver, lh6r. !! of [email protected] morc o. .nrly!i!. tt Indtc.t.s lh.t .tt tho iet.t.. !.m bsdnrg h.v6 the s.m. th6y werc coll6cr.d lrom th6 6am6 q6og.lphic.t ar6!. !r. .l.o some ditl.f6nc.s molir3 pr$€nl on lG. th. whol€ q6nom. ol In abundanc6 .nd ofqsniz.tion ot 4. /rtbl iet.t€s, 96notic difiEr.nc$ 3)dar.d in thot g6nom€. indtcadng somo EHf,PffEB A Dlscusstot{ A numb€. ol pathogonic lungi socroro toic n6tdbotic producG which have dn important rclo in parEsire virul6nce, Information on lh6 ch6mistry .nd mod€ ol action oI ruch compounds is 6n impo.r.ot srop in undeEr.nding the dise.s6 3u3c6ptibility and rGasranc€ phonomonon in pl€nts. S€v6ral phytoloxic fungal m6tsbott€a hav€ b6en roportod bur ooty a lew ot th6ss hEve been invsslig6ted lo the ertsnt lhat rh.ir rotss in pt.nr dise.s66 .is borh convincingly esrsblshod rnd ctearty dellnod (Pringt6 6nd Schdfler, 1964; Sr.obel, 1973i Strob6t, 1973; Rudolph, 1976i Schatfor.nd YodeL 1972 and fodea lgoot- Arr lungal roxing srudi6d 60 16r hav. tow mol€cul.. weights but 616 ch€mically divers6- Then chomicat idonrny includes rither rin€ar or cyclic p6ptid$, toeonoids, sus.rs, .od potyl6ron3 lDurbin, 19811. Som6 ol rhes6 rorins rllecl only plaats thar 616 susc.ptlbto to the toxin.producinq pathogsn. Whil6 olhers ar. non specilic and may be roxic not only lo host planl. but .lso to no. host plants. mic.oorganism6, and animals.s well(Yodea tgaol- Pu,itied, singlo spoiod lungus w63 Btored in liquid oinogen or at , TOoC in order ro svoid g€noric varistion during rh6 course anvestig.tions. Dilloronl mthod3 lor ol th6 th. bio.3..y ol phytoroxiru hwo bo.n propo€ed (Budolph et al., 1978). Including determindtion ot rool elongation inhibition, callus g.owth inhibition biossay, deiachod leavos bioass.y, in ord€r to bioassay doroct phyrotoxic lcllviiies, w.s compounds protoplast. dev€lop6d. Th6 a36ay ! v€rv ronshlv€ 6nd conv.nlgnt is bas.d on lhs ability ot phytotoxic ol c6ll wsll 3ynth63i3 in chickp.e culrur., rs d€l.rnined by lluore6.6nt microrcopy, Tho ro inhlblt reg6n.raiion p.otoplasts/calls virbiliry assry has boon u66d ro *tablish synth..i! ot 3€verar phyroroxic compounds durang qrowrh ot /4. EDnrt in Cz.p6k Dot liquid modium sopplemenrsd with an aqu€oui exrracr ot chiclDea sse&, Ih6 plosmd membr.ne is an obvtoos csndidsto tor rhe sit. ot th€ prim.ry losion caused by 3r,ch roins .nd rhe.etor€ . ft.ns of me6suring prasha mBmbrane dyrtuncrion ih protoptssr coutd fo.m rh6 basis ot assay. Proloplasts/celrs consritured s more . toxin suitabt€ mateiot. Tho adv.nta963 ot Ging protoplBrs owor whol€ pt.nrs .nd curtings for 5!.6yins toxlns h.v6 b.on dlscu$€d in dera tB.oim.n and crtun, 1 gAt ; Errl., j 978; Ggndloll ot 6t.,t987and StrEng€ 61 6t.,1982). Initiat .xporimenb had shown tnat proroprasls wer. dilficutt lo isot.t. 6nd were o8ity ddmag.d during pipetting. C.lls wero tavou.6d tor rhe toxicity bioassay bocauso lh.y coutd be isoral€d mor6 quickly and w6'e mor. robust. Broiman and c6tun found th.l lt98l) c€ll suspensiof,s ol lobscco w6.e tess seNiriva than p,otopt.sts ro a toxin produced by P, cittophho@. One €xpt.nation tor this difor.nc€ might b. rhrt suspension. usuatty conrain clumps ot c6tb which coutd h.v€ hindgrod dillusion ol the lorin wh6re6s th€se wer6 removed by fihration. Cells lhat a,6 derived directly f.om t€af tissrres may s,so oe mor6 .€pres€nt.rive ol ihe inr.ct plant thro c6ll. whach w€ro grown ov6r s.voral generation. in tis3u6 culruro .nd show cyiotogicat changes dnd g.nedc Ou. srudios show rh6 presonc6 ot rhr66 phyrotoxic m.rabotiros produc€d during /h v,rro growrh ot rh6 fungus {Fig. 11). TLC , Hpt-C 6nd spectropholomdrric studie rov€3led th6t two out ot thfee compounds w6r6 id6nticdl with solanapyronos A .nd C which w6re provioosty r6port€d lAtem ot al. 1989) using Kabuli chiclpea cuhivar tLC 200 in vn,o gtowth o, Ascochyta rcbiei. Mat n.t at. (19781 did not char.cr6riz6d rhe rortn6 f.om Ahe.na,ia tulani but rh6y g.ve 8t v.l'r.$ lor th6 compound3 on .ilica gol. Thes6 compounds hav. 6l.oady been idenlitied as phytotoxins. solan.pyron.r A and C $socaat6d with €rrly blishr oI poratoes 8nd lomatoes .aused by Aftetnena so/rri llchiharc 6t chromatoqr.phed a1.,1983). Two cfosofy rcldted lu^gi, Ascochvta pisi end Ascochyta fabae produce . phytotoxic compound, ld€nlillod as ..cochitin6 {Lopoivre, l9a2l which offects 6 wid6 Gn96 ol orgdisru lrom 86cte.i6 to high6. plants by intorl€ ng wiih collpem€.bilaty {B€rtini 1956; Oku rnd Nakanishi, 19661, Asco.hytd pisi {Lib.l induc€. nocrctic l.sion. on p6a leafl6rs, while Ascochvta taba.lgp.g.l B lh6.s6nr ot "&own Spors" diseas€ in the brc.d bean- h h6s been demonslratod lhEt rh. susc.ptibiliiy ol p6. cuhivar3 I. dnectly relaled to lheir sensativity to oscochitine snd ths virul€ncs of lungal isol.i6s wd. simil. y rcbrod to th.n ability ro iynth*iz. rh6 roxin i, via@ (feooiwe, 19821. Mat6m 6t al.,{1978) reportod rhe isol6tion ol two toxini iiom culture liltrates or lfir.ra.b so/rr,; polato. On the ol th*6 obseN.llons, tho lungus was srcwn b636s th6 causal agant of eldy blight ol on czapok Dor liquid m.dium suppl6m€nr6d wlth .xtracr ol chickp€. 6..d3. lt wrs obsoded durino exp.rlnents th.t no roin wrs d.iocied when 6xtr.ct of chlclF€a seeds was omirted from th6 m6dlum. Exltaci ol host plants hd6 previoGly be6n r6porr.d ro induce ioxio ptoducrion by pl.nt p€rhogenac lungi /h vllro (Sobeson and Strcb€!, 198€l snd in ono in3t.nc6 the inducing compound has been identin6d (Pink€ton and Strob€|. 19781 Elisht ol chickp6a caused by dis6... to contrcl lo. ptoduction ol . numb6. oi L Eb€t htc prcved to bo a difficulr re!son5. Amonq lh6s6 .r€ pycnidia conl€ining copious numbots infectgd plants which c6n l€ad to .rplosiv6 th. t.pid ot pycnidiospor.s 6prd€mics, lack ol on r.oo 1r0 lh. pl!n1., dilicultl€r In m..e.ing loch p.rtirl r.!i!i.Eo l| m.y oxirt, .nd . l.ct ot con3.n3u. .. lo it! modo ol inh6rit.nc.. Duting th. i€Bi.tanc6 in 1977 78 .6r$., wh.n rc!.6rch on A.cochytr blighr lCAnDA, ! s'6at n€.d w!3 l6lt lor scrGlnios r6chniqu.. Ourlng . ol chlckp..3t.rl.d 3impl., .lllci.nt .nd r.li.bl. li.ld- lho 1977-78 3663on, wh.. r 3p.!y with pycnldlo6por. .u!p.n.ion prcp...d tom rh. nlrurrlly lhLca.d phnt. undor naru"l rslnlall conditiom did not p.ovld. sullicint db.!6. pr.!.uro (l-uthr. .t .l.,l93Aj Serr* &d H.tlr, 1951) coupl.d wlth .uppl.mnlny lprinkl.r nrig.don w.3 comid.r.d. Th. moihod w& l.!t d .6Een and rh€.66ulti w€r€ 6xc.ll.nt. slngh l.chniqu. which involv.d . ot.l. during ih. 11981) d$.dbod iho combl.liion ot int r-planting ! 1974-79 3o&cptibl6 sp...dd lin., $.tt.ring b.two.n .ow inf6ct6d d6bft coll6.t€d from rhe provious 66.$n, sprayios pycnldlo.por6! h6rv4t d lrcm th6 hl€ct€d plrnl. ol . 3u3c€ptibl€ lin6, and Foviding lpdntlor lrlgdaon. Th. .ttlcloncy ol dlll6ront m.lhodr ol inoculdion with .nd without ryrintllr trigdion w.t fh! inocul.tio. moihods comparrd w6r6 lll dls6$6d dobri!, llll pycnldlo.por. .u.p.n.lon lrom por. cult!,. oi lh. tungut, .n.l li{} ft..hly comD!..d. I h.vo .l.o Invo.ilgrt d lh.t i.oculom p.oduction on .€.d3 b r convoniont m.thod fo. obt.ining purc .nd hlqh inf.ct€d pl.nt6. chlclpoa conconrrEtion ol ato.. !u3p!n.lon. SCo.. !u!pon!ion! obralnod lrom oth6. lourco. such a. inr.d.d pl.nt m!ro.i.l ud liquid or rgar m€dium l. u.udly cont.minatod with d6bd!, m6dl6 or turt! ol myc.lium. Ths33 crn block lh. norrlas of sprey guns wh€n sprlying .por$ on .cr66nlng pl.nl6 in iho llold or gr..nhou!.. In conlr!3t, .por6 .u.p.n!ion obrdln€d trom inocultlod chickp.r 666d i! v6ry fiuch co.cdn&.t d .nd lr.6 lom .ny luch conlamin.tion lFig. 2). Copiou! lporulEtion ol lh6 fongu. und.r f.vourtbl. condliion3 h€lpsd lor rlpid dsv.lopMni ot di36a36 in lho liold or 111 For th€ v.ri6bility ol th6 prthog.n for rhet palhogenicity, 36v6ral repo.l3.16 rvailablo rbolt the v6riability in isoldtes oI A.rrbltl lBedi lnd Auil., 1969j sino ot al., 1981; Gowen 1983; and Alam, 1984; Feddy vt.nd 6r.wal, 1974; oor$hi lnd sih.n, 1984; s.ngwar 1989 and You!.i et a1.,19931. Six r6ring 5cal6 h6v. b6en dovised 6nd used by v.rious workeG Ior scorihg bliOht soverity lAuil6, 1964r Auila End Bedi, 1967j Gr6w.l .nd Vir, 1974 and Morall and Mc(6nzi6, 1974'. To lacilirar6 rapid €valuaiion ol lin.s und.r pot culture conditiom, F€ddy .nd N6n. {1979} 6volv6d a g-point scal6. Bas6d on Grswal's (1981) dis6as. /rrt i.tins sc6le, th. nin6 iool.t6s of,4. t, purilj.d trom infeciod chickpea plants collscted ftom diflorcnr reoaohs ot Pakisi.n were caregorlzed .3 pathog.nic lrom grade 3 io 9 {Grawrl, 1981) by lollowing scal€ 1 to 9lor disea36 rrting. R.c6ntly d n6w sc.l6lor scoring inl6ction, lha lin6.r intoclion indot, ha6 be€n 6stablEh6d whach dpp.ars to givo consistenl ,osuli6 lRi.hi 6t rr. 1990). Thi6 should b€ ot ben6lir in 3creeninq lor r$Bt3nc6 ro th6 dbe.s6 6nd in inhgriranco sidi63. How6v6r, numerous t6rl3 ol l3ol6t6s at the Intornation.l centr. lor Agricultur.l Res..rch in th€ Dry At6.s IICARDA) have giv€n no ovidooco ol rdc6 6p€clticity. For exdmple wh€n sh isol.t€3 wer6 Inoculalod on ning gonorypes of chickp€a, rh6 lowost 6cor. on hish66t 7 {Anon, singh, et.l., 1990)- Thos no th6 1 9 Ecslo cult'vr showed compl6r€ r.sist.nco. {1998) Ecrc€ned 165 chickp6! lin€s ascochyr. blighr. Non6 w.s w.6 3 and ih. r.t.d .s lor r66i5t.nco to hlohly r.sistant la rating ol l, ascochyt. t6bbi. Thr€€ lin.6, lCC X 810800, 1CC x 13816.nd PBG 1, lnd 21 wer6 moderll€ly ..si3tad lratlng of t€Bilno. Flrliermor€, th. v.tiation th.t wt6 r.cord.d w6r6 tat€d a6 resbtent {rstino 2 3l 4'5) In all th6 years to 112 .ugg..ted lhat 3om6 lsolalB wsr€ dor. .ggr.ssiva on rll cultivrrs tha. orh6rs, and rhat som€ chickpea g;notypos wero moro susceptiblo lsolaros then others. On6 hypoth€sis which is to all compatible with thes6 observrtions is lhat th6 mor6 aggrossiv. isolares wore more roxigonic and the more susceptible g€norypos we.e moro loxin sonsitive. Th6 r66ult3 repod€d domonst.ste ihe capacity ot nine isolatos ol th€ lungue to .ynrhssizo phytotoxac compounds A v,?o. Moreov€r, the isolatos variod not only in the .mounr. ol lh6 loxins prodvced bul a|3o in lhe kinds of toxic compounds. In this reqa.d, CAMB 10 i3 oublanding bec.us. it synth$izod cytocholasin D, s new phyrotoxic compound isolated from 4. .arlel rdth€r than lhe sol.napyrones, As with th6 solonapYrone toxins, symptoms caus€d by plscins chiclp€a coNngs in sorution ol cytocharasin O {Fis.29) wero rypicslot rhos6 caus6d by inl€ction with th€ longls lAl.m et al. 1989 tnd Hohl 6t sl, 19911. Thus 6t ihe biochomicol lev€l ther6 is contid€rabla hete.og€neiry among lungi isolares, Th€ cotrelstioa co6llici6nt oI toxicity wilh solanapyron€ concenlration lor 6i9hi isoldt€s ol th. lunqos was high {O.859J wh6n lh. culture litrarel w6r. obtaanod lrom modia in which exlract ot cv-0153 was incorporstod snd the assav c6lls wor6 obtdin6d trod soranapyronBs coutd .ccounl cv Cvpr.ss3, suqsostlng that lhe tor mosl ol tho roxic oilecl. Howevor, wiih oth6r combinatioos ol cullivsrs osed lor modia and tor sssav cells, tho cotrelriion coellicienls wero lower. Her€ oth.r unknown r&tots mav hav€ interlo.od wth ioxic scrlvhy or conttibot6d io il L.re, on chen and srfanse i19911 publi3h€d . dolin.d 6 medium in which 6olsnapyron€5 A, B and C w6,€ produc€d Cetions w€t€ th6 crucial gddilions ro Czap6k Dox nutionts r6quir€d lor solanspYrono ptoduction and' 113 ot the36. 2inc w63 6n 6$nrid r€quirem.nr. Mo6over, ..mov.l or cstions from s6ed 6rtr6ct bv 6 cstion stchanq€ t6sin d6slroY€d its abilirv to .upport sobnapvrone synthosis (Chon .nd Slrang6, 19911. lt will now be int.restins ro d6l6rmane whethor th€ samo cations p.omota soludpvrone ptoduction bv orher isolates of r. .rDi.i.nd wh€lher cttion tvaildbility ol tho soil in whlch chickpea is grown influenc63 the Eggrossiv6ness of lhe palhog6n bY al. ell.ct on solanspyrone ptoduction. Evon lhough a mditied h6diom w.s lo. the maximum production ol Oox liquid hedium to .void lnv discov..ed (Chen 6nd Srr6nge, 19911 solanspyion66 bur w€ used Czap€k va,iation and lor th. continuiry ol our prsvious exporimonts. h has tlgl l--9, A. rabiei, in .1i11 curturo produc€s sol.napyron.. A, B & C lod cvtoch.l.sin O thtt int€iact with vtrious chickpoa expllnts to produco sYmptoN simil.r to thoss ob56N€d in blight' sticken chickpea plants. As tho toxins woro svothesi2od io mdit which th6 conr'inod extr€ct ol 6hickp6a, ir is lik6lv lhal thev 5.e .lso produc'd 'n plant, Phylotoxic compounds hav. recenrt beon lound in li'ld inleclod doterial liom r6a cuttivar! {Shahid and Biazuddin 1998) Howevor, Hohl ot al-, t1991) we.e unable to 3how the pres€nco of tho solanapvron* ro inl.ct.d l€al tissoss- Tho lev6ls o! sol6napvron* A, B & C lnd cvtochalssin D extrsctsd ttom th€ lield inl6cl6d plants varving in disasse su€ceplibilily showed 6 comPlet6 6bsonce ol solanapvrono A in 6ll lhe lo the fungrs Th6 absenco ol 3ot.n.pyron. A m.Y not r.p....nt . tru. l.ck oI tvith$i3 but rtth'r rtpid inactivaiion immsdiatelv 6tt6r produciion- lnrsiestinglv' the l'veb ol plants whe$er susceplible or rosi.tdnl to dnec{v corclate with th6 susceplibility ol lhe chickpea cultiva. Th6 mo.e susc€ptibl€ b rhe cullivor' lh€ higher tho cytoch.lasan D s66ned 114 c6nc6ntraiion ol cytochal8ln O and ln itct thdr6 wa3 compl.re ab$nc€ ol cytochalasin O in dig.as6 rosistanl chickpoa cultivaG, Clvt72xlLc3279 .nd CM72xltC116. lt is poinled out lhst the tbs6nce or cytochtlasin D .pp6ar6 to be due ro boih low productioo 6nd rspid in6ctiv.lion This notion suppo(€d trom rho observations pr$ented in Figlre A which is clea.lY domofttrate thar all phyrotoxic compounds ar6 .6pidly inactivat€d rohothor produc€d ,h v,1/o or dkectly in thq snd pl.nr. Funh€., thert was l$e production mt€ rrpid Indctlvatlon in di6oos6 r66ist5nl cuhlv.rs wh6n compa.6d with disodse suscepribl€ cultivars. An invostigarion of th€ toxic molabolites producod by lhe /4, hateromupha tsch. 6t S.cc.) Curizi l.d to rh€ ptlification ol . n.w cytochalasin n€m6d ascochslssin (CapaBso ot 31 19881, tog€thsr with 6 lsrg€ amoont (Capasso.t ol cyroch.l.sin B snd 61. 19871 and smaller .mounb ol cyroch.lasin A dgoxrphomin lBinder.nd Tamm, 1973) obtdinod lrom tho liltratss ol lhree week old still coltures incubated st 2loc bul sttempts ol isolare the cytochElasin lrom natutally inloct€d olo.ndd l6av6a we,e unsucc.sslul (Boltalico et al. 1989). Ou,ing lhe study ol tin.tlcs ol production ol dill6r€nl phvtotoxlc ,4- /rb@ r6veal6d rh.t rh6 production ol phytotoxic compounds wts limil€d io . ceit.io stag6 compounds during 25 dsy in viho gfowrh ol th6 lungus ol th6 g.owlh cycl6. Thus hrimuh p.oductlon ol rh6 vsrlous phvroroxic compounds w!! prodlcod b.tw.6n day 11 ro 15 tollow.d bY t gradutl inEcrjv.lion. lt wss intot6.ting to not6 th.t induction ol th6 vtriou. phytotoxic compounds 6nd thet subsequ€nr inactiv,rion exhibitBd diE.imllar prcliles. How6v.r, in all c.sos, ptoduction of solsnapv,one A prec6d.d that ol soldnapyronos B rnd C. Furth€r, ths l6v€l ol toxicitv, ts observ6d bY rh. It5 is hot compleraly accounted ror rh. lour phytotoric compound. (Fig.. 33, to 35t. lt wE in!6r..ling to noro ih.r protopla€l bioassay (Fis. 2), subsequ.nt incub.tion ol ihe runoal cultur6s l€td ro a gradu.l los3 of th€ roxicity. Thls virtuallv no toxicitY wr5 o&.Nod alt.t 21 day incubarion in atl the isot6rss.xcepl CAMA 10 in which th€ phytototic 6ctivitY stabiliz6d ar 45% ot the iotdl dcrivlty. Thes€ dal6 clearlY show that 3om€ of th€ phytoloxic compounds und6rgo inactivation du ng prolonged incub.tion 6nd tho observed d€qradation sppeErs to follow 6 simil.r prolile a3 th€ fungal mycolium mass, Th€ ob.rved dedoas. in the mycslium w€isht .lt6i oprim.l growth m.y bo .nribut.d to run96l dsotadatlon .nd dr.th. appedr. thsl th6r6 m6y b6 Ih$ it additional phytotoxic compounds v6t unidehtiliod. Th6 ob66ruatlon thst.lldiE€asod plsnts Ertiliciallv Inleclod wlth diflBrent isofares ol As.ochytd .dDltl, eapeciallv CAMB 7 5nd CAMB a, conbined varying levsls ot cyrochal..ln D is in€tplicable The r6sutrs r€porr€d in Tabl6 1l d€monsrrarod that 6om€ lungal isol6ros w6re more a9gt63siv. on borh, rho sulcoptable aod rosislrnl cultivars. Iheso obse.vations .re in .gre6m€nl with the studi€E ot TCAFDA {G.ewal, 1981) on two hundr.d 6nd .ov.ntv oight isolat€s ot A t'biet th't wsre groupsd into 13 torms on the i.sis ol thet p.rhogenicitY, growth and sporulgtion. Th€ obsorv€d varisbilitv appo.r€d to b€ importtnt ro control A v6.y import.nt obseNaiion for th6 po6sibl6 rol€ oI solshapvron's roxins during p.thog6n.si! Is th6 r€llt thal , .aOlbl produc€ and sect€r' torins. These compounds mlght bs .6l6v.nt ior th€ Interaction boiwe€n .dbrel and chickpsa plonts bY suppr.6sing tho ,4- g6nsrar roBiBttnce moch.nisms in susceprible plant. as compsred io r€6lstant on6 Mdt ol the delen.. rercrioN .pp66r ro sbrr .imullaneolsly upon inloction. chickp.a lcica, a,iatintm t.l ol rh. 6nd Ascochyta nbiei anro.action, ptaroc.rpan accunularion w6a 6hown ro bo lccumulated by tho lormation g.oup5 16 parhogenosis related Frot€in lDaniel incr€ased polyph.nol d6posilion (Hohl, ol v.rious .nd 86.2, l99o) et al., 1990). 6nd Pt6,ocarp.ns medlclrpin and mrbcki.in 6r€ lh6 phyto.lgxlru ol chickpea which 6re roadily .ccumulaied resardod as in pl6nls. Constiruriv€ rntidicrobial isollavon$ m.y be univorsal protecliv€ barriers rgainst microbes wh€re.s pl.nt dsl6ns6. dirocled .t sp.cilic prlhogan3. Succ46tul Invasion ol lh. pl6nr maY, h rurn, involvo pt€rocsrpan phytoalexins ar€ active lnvadlng ihe ability ol rh€ lungsl pathogen to deloxily rh6 d6lEns6 compound ol lhe host lB6rz et al., t99O), A vaii€ty ol d.ls indic.ted th.t palhog€d 3r. .bl6 ro ahor pl6nt ch€micdl dst6nses by producing msr6bollt6s less toxic ro mic.obo!, confeding to the pathogon a hlgher d6o.e6 of vnubnc6 Ih€ absenco oI solanapyron€e A, B snd C in artilicidlly inlect6d pl8nt5 with iBolat€s CAMB 2, CAMB 3, CAMB 6, and CAMB 7, mdy not ropr.senr a rru6 l6ck ot inducrion bur rathot rapid inactivation ol notion is support6d by the r€sult6 $. roxins. in Figs. 33, 34, 35 which This cloadv d6monslrai6 rhat 6ll phytotoxic compounds, .6gardl63s ot pathog€nicltY ot rhe lungus, rapidly d€rorioralod, Th6re is, n6v6nhele$, l*6er production of rhe phytotoxic compound6 in llngus r4isrant cultivats comoar6d with lungus susceptibl6 6!ltiv6ts. A corelation b6rween ihe sbiliiy io loim th€ loxirc It v,!tt culllro and porhogenicity wa3 estEblish.d lKoch 6t al., l99l) bur the 3trict d€p€ndence oi rhe aggrotsive ch.racter ot , giv6n bolalo on loxin producrlon h6s bedn questioned Tho .v.lualion ol lungal toxins lor pathologic6l lunctions and tho use ol sr,ch totins resisranr pldnt .s a mean ol selsction tor matei.l is a technically lea8ibl6 and othorwi5e a sirategy. Th6 besr exEmpl6 is viabl€ E host spocitic toxin. producd by lh.lungus b lungalr.Eist.nc. in o.t pl.nts Cochliobolus viclo.iae l\oder, l9AOl 36lecl {whoeler 6nd tuke, 19551. Srudiss with orh€r toxins to solect dis€.se resistant pl.nrs hav6 yaeld6d similar .6ulls {Yod.r, 19All. However, if sensltivhy ro tho toxin is lund.m€nt.l lo p.thogeniciry of lhe parsit6 rh.n ser€clion lor toxin insgnsilivity may givs plants with duisbl€ rcsistanc6. Aut ih th€ pr€sent srudies, c.usativ6 blighr 1016 ll h.3 not been possibld to €stablkh a lundamsntal or ot the solanapy,onss or cytoch6l.sin D in th€ on3ot ol lun96l dl!.a36- Ther6lor6, it csn b€ conclud€d thar theso phvioroxjc compounds may not bs us€d lo 6el6ci resistdnl plont mare.i.ls. Th. deteclion .nd classllication ol p.thotvpos immediat.lv .iier ot th. lungus Moreov6t, resistanc. br6eding c6n b6 l.citilaled by ostablishing Iast tnd reli.bl€ inlection, is very important lor th6 control sysr€ms lor parhotypo classiticotion which do not.6quir. tsdious inoculatlon The idenlllicalion ol PathotYPes b*sd on morphological, chomical is laborious and ralher time consuming and its rsproducibiliiy is ort6n sull3rs lrom .evoral dis.dvanttqes. poohnd Some n€w invdtig.tion. h5v. b..n cari€d out at th€ moi€cular l6v.l. In r5c€ni y6ars, s6voral groups hsve demonstr6ted rhat highlY and we.kly virut€nl isotarog of lungi ctn bo dislinguished on fi. bases ot DNA polymorphisms lJohnson & lewis, 1990 and Koch et al. 1991)' amplitic.tion prcducts goner6t6d bY random amplili6d polvmorphic DNA primeis lcoodwin 6nd Anni6, 1991 and Schatter 6nd WostemeY€., 19921, .t .1 19921, iDNA s6ouenc6 lMorales et !1., 19931 and the pres6nc6 ol . Paiticular rop.titive lins.rprinting with oliqonuclootid! prob.. tMaY.t DNA soquef,ce {Mor.les or al., 1993) is to An importahr dpplication of theso t€chnlqu6s is in popllation blologv estsblish tha id€ntitY 6nd r6lalodooss ot lndivid!'ls in popul'tions iBurke and &olord, 1987; Wostn6.t, l99O; Wetton 6i al, 19871 Tho66 lechniques 6r€ slso ideolly 3uitsd for studYing clonal popul'tion3 {Schor€r ol , 1991: Tu'ner ol tl" 5nd sr€veno, 1947i Borom€o et al , t99oi t€vv 1990) by marching DNA hybridizstion p. 'r 6ins on southern blots lt is rel.tlv.ly ersy to determino ths clonal idsntitv ol each individu'l bv DNA ol ggnotic div6rsitv dtpends oo s€v€ral lacior3' l'" lrom th6 random gsnetic drill lollowinq lh6 3ol€ction, immiqrstion The €xi€nt gonetic €nvironmont and meiotlc .ecomblnalion woutd lncr'6!€ vditbilltv lt lor can be concludo that DNA lingorprinting olt€r! soversl 'dvantago6 lt population g€n€iic snalY.i! tnd th' study ol host/pathogon co_€volution qrowth tai6' paihogenlc moro sensitivo than measur€menl ol sporulation' is lollows lnd physiologicll parareto.s. Oligonucl6otid' ling'rp nnng the psrricul'r par'm€rtrs ov6r 6mtrl rccurarc monitoring ot lh6 mov6ment oi ol palhogsn populations and larg6 goograPhic€l disl6nc6s, the dovolopment G6istling6r 6t al' over iim€, snd in r6spons6 lo dilter€nt host culliv'rs d6tecr' consldottbre l199Ol r€ponod that Oligonuclootido lingarprinting moleculat genotic div€rsitY boiwsen /4scociyta isolates countvwis' at th€ &e inct66s' ol regionat di6tanc6 lt bY lingerprintino tr6 r6vo6lod that all simplo .6p6titivo sequonc* t6ted lev€l .nd this divelsitv Incr6as6s wirh 119 to rom6 dogreo in all isol6tes ol A. robi.l- Ihis c5n b6 seen bv visual insp6crion or rn€ gen€rat6d prolilo ot lhe sttains isolat6d lrom ih6 pr€senr s.me geogtaphic area Mev€r 6t al. (1991) also reported thdl there was mor6 similo,iry borween the strains isolatod liom closff geographical 6tea lh6se obs€rua(ions indicsled that it is th6 €nvtonmenral oltect th.t r6voal s€netic dirt€rences 66 w6ll as similaririe6 botw.6n isoral'E ol A rcbi.i. F om lorcoino discossion, it can bo concloded th.t ths lingelprioting is very usolul t6chnique lor snalvzihg gen6tic div.t3iiv ol lunq'l isolat€s in conrr.t to conv6ntlon.l m.thod!. und.r rh€ pr6..nt 'xp'rld'ntt tll isolates ol /. rablbi showed more o, loss the same banding patt€rn in Iino€rprinting analysis lr indic.res lhat .ll isolates h.ve the stm6 genomc srroctura as ( b collect€d lrom lhe s.mo g6o0taphic'l area But ihoro are also some dilt6renc6s in abond6nce 6nd organizElion ol motils prGsent on th. whole genome ol I iablei i3olales indic6ting som€ gonetic dillerenc€s ,lnll .t€ found mosr stlitabl6 r63liiction in,4 /ablei oul ol 3it prob€s t€sled m exist€d in thear genome l€ql and enzymgs tor DNA linserp.intlng p'oduced oligonucleoride linqorprinting, (GATAt4 and IGGAT)4palt€rns mors distinct monomotphic bands, 66silv det€ctable in DNA liogerptinting analysb ol4. /rbie,. EHf,PTEB UE REfEFENCFS Alam, s. S., Bjlloo, J. N., Slawih, A. lvl- Z-, William, R- N., Sheps.d, R. N. and Slrange, R. N. (19891. Chickpga blight p.oduction phyfotoxins solanapyrooo3 A 2At 2627 2631 .id C by €l the A*ochyta .abiei, Ph|tochem. . Alah, s.S., Sl,a@e, R.N.6nd Outeshi, S.H. t1987). tsotsrion ol4scodryrt faOlel and a conv€ni€nt morhod tor copious inocutum producrion. fre Mvcolosist. 21.20 Ali, S., Muller, C.R. and Eppl6n, J.T. {19861. oNA tinserprihtins by oligonucleotide probos spocitic tor simplo r€pear6. Hum. Genet. 741 239.243. Atanasoll, O- and l(ov6chevsri, l_C. 11929). lPara3itic tuogi n6w for B0lg6rial Ftst conrribution Butt. So.. So. da Rulgaia Sofid 3t 45 32. Aujla, S, S. (19641, Siudy on ef6ven isotares ot phvttosticta nbtei lpa.6.l Trot, the causal lgent Phytopathol. 1 7 ol qram btight in th6 ptnieb, tndi.n : 83-87. Auir6, S. S. and B6di, P. S. {19671. Rerarivo reaction ot ditt6renr v.rioties sr.m to bfrghr dis.@€ inatt.r.d tty cauEal ag6nt 4:2r4 216. phvtostict. olg.am blight in th€ Punj6b. J. ot 6bi.i lp6$.t Troi rh. Res. puniab Agtic. Univ. Barr, W , Bless, W M€i6r, , BorOer D, Ollo, ch P.pendotl, G., Gunia, w, M.ct€nbrock, U , and Soper, E. ll99o). Phytoaloxins as pa.t oI ndlced defenc€ r€actions ih ptants: their olicilation, lunction and d€iabolism. ln: Bio€ctive compounds lrom plants (CIBA Folndation Sympostud 1541. pp. 14O 156, Wiloy, Chichoster. B6di, P.S and Aujl6, S.S. (!969). v.ri6bilitv in Frvl/ostdl, f.brei lPdss ) Troll , rhe incit.nt ol blight dasease ol gram. J. Fes Alniab Ag.i Uriv 6:103 106. J 11986l ln: Chitin in natlte and lechnologv lvlussorsillis, Jeunlaux, C snd Good6v, G W- eds. pp 234'230 PleDun Press, New Yo*. Benr.sconi, 6hd Pil6t, P.E B€rlini, S. (1956). S! di un composlo ad szio antibiorica Ascochvta pisilli6. Ann.SDerttt ,4gl. (Romal Bindcr, M. atld ramm, C (l973l N S lr: 545 556 e6le l13l 24 Oxa Ir4l Desoxaphomin, das cvtochaldsan. ein moglich€r bioqenetischer vorlauler cyrochalasane. prodotto da dd t/e/, chnh actd S6:946977 Boehringet Mannheim {l991'. DIG oligonucleotide labelling and detection kil, l.bo.atorY manhusr' Bo(om6o, E. S., Nelson, PoPUl.tion slruclur. Phvto|atltol. Ao: gar n., Bonm6n, J. fv4 and Leuns, H- 11990) ol rh6 rice bl6si lungls ln lhe Philippinos r22 Bonalico, A.. capasso, R,, Evid€nie, A,, Logrioco, A., B6ndazzo, G. and Vuro, M. (1989). Produciion ot cytochalasins bV isolates oI As.ochyta hetercmoryha kom oleande, €nd neqative evid6nce lor lhoi role in lo.t spor diseas€. NATO ASI Series, vol. H27. Phytoloxins and Plant Palhogenesis. Ed. Graniri 6t al. springerverlao B6.lin Heidelberg. Sreim.n, A. and Galun, E. 11981). Plant protoplssts as lools in qu€ntitaiive assays of phylotoxic compounds lrom Phytophthorc citophthorc. Physiol. Plant Pathol, 19:181 191. Brown, J.K.M., Odell, rvl., simposon, c.G. and Wolle, M.S (19901 Th€ use oI DNA polvmorphisms lo tesl hypothesis about a populalion ol Erysiphe ganinis l.sp, hodei, Plant Pdhtol. 39:391'441 8rown, S. S. and J. A. Spudich, 11981). Mechanism oI action or cyiochalasin: evidence thal it binds to aclin lilaments ends- J. Cel/ Biol. AA: 487 491 Aurke, I. . and &ulord, M. W. (1987). ONA linq.rpri.iing in bnds /v,&'e 327:149'152 Burler, E.J. {19181. Flngi and Diseases in Plants Tfiacker Spi.k & Co PM High Calc!tta, India pp, 244 251. Caetano Anoll€5, G., Bsssam, BJ snd Gressholl, {1991a) resoluiion DNA ampliJicaion nngerprinting using verv short 'rbntary oligonucl6olide prihers. 8/bte.r- 9. 553 557' caetano.Anolles, c., Bassam, B.J. 6nd cressholl, P.M. amplificarion fingerprinling a ll99lb). DNA strategy for genomo analysis. P/trr Mol.Biol. qep. 4t 294.39i.. capasso. R,. Evidente, A., Randa22o. G., nitieni, A., Boitalico, A., and Logrieco, A, (1987). lsolation ol cylochalasin A V!ro, M and B from As.ochyra heteromorpha. J. Nat. Products sot 989 99o. A-, Fiiieni, A., Rsndazzo, G., Vurio, M. and Bottalico, A. (19881. Ascochalasin, a new aytochalasin from Capasso, R., Eviden16, Ascochla hetercNryh.lsch, €t Sacc.) Cutzi J NaL P@ducls 51: 567-571. Cadson, P,S. 11973). M€thionine sulloiimine resisiant mulsnts of lobacco. Sc/:, 180:1366-1368. Chon, Y.rv4. and Svanqe, FN. i1991) Svnthesis or tho sol6napvrono phytotoxin6 by AscochYta rcbiei in response to m6ial ctlions and dovslopment of E denned m6dium loi toxin production P/"r P'r'ol 40: 4O1 4O7. ST 11995) Dift.renti.ring homorhallic Volvariella mushrooms bv RFLPS t.d AP_PCR l4vcological Res 99' Chiu, S.W., Chen. M. and Chang6, 333-336. Collm6r, A. and Keen, N.T 11986l Th€ rol€ ol poctic 6nzvmes in plant pathogenesis. ,4t . 8eL Prvtopathol 24t 3A3'4O9 Coops., J. A., Bryan. J., Schwab,8., F,ied€n, C., Loftus, D. J.6nd Elson, ..t|3. J. cet Biot. ot recognilion in planl diseass.,4n. Bev. E. 1.11987). Microiniection or solsolin inlo livins 491.50r. Daly, J.M. (1984). Th€ role Phvtopathol. 22t Dani6l, S. and 27 3-307. Bar, W. 11990). oi Elicitor indlced metabolic chanses in c€ll L) cultivtrs .osistant and susceptibfe ro ,4s.ocl'yra Qbiei. ii Dillerc^rlal inductiofl ol chalcone synlh.se mBNA aclivity and analvsis ot It viro ir.nsloled prolein cuhurss chickp€€ lcicet aietinun patterns. P/r,ra 142: 279-286. DaNill, A- G. and Albersheim. P. 11984) Phytoalexins and thoi. elicirots- A defens€ againsr mifiobial inl6ction in plsnt. ,4rn. Rev. Phvtoprthol 351 243 215. oescenzo. R.A. and Harinston, T-C- (1994l Use of {caTls as a ONA linoetprinting probe tor lunqi. PhvtopathoL A4:534 54O (1986). The phytoal€xin r€sponso: elicitation signalling and ot hosr gene €xpression A,bl Rev 61:239 291- Toiin in Pl.nt Dise.56s. New Yo.k Acad6mic 515 pp, Ean6, E.D. (1987) Phvlotoxin srudies with plant c€lls 8nd pfotoplasts ln tnretnalional Frcntiers ol Ptaht Tissue Conure, Ptoceedings ol the lvrh Ed bv Congtess ol Plant Tissue .nd C€ll Culture, calsary' Atb€rta T.A. lhorPe, PP.363'372. t25 J. {19861- Ebel. 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S (19801- Cvtoch6lasin block actin filamenl wilh F €long6rion bv binding to high aliinilv sit6s associated actin J 1iol. Chdn 255. 838 834Flor. HH {1971) Curent stalus ot genejor gen€ co^cepr 9.275 296- A'n Rev G6mborg, O.1., Miller, R.A. and Ojima, K. 11968). Nut.ienl ,equnemonls ot ol soyb.an roor cells. Exptl. CeI Res.50: 144 151. Geisnihg€r, J., M.qbool. S., (aiser, w-J- and Kshl, G. {1996). D6t€ction nicrosatellito ling€rpJinl mark€rs 8nd lh6n msndslian inhorilanc6 Ascochyta rcbiei, Mycol, Res, 9: 1113 1121. Gendlolf, E.H., Scheffer, B.P. and SomoNille, SC (19871 An improved bioassay lor victorin based on the use ol oat protoplasrs Physiol. and Mol- Plant Pathol. 31,421-427. Gengenbach. 8.G., Green. C,E .nd Donov.n, C M. 11977)lnhoritanco or selected pothotoxin r€sittancs in maize plsnG regen6ralsd lrom cell .unu's. Ptoc. Natl. Acdd. 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