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
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