J. Phytopathol ogy 136,238-216 (1992)
ô
ttô, it""f
PaËy Scientific Publishers' Berlin and Hamburg ISSN 0931-1785Facuhé d.es Sciences
Agronomiqwes,
5030Gemblowx' Belgium
Effects
of Commercial FattY
Acids
on Cutinase
Release
by Ascochyta
pisi
B.
N.tsn'qout,
P'
Lrlotvtr
andJ'
Sr'rrl'q'lAuthors'addresses:B'N'c'snaout,EcoleSupérieured'Agriculture'7l19le'Kei'Tunisia' P. Lepotvnr ,,ta J. srn,it-,
rlt"tia'at'
ScienËes Agronomiques' 5OlO Gembloux' Belgium'Wttb / Jtgures
Recei'ued October 1,1991; accepted December 12' 1991
Abstract
TheeffectofarangeofcommerciallyavailableCl5andCl3fattyacids,onthereleaseofcutinase
b" Ar;:;i;;;-;,r, *"r
r,îdi.,i.
iîir."l6'p.ric
acid.wai us.d a, sol. carbon source, cutinase activitv was released in the culure*.ai"-,
.riJ*'"s
markedly enhanced by post-treatment of a' pisi.cultures with acetone used as.n.y*.
.",.ra,or.
Without acetone, lar, a,]iinata was naturally released in .ui.,*
_.di,.rln containing juniperic acid at 0.5 %,rhln
ata.ol o/o or 0.05 %. lJpon post-treatment with acetone, the same l.u.t of.i,inrr.
was released with âll three concentrations' thus suggesting that;;;rr-.;r-ina,.r..a,
U*
""r
..-pf.*ly
..1.,.ed in the presence ofO'5 o/' juniperic acid' lflhen
ricinelaidic or ricinoleic
..iJr-;;;; ;ipfl.â"n,.d
at 0.5 % to cutinin
the culture medium' they strongly inhibited ,h. ,.1."r" of.,r,i."r.,
"u..
with acetone Post-treâtment comparable inhibition by ricinoleic acid was"tro
our.*".i
*ir." ;r"ip.r;.
acid wasusËd a, .utinas" inducer, thus suggesting that
;;;;;l;;;
..1.rr.,brt
also thep'oiu'tion
of cutinase were inhibited'Zusammenfassung
EinfltssevonkâuflichenFettsâurenaufdieCutinasefreisetzung
durch Ascoclr Yta PisiUntersucht wurde der Einflujl von kâuflichen C16- und C13-Fettsâuren_auf die Freisetzung von Cutinase durch Ascochyt,
p;;.'\il;J
j.tnip".râu." als einziee Kohlenstoffquelle vorhanden vrar' wurde Cutinase in dasfrf,rr-.air.
Ër.iglt.r.t, und dieswird.
du,ch einà Nachbehandlung derA. ptsl_Kulturen
.i, a..ro,,]"li;r;;;;?;1,";,
stark erhôht. ohne die Acetonbehandlung wurde*."1g.. c"ii."se
in dasKitur-edium
freigesetzt, w.enn Junipersâure in einer Konzentration von0,5 7o statt 0,01 % oder
0,0;;;-;.h";d..r
ïrr.
Nach einer Âcetonnachbehandlung jedoch wurde die gleiche Cutinasemengetei
allen Konzentrationen freigesetzt, was darauf hindeutet' daf3 das Enzym induziert, aber im Vo.h.nd.nr.in von 0,5 7o Junipe"rsàure nicht vollstàndig freigesetzt war'Effects of Commercial Fany Acids on Cutinase Release 239
\wennO,5%Ricinelaid-oderRicinolsâureCutinimKulturmediumbeigemischtwurde's.urdedie Freisetzung
'on
Cutinase;;';;";;h*.;;;, *ln
"r.t.'
einer Acetonnaihbehandlung' Auch s'ennTunioersâure
"1,
Crrln"r.injr.i.r.? rl"r*"nd.,
wurde, s,urde eine gleiche, durch Ricinolsàure
verur-Ï.rt'..,ù:iff;;;;;;;;;;i.,
a.,,.,
darauf hin, dafi nichtn"ii"
F'ei"tzung' sondern auch dieProduktion uon Cutin"se gehemmt wurde'
Cutinase
is
an
esteraseable
to
hydrolyze cutin'
a Poly.mer9f
Ct'
and
Crsfattv
acidswhich
i,
tt'.
-;,]
.o.npon.n,
of plant cuticle.
Cutinase isproduced by
il,LiiÏ}ii,li;,t;;;;;;;;,,,iàn
in
thehost-plant.
rhe
mostimporranr_works
;;;;.;;;î
in,
,îu;.i,
i.'.
t'*-'rized
bv
Kototru*uoY
(1e8s) and
KÔ'rn
(lee1.).cutinase
is
known to
be induced
in
fungi by
using
cutin
as sole sourceof
carbon. Fungal
.urinr,.
;; ti;;
induced
by"lo*
concentration-sof
fatty
acidsextracted
fro- hydrj;;.d;;;
(Lr:r
and.(oto"'*tDY
1978'wot-osHun
andKor-.crruxuoy
1986,i;;;r;;;.i.-rsss)'
irs activity.was
severelyinhibited
by
reasents
directed towards active serine' such
"
diitoptopyl
{luorophosphate
1OËf'; (Punor
and
Kornrrur'unv
1975)'In
apreviou,
prp..
(Nasnaour
ei.al'
1990)'Ye
have sh.oyn
that two
peanathosens.
urrorpti[,,'tù'pi'"'itlfnerk' tr
Blox';
Vestergr'
lA'scocltl'ta pinodesÏ;;.;i'';i'i;;;ii;;"-p"'tliu''
"1""d
cutinàse
actiritv
when
gro*n in
amedium containing.u.i,]
",
sole sourceof
carbon;
this
cutinase wasinl.olved in
the
successfulinfection of
the host'
In
the presen,*î.f.,^*-
ri,rJi.a
the
effectsof
somecommercial
C16 and Cisfatty
acidsàn in
ritro
releaseof
cutinaseby A'
pisi'
@
Materials and
Methods
Growth
conditionsSpore suspensions of ,4 pisi (strain
LG'
kindly.clonated b'vDr
Slrnt'INRA'
France)I'ere rubnrerred in rhe culrure n,edja'{de.cribe d in C,tinase intltr,tion par'rgraph) in. Erlenmer er tlask'' to
reachafinalr,olunreofl0mlcontaining5xlOsspores/ml.The.ialswereincubatedonârotârv .haker at 16'C. wirh a phoroperiod of to h Jight'
For cutinase
r.rl"i,y'ii*'""-tn"
''l
"prsi-culttresof
15 days q'ere filtered; filtrates were coilected and m,vcelium",r;';.r;r;;;à;i
i.
?r.tt, -1,,"eral solutionof a modified czapeck-Dor's medium
(Drcxlt'lx
-a
pottt
lsôà) conraining 20% 1r/r;
of acetone used as potentialenzl'me extractor
(L,lunrxr
rrsz;. Afterz 1-', ,1-'.,u,p.,.",io-n*,,
fil..,.d
â3]il1 to discard the nrr'celium.For esterase
".,itit1"'tt'*tt-tn""4
p'slttltu"'
o'ere int'Ëatcd for2'{ to 72
h
At the cnd of incubarion period..li,rillcdïr,.r. o,,..,oi.
ur.d as enzrnre exrrrcror. *ere addedrlo"o '
r) Incubation s.as continued
f";;';,'r;;.ulrur.,
*.ere finally fiJtered to discard rhe m1'celium.Cutinase
induction
To induce cutinase, the mineral solution of the modified Czapeck-Dox,s mediun-r (Dtcxiu,tN
and paru_ 1986)
s,as,.rr."li).ii. fH
) *irrr prr.rphoric acid, ".d o'"s supplemenred'irh
0.2 %(r'/
v) cutin and/or differen,.on..n,ri,ion,
of,h.
ioilo*'ing co'-rmerciallv auailableC't
and C15 fattyacids:
-
palmitic acid (hexadecanoic acid) from Sigma' .- i".rp..i.
acid'(16-h1-drox1'hexaiecanoic"tid;
ftorn Aldrich-Chemie'-
,t""ri.
acid (octadecanoic acid) from Sigma,240
Nasnaout, Lr'Potwr' and Seual-
I2-hyàroxystearic acid (DL-12-hydroxyoctadecanoic acid) from Aldrich-Chemie'-
.l.ir!f.iai.
acid ([+]-12-hyd.o*y-tt'n'-9-octadecenoicacid) from Sigma'
-
ricinoleic acid (tÀl-iz-hyiroxy-cis-9-octadecenoic acid) fromSigma'
cutin vras purified from peelings of apples cv. "Golden Delicious", as describedby B'r.reR and
norun^* ftrzs;l 1.he.in.."i
.oluiio.r*rr,.,r.d
as cutinless control medium'All
media ''ere autoclaved for 20 min at 120"c'
cutinase activity
cutinase activity released by
A.
pisiwas measured by microtitration of the fatty acids releasedfrom a cutin subsrrate. This -ethJd
*"r..r.d
wirhA. pisig.o-n on medium containing iuniperic acid as sole sourceol
carbon. ,4. pisi culture filtrates were dialysed for 48 h àgilnst drstllled water' ànolvoohilized. Lvophilized filtiates were incubated for 24h at 35 "C in the
iutin
reaction medium ofi.;i;ï;;;'iiuôïll.,a.
oiz
a
or 1mlvl phosphate bufferpHl,l
ml of cutin suspension at;ô;;r;i
"na'"
arâp"f
;"il.;;.
The 'eaction-tdi'-'-
*'s
'upplemented,or notwith
10mM à;lroJroo, t fluorophosph:r.iOefl.
a cutinase activity inhibitor (Pr:not andKolnrn'ruDY
1975)'il";:rti;J;;.i;;;'iil
T,*r, ,t."
filtered,"nà pH of thc iiltrate was adiusted
to J'5 with phosphoric acid.
'"--'r"rry
acidsin
the filtrate (3ml)
were extracted 3 times with diethyl ether, followed by evaporation ,rnd.,- u".un..îi.y J.*.r'rr."
dissolved in methanol and titrated with NaoH (from the|;ii"l ;H ;p
to pH 8.2).p"t-;ii.
acid was used as standard, and cutinese activity *'as expressed as"ritr
1i-l;z-i of filtrate(l
U
:
r prg of palmitic acid equivalent/h)' Esterâse activitYcutinase is also evaluated by measuring esterase activity ,urrngpara-nirrophenyl.esters.as model substrate
(Korarruruov
1985).In
the present work, we evaluated esterase aclrvlty usr.,'g P4r4-nitrophenylbutyrrt"(pNpBj "r'rrrùrrr"," indpora-nirro,phenol IPNP) asfinal product,asrevealedbymeasuring absorbance
r,4#;;. TÏ" ,n.y:*iX.e..tion
was c.rrried out in 7 ml of 3o mM phosphate;;fi;
;Fî
i, *i*.d -irt
r "rt ori.
p,rl'cultr.e fiçrate and 2 ml of 1 mM PNPB in the phosphateirii.. ipi-i-i*
rw et a;.1978). Esterâse activity was expressed as units (U)/ml of filtrate (1 U:
1 irlvl PNP/min).Results
Induction of
esteraseâctivity
with fatty
acidsAmong the
fatcy
acidsused ar
0.05 o/oor
0.5%
assole source
of
carbon,;.,,'1pA.l.ia
f
ro".j
to
bethe
only powerful
inducer
of
esteraseactivity
reieaseabcdgl
Fig. 1. Esterase activity released by A. pisi,after 24 h of culture in media containing o'05 % or 0'5 % - -"oi
diff.rrn, f"tty
"É;d, used as'sole carbon
source, followed or not by acetone Post-treatment
a: mineral solution (MS) b: N4S+palmitic acid c: IMS+juniperic acid d: MS+stearic acid e: MS+12-hydrox/stetric ac f : N4S+ricinelaidic acid g: f,4S+ricinoleic acid without acetone N acelone Post'treatrnent | : stmoara deviation
F-,*"tuGtrâr6""iô-5%l
sEffects of Commercial Fatty Acids on Cutinase
Release
241af.ter 24
h of
incubation, when
A'
pisi
cultures were
Post-treated
with
20 %;;;
pig.
fj.
Without
acetone po_st-treatment, esteraseactivity
.eleased after;4;iyÀ.i*i,
wasalmost
nil
for-all fatty
acids used'Induction of
esteraseactivity
by juniperic acid 'Whenincreasing concentrations
of juniperic
acid,were
uttl
tt,
t"lt
:Î:-!-":
source,
high
esterase"actitrity was
released-aker 24
h
by
'4'
prsl' when
cultures;;Ë;rï;eated
with
"..,o.,.
(Fig.2).
No
significanr
esreraseactivity
was
released
without
acetonepost-treatment'
4.5 : 4.0 E .' J.U Fo 6 2.s @
0
:.0 o 6 LrJ 1.5 without acetone ô.0 ô f 0.4 0.5Juniperic acid concentratjon (%)
Fie.
2.
Esterase activiry released by A."pisi afrcr 24 h of culture in mediacon-t"itrittg
increasing concentrations ofjunipeiic
acids usedas sole
carbonsource, followed or not by acetone
post-treatment,, (T: standard deviation)
1.0 0.s 0.0
Frtl,r"uf
I acetone_.1 :.ofJ post' - _1----:i:Ë:=---::::?
0.05 %o.o1 a/ô0.5 %
I
I ltreafnentlTzLoI=È,
II,'È
I
-ï
,ii
I :str
tt'V.J
,ll
,!o.ost"--
1
,,,iI//
t/
// I ,rl
,', tltt,oI
ll
/ I
-l
o.o, 'n',/
t ,---/,fio.s"t"
1,/
-+t
r"'
/ --'' t -,/
o/-7-,--:!'
oo"a----'l---'---Juniperic acid concentraiion (%)
:40 E I J.s .> J'U F L (u 2.5 o (Û ^^ o a 1.0 0.5 0.0 72 o2444 Time (hr) Fig.3.Ester:rseactivityre)eased byA.pisiafter.cultureinmediacontainingdifferentconcentrationsof
;#;;;;;t;
"'.i
",',ot.
.*#i;;;:;:i;T:ffi":l
""t
bv acetone Post-trearment'(r:
standard'72
Time (hr)
J. PhytopathologY, Bd. 136, Heft 3
242 Nasnaour, Lrpon'r.r and Seuar
When the culture period
in juniperic
acidmedium
was extendedup
to 72h.
the
releaseof
esteraseactivity
increased, and washigher
with
0.01'/"
or 0.05'k
than
with
0.5%
juniperic acid. Without juniperic acid (control),
no
esteraseactivity was
released (Fig.
3).
Acetone post-treatment resulted
in
the
similar
releaseof
esteraseactivity
for
the three concentrations
of juniperic
acid.Cutinase
âctivity
induced byjuniperic
acidThe
enzyme
released
by A.pisi
grown
in
culture medium
containing
juniperic
acid
(with
or
withor.rt
acetoneextraction
from
the
mycelium)
showedcutinolytic
(cutinase)
activity, and was inhibited
by
adding
DFP, a
cutinaseinhibitor, to
the enzymatic reaction medium (Table
1).
(
Table 1
Cutinase activity of A. pisi, as measured by the method of microtitration of fatty acids released from cutin substratet)
Treatment Cutinase activity (U/ml)
Control without lyophilized culture filtrate Lyophilized culture filtrate
Lyophilized culture filtrate
*
DFPLyophilized acetone-treated mycelium filtrate Lyophilized acetone-treated mycelium filtrate
*
DFP0.15 0.60 0.1 8
0.84 0.15
t) A. pisi culrures grown
for
15 days on juniperic acid medium, were filtered and the fiirrate wascollected. The mycelium was resuspended
for
2h
in
fresh mineral solution containing 20 % acetone, and then filtered again. Both filtrates were dialysed, lyophilized and mixed with a cutinsuspension for 24h, in the presence or not of 1O
m}l
of the cutinase inhibitor DFP. The releasedfatty acids were extracted in diethyl ether, dissolved in methanol, and microtitrated with NaOH.
'
Inhibition
of
esteraseactivity
by{atty
acidsThe addition
of
0.05 o/ofatty
acidsto cutin in
the culture medium
did not
modify
thelow
esteraseactivity
released byA.
pisi
after 24 h.Post-treatment
with
acetoneinduced
alarge increaseof
the releaseof
esteraseactivity
byA.
pisi grown
on cutin
alone (control), while addition
of
stearic
acid
to
the cutin
medium
reducedthe
reieaseof
esteraseactivity by
half.
Addition of
0.05 7oof
other
fatty
acids
to cutin
had
little
effect (Fig.
a).Addition
of
A.5%
fatty
acidsto
the
cutin culture
medium resulted
in
low
esteraseâctiviry in
all
caseswithout
acetonepost-treatment. Post-treatment
with
acetone
largely
increased esterase release and reachedalmost
the
sameievel
for
cutin
alone,
or for
cutin
supplemented
with
palmitic, juniperic
or
12-hydroxy-stearic acids.
Incubation
of A.
pisi
in
cutin medium
supplemented
with
O.S % stearicacid,
releasedonly
1/3
of
the
esteraseactivity
releasedin
the
cutin
alonecontrol.
Vhen
0.5ok
ricinelaidic acid
or
ricinoleic
acid was
addedto
the
cutin
medium,
releaseof
esteraseactivity
dropped
to
almost nil.
ç
72h,
.a5 % leraseimilar
,ln1ng orved ;inase 'ml) ie v/âs 2.0% r cutin :leased aOH.I
not
with
rown
dium
fatty
rlow
with
'1for
oxy-.5%
rlone:urin
6
4..
qabcd. by A. Pisi after 24 h of culture o.s
U
àt
different fattY acids'treâtment
5.0
Acids on Cutinase
o.o
Inhibition
of
esteraseactivity by
ricinoleic acidAfter
24h
of
culture,
little
esteraseactivity
was releasedby
A'
pisiqt?*n
i1
cutin
mediurn
,,rppt.,itï*J
*ith
inc'ea'ittg'concentrations
of
ricinoleic
acid(Fig.
5).upon
post-trea[menr
vrith
acetone,tie
releaseof
esteraseactivity which
was
high at
low
..".;;;;;;i;"'
àt
t;t,noleic
acid
in
the cutin medium (up
to
0.1,
.k),decreased
rh;Ë;;;;;;*,g
ricinoleic
acidconcentrarion,
to
reachalmosi
nil with
0.4"k
and0'5
%'
abcdel
Fig. 4. Esterase activity released
supplemented with 0'05 7o or
Fie. 5.
Esrera:e activity released by A."oisi a[rcr 24 h of cuhure in media con-taiiing cutin 10.2'b)
supplemented q'irhincreaiing concentrations
of
ricinoleic acid, follon'ed or notby
âcetonePost-treatment,
(I:
standard deviation)When the culture Period
wasobtained (Fig.
6).in media containing cutin (0'2 %) followed or not bY acetone
Post-acetone post-"treatment
"1..
without " -
,
acetone
'-
+---'\--.----l
---'
0.1
a.2
o)
.( BËinoleic àôid concentration (o/dadded to cutin susPension (0 2 9i')
0.5 5.5 4.0 3.0 2.5 2.4 1.5 0.0 1.0 0.5
â
E)
',
F o (6 o o t-rlEffects of Commercial FattY
a: cutin (0.2 7d (C) b: C+pâlmitic acid c:C+juniPerjc acid d: c+stearic acid e: C+1 2-hydroxystearic ac f: C+ricinelaidic âcid g: C+ricinoleic acid I without acetone N acetone Post-treatrnent | : standard deviation
ForlcentraÏon -ot oos
9âl
s(_. ç '5 J.0 F o G -. 6 l! t.s 1.0
Nasraout, Lrporvru and SrIllal
Bicinoleic acid concentratjon (7d added to cr,rtin suspension
(0.2n
ô'
Time (hr)
48
Time (}tr)
Fig. 6. Esterase activity released by A. pisi after culture
in
media containing cutin (0.2 %)sup-plemented with different concentrations
otr.*:ïn:.;,11,f;:ti*.0
or nor by acetone posr-rrearmenr,Juniperic and ricinoleic acids as carbon source
for
A.
pisiCulture
of A.
pisi
for
72h in
amedium containing
A.05% juniperic
acid ascarbon source,
releasedan
esteraseactivity which
was
enhancedby
post-treat-ment
with
acetone.
In
contrast,
when
0.5%
ricinoleic
acid was used as
soiecarbon source,
A.
pisi
did not
release
a
significant
esreraseactivity.
When
supplemented
to
medium containing
A.05%
juniperic acid, ricinoleic acid
at 0.5%
inhibited completely (without
acetone)or
strongly
(with
acetoneposr-treatment) the
releaseof
esteraseactivity
(Fig.
7).Fig. 7. Esterase activity released by A. pisi after 72h of cuhure in mineral medium containing O.O5 %
juniperic actd and/or 0.5 % ricinoleic acid, followed or not by acetone post-rreetment
5.5 5.0 : 4.0 C I 3.5 o 6 2.s o E r.o 6 l.! t_5 1.0 0.5 0.0 TOo/. 1 -æ|lot'a1. .4+---tOos'Â ta' ! 1.
ç4o
6-=-3-o05o,'o a: mineral solLjtion (MS) b: MS+juniperic acid (0.05 %) c: l\,,1S+ilcinoleic acid (0.5 %) d: MS+juniperic (0.05 7d and ricinoleic (0.5 7d acidsI
vrithor,'t acetoneN
acetone post-treatrnent standard deviaïonEffects of Commercial Fatty Acids on Cutinase Release 245
G'
Discussion
Cutinase has been
usually induced
by
cultivating fungi
on
cutin
as
solecarbon
source(KorarruruDY i985, KÔuan
rlfl1'
1n
iht
ttit
of F'
solanif
'
sp'pisi,
cutinase was alsoi;;;
tt
tttl"
fatty
acids(LrN
andKole'rruruox
7978'
..silo,os'u*andKole,rrurulvlg86,Pontleetal.t'eat)'Inthepresentwork,we
used comme.ci"l
frttf
;;;il "'
t"'bo"
toutt"
fo'
À'
pisi'
ind
showed
that
juniperic
acid is
.
gooâ
i.,d,r"",
of
thein
,it
o-r4"^r" of àrt.r"se activity by
this
tun*ïr,n*
the
method
of
microtitration
of
fatty
acids
rereasedfrom
cutin
substrate,
*e
co,tfi'n".]
;h";
;ht
'nty^^'iJ'^*i;t
induced
by
juniperic
acidl*ith
or without
acetonepost-treatmènt) is indeed
cutinase',*"'ïÏ,rrl'."î;;;;-;",
released
by
A. pisi
)fr.
z+h of
culture
in
a
mineral
medium
supplementeff;l;;ù. i:
"Ji
c"tit""
activity
increasedwhen
the
culture period
*.,
.",lnJ.
Â't"
ih,
but
less cutinase was .eie"sedin
themedium
.;;;;il;0.5
%
j"i;;;i.
"tid,
thanwith
o'01'% or
o'05
%' After
acetonePost-treatment,
the
level
Jf
"lt""d
cutinasewas
the
samewith
0'01"/o'
O'05
7o
or
0.5
%
juniperic
acid' These
resul"
"'ggt"
th"t j""-ipttic
acid induced
thesynthesis
of
cutinase atail three concentrations,
whiie
ttre releaseof
this
enzymei;i;r;;iiil;;^hÀi;.Juy
6.s
% juniperic
acid, and resrored
by
post-treatment
with
acetone.Tostudythepossibleinhibitionofcutinaserelease,themineralculture
medium
*",
,uppl.-*ttd
*i'h
amixture of
tt'ti"
and commercial
fatty
acids'Ricinoleic
a.,drici.,.laiài"
".ldr
ato.5
o/ostrongly-inhibited^cutinase
release, evenafter
acetonepor,-a.""t*"nt,
but.were
without
effect
at0'05
%'
Among
"U
f"*yîtiJ' lt"ta,
ricinoleic a"id
*"s
the
strongest
inhibitor of
curinase release
by
A.
pisi.At
0.5'7o,
it
severelyinhibited
the
releaseof
cutinaseinduced
by
cutin
",
,iil;;;r.il"rr.i
^,
,o1,r.. of
carbon,
but
was
without
effect
at
o.o1%
,,
ô.6;'ù:.
This
indicates
that'
in
contrast
to
iuniperic
acid'ricinoleic
acid
might-i'JiÉi;;";
only the
;.i;;;;,
but
alsothe induction
of
thetn"ËL
overall
results
suggestthat
some
commercially
available
C15and
C13fattv
acids are able,. i.a".?ït
inhibit
thein aitro
ttlt"'e
of
cutinaseby
A'
pisi'
:i,#ï'i;;;;
;;Ë';;;;;;i;;;"
"r
the rungar cutinaseproduction during
t'lr.
pro..r,
oi
ittf..tiott
of
the
host-plant'
Vethankthe..AdministrationGénéraledelaCoopérationauDéveloppement,,(AGCD)'
Brussels, Belgium, for support to B' NasRaoul'
Literature
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y.
HrNrs, 1e85: Detectionof
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;.
;i;i;;;iïl.p1a
'ia
sËnsiriue plate asiav for the detectionof cutrnase
"'-*';;od;;;J'uv
pl"" f"thoi"'i'
fungi' Phvtopathologv 76'47*75'
246
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ind
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