Effects Of Commercial Fatty-Acids On Cutinase Release By Ascochyta-Pisi

J. Phytopathol ogy 136,238-216 (1992)

ô

ttô, it""f

PaËy Scientific Publishers' Berlin and Hamburg ISSN 0931-1785

Facuhé d.es Sciences

Agronomiqwes,

5030

Gemblowx' Belgium

Effects

of Commercial FattY

Acids

on Cutinase

Release

by Ascochyta

pisi

B.

N.tsn'qout,

P'

Lrlotvtr

and

_J'

Sr'rrl'q'l

Authors'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 of

O'5 o/' juniperic acid' lflhen

ricinelaidic or ricinoleic

..iJr-;;;; ;ipfl.â"n,.d

at 0.5 % to cutin

in

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 was

usËd a, .utinas" inducer, thus suggesting that

;;;;;l;;;

..1.rr.,

brt

also the

p'oiu'tion

of cutinase were inhibited'

Zusammenfassung

EinfltssevonkâuflichenFettsâurenaufdieCutinasefreisetzung

durch Ascoclr _{Yta Pisi}

Untersucht 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 das

frf,rr-.air.

Ër.iglt.r.t, und dies

wird.

du,ch einà Nachbehandlung der

A. ptsl_Kulturen

.i, a..ro,,]"li;r;;;;?;1,";,

stark erhôht. ohne die Acetonbehandlung wurde

*."1g.. c"ii."se

in das

Kitur-edium

freigesetzt, w.enn Junipersâure in einer Konzentration von

0,5 7o statt 0,01 % oder

0,0;;;-;.h";d..r

ïrr.

Nach einer Âcetonnachbehandlung jedoch wurde die gleiche Cutinasemenge

tei

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'enn

Tunioersâure

"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 nicht

n"ii"

F'ei"tzung' sondern auch die

Produktion uon Cutin"se gehemmt wurde'

Cutinase

is

an

esterase

able

to

hydrolyze cutin'

a Poly.mer

_9f

Ct'

and

Crs

fattv

acids

which

i,

tt'.

_-;,]

.o.npon.n,

of plant cuticle.

Cutinase is

produced by

il,LiiÏ}ii,li;,t;;;;;;;;,,,iàn

in

the

host-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 source

of

carbon. Fungal

.urinr,.

;; ti;;

induced

by"lo*

concentration-s

of

fatty

acids

extracted

fro- hydrj;;.d;;;

(Lr:r

and.(oto"'*tDY

1978'

wot-osHun

and

Kor-.crruxuoy

1986,

i;;;r;;;.i.-rsss)'

irs activity.was

severely

inhibited

by

reasents

directed towards active serine' such

_"

diitoptopyl

{luorophosphate

1OËf'; (Punor

and

Kornrrur'unv

1975)'

In

a

previou,

prp..

(Nasnaour

ei

_.al'

1990)'

_Ye

have sh.oyn

that two

pea

nathosens.

urrorpti[,,'tù'pi'"'itlfnerk' tr

Blox';

Vestergr'

lA'scocltl'ta pinodes

Ï;;.;i'';i'i;;;ii;;"-p"'tliu''

_"1""d

cutinàse

actiritv

when

gro*n in

a

medium containing.u.i,]

_",

sole source

of

carbon;

this

cutinase was

inl.olved in

the

successful

infection of

the host'

In

the presen,

*î.f.,^*-

ri,rJi.a

the

effects

of

some

commercial

C16 and Cis

fatty

acids

àn in

ritro

release

of

cutinase

by A'

pisi'

@

Materials and

Methods

Growth

conditions

Spore suspensions of ,4 pisi (strain

LG'

kindly.clonated b'v

Dr

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-culttres

of

15 days q'ere filtered; filtrates were coilected and m,vcelium

",r;';.r;r;;;à;i

i.

?r.tt, -1,,"eral solution

of a modified czapeck-Dor's medium

(Drcxlt'lx

_-a

pottt

lsôà) conraining 20

% 1r/r;

of acetone used as potential

enzl'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'sl

ttltu"'

o'ere int'Ëatcd for

2'{ to 72

h

At the cnd of incubarion period..li,rillcd

ïr,.r. o,,..,oi.

ur.d as enzrnre exrrrcror. *ere added

rlo"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 auailable

C't

and C15 fatty

acids:

-

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-octadecenoic

acid) from Sigma'

-

ricinoleic acid (tÀl-iz-hyiroxy-cis-9-octadecenoic acid) from

Sigma'

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 released

from a cutin subsrrate. This _-ethJd

*"r..r.d

wirhA. pisig.o-n on medium containing iuniperic acid as sole source

ol

carbon. ,4. pisi culture filtrates were dialysed for 48 h àgilnst drstllled water' àno

lvoohilized. Lvophilized filtiates were incubated for 24h at 35 "C in the

iutin

reaction medium of

i.;i;ï;;;'iiuôïll.,a.

oiz

a

or 1mlvl phosphate buffer

pHl,l

ml of cutin suspension at

;ô;;r;i

_"na'"

arâp

"f

;"il.;;.

The 'eaction

-tdi'-'-

*'s

'upplemented,or not

with

10mM à;lroJroo, t fluorophosph:r.

iOefl.

a cutinase activity inhibitor (Pr:not and

Kolnrn'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

acids

in

the filtrate (3

ml)

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 activitY

cutinase 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,asrevealedby

measuring 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 or

i.

p,rl'cultr.e fiçrate and 2 ml of 1 mM PNPB in the phosphate

irii.. 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

esterase

âctivity

with fatty

acids

Among the

fatcy

acids

used ar

0.05 o/o

or

0.5

%

as

sole source

of

carbon,

;.,,'1pA.l.ia

f

ro".j

to

be

the

only powerful

inducer

of

esterase

activity

reiease

abcdgl

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

s

Effects of Commercial Fatty Acids on Cutinase

Release

241

af.ter 24

h of

incubation, when

A'

pisi

cultures were

Post-treated

with

20 %

;;;

pig.

f

j.

Without

acetone po_st-treatment, esterase

activity

.eleased after

;4;iyÀ.i*i,

was

almost

nil

for-all fatty

acids used'

Induction of

esterase

activity

by juniperic acid 'When

increasing 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

esrerase

activity

was

released

without

acetone

post-treatment'

4.5 : 4.0 E .' _J.U F_o 6 _2.s @

0

:.0 o 6 LrJ 1.5 without acetone ô.0 ô f 0.4 0.5

Juniperic acid concentratjon (%)

Fie.

2.

Esterase activiry released by A."pisi afrcr 24 h of culture in media

con-t"itrittg

increasing concentrations of

junipeiic

acids used

as sole

carbon

source, 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/ô

0.5 %

I

I ltreafnentlTz

LoI=È,

II,'È

I

-ï

,ii

I :str

tt

'V.J

,ll

,!o.ost"--

1

,,,i

I//

_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

acid

medium

was extended

up

to 72h.

the

release

of

esterase

activity

increased, and was

higher

with

0.01

_'/"

or 0.05'k

than

with

0.5

%

juniperic acid. Without juniperic acid (control),

no

esterase

activity was

released (Fig.

3).

Acetone post-treatment resulted

in

the

similar

release

of

esterase

activity

for

the three concentrations

of juniperic

acid.

Cutinase

âctivity

induced by

juniperic

acid

The

enzyme

released

by A.pisi

grown

in

culture medium

containing

juniperic

acid

(with

or

withor.rt

acetone

extraction

from

the

mycelium)

showed

cutinolytic

(cutinase)

activity, and was inhibited

by

adding

DFP, a

cutinase

inhibitor, 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

*

DFP

Lyophilized acetone-treated mycelium filtrate Lyophilized acetone-treated mycelium filtrate

*

DFP

0.15 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 was

collected. The mycelium was resuspended

for

2

h

in

fresh mineral solution containing 20 % acetone, and then filtered again. Both filtrates were dialysed, lyophilized and mixed with a cutin

suspension for 24h, in the presence or not of 1O

m}l

of the cutinase inhibitor DFP. The released

fatty acids were extracted in diethyl ether, dissolved in methanol, and microtitrated with NaOH.

'

_Inhibition

_of

_esterase

_activity

_by

_{atty

_acids

The addition

of

0.05 o/o

fatty

acids

to cutin in

the culture medium

did not

modify

the

low

esterase

activity

released by

A.

pisi

after 24 h.

Post-treatment

with

acetone

induced

alarge increase

of

the release

of

esterase

activity

by

A.

pisi grown

on cutin

alone (control), while addition

of

stearic

acid

to

the cutin

medium

reduced

the

reiease

of

esterase

activity by

half.

Addition of

0.05 7o

of

other

fatty

acids

to cutin

had

little

effect (Fig.

a).

Addition

of

A.5

%

fatty

acids

to

the

cutin culture

medium resulted

in

low

esterase

âctiviry in

all

cases

without

acetone

post-treatment. Post-treatment

with

acetone

largely

increased esterase release and reached

almost

the

same

ievel

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 % stearic

acid,

released

only

1/3

of

the

esterase

activity

released

in

the

cutin

alone

control.

Vhen

0.5

ok

ricinelaidic acid

or

ricinoleic

acid was

added

to

the

cutin

medium,

release

of

esterase

activity

dropped

to

almost nil.

ç

72h,

.a5 % lerase

imilar

,ln1ng orved ;inase 'ml) ie v/âs 2.0% r cutin :leased aOH.

I

not

with

rown

dium

fatty

r

low

with

'1

_for

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

esterase

activity by

ricinoleic acid

After

24

h

of

culture,

little

esterase

activity

was released

by

A'

pisi

qt?*n

i1

cutin

mediurn

,,rppt.,itï*J

*ith

inc'ea'ittg'concentrations

of

ricinoleic

acid

(Fig.

5).

upon

post-trea[menr

vrith

acetone,

tie

release

of

esterase

activity 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

acid

concentrarion,

to

reach

almosi

nil with

0.4

_"k

and

0'5

%'

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'irh

increaiing concentrations

of

ricinoleic acid, follon'ed or not

by

âcetone

Post-treatment,

(I:

standard deviation)

When the culture Period

was

obtained (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/d

added 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-rl

Effects 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.

pisi

Culture

of A.

pisi

for

72h in

a

medium containing

A.05

% juniperic

acid as

carbon source,

released

an

esterase

activity which

was

enhanced

by

post-treat-ment

with

acetone.

In

contrast,

when

0.5

%

ricinoleic

acid was used as

soie

carbon source,

A.

pisi

did not

release

a

significant

esrerase

activity.

When

supplemented

to

medium containing

A.05

%

juniperic acid, ricinoleic acid

at 0.5

%

inhibited completely (without

acetone)

or

strongly

(with

acetone

posr-treatment) the

release

of

esterase

activity

(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 acids

I

vrithor,'t acetone

N

acetone post-treatrnent standard deviaïon

Effects of Commercial Fatty Acids on Cutinase Release 245

G'

Discussion

Cutinase has been

usually induced

by

cultivating fungi

on

cutin

as

sole

carbon

source

(KorarruruDY i985, KÔuan

rlfl1'

1n

iht

ttit

of F'

solani

f

'

sp'

pisi,

cutinase was also

i;;;

tt

tttl"

fatty

acids

(LrN

and

Kole'rruruox

7978'

..silo,os'u*andKole,rrurulvlg86,Pontleetal.t'eat)'Inthepresentwork,we

used comme.ci"l

frttf

;;;il "'

t"'bo"

toutt"

f

o'

À'

pisi'

ind

showed

that

juniperic

acid is

.

gooâ

i.,d,r"",

of

the

in

,it

o-r4"^r" of àrt.r"se activity by

this

tun*ïr,n*

the

method

of

microtitration

of

fatty

acids

rereased

from

cutin

substrate,

*e

co,tfi'n".]

;h";

;ht

_{'nty^^'iJ'^*i;t}

induced

by

juniperic

acid

l*ith

or without

acetone

post-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

increased

when

the

culture period

*.,

.",lnJ.

Â't"

ih,

but

less cutinase was .eie"sed

in

the

medium

.;;;;il;0.5

%

j"i;;;i.

"tid,

than

with

o'01'

% or

o'05

%' After

acetone

Post-treatment,

the

level

Jf

"lt""d

cutinase

was

the

same

with

0'01

"/o'

O'05

7o

or

0.5

%

juniperic

acid' These

resul"

_"'ggt"

th"t j""-ipttic

acid induced

the

synthesis

of

cutinase at

ail three concentrations,

whiie

ttre release

of

this

enzyme

i;i;r;;iiil;;^hÀi;.Juy

6.s

% juniperic

acid, and resrored

by

post-treatment

with

acetone.

Tostudythepossibleinhibitionofcutinaserelease,themineralculture

medium

*",

,uppl.-*ttd

*i'h

a

mixture of

tt'ti"

and commercial

fatty

acids'

Ricinoleic

a.,d

rici.,.laiài"

_".ldr

ato.5

o/o

strongly-inhibited^cutinase

release, even

after

acetone

por,-a.""t*"nt,

but.were

without

effect

at

0'05

%'

Among

"U

f"*yîtiJ' lt"ta,

ricinoleic a"id

*"s

the

strongest

inhibitor of

curinase release

by

A.

pisi.At

0.5'7o,

it

severely

inhibited

the

release

of

cutinase

induced

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

also

the induction

of

the

tn"ËL

overall

results

suggest

that

some

commercially

available

C15

and

C13

fattv

acids are able

,. i.a".?ït

inhibit

the

in aitro

ttlt"'e

of

cutinase

by

A'

pisi'

:i,#ï'i;;;;

;;Ë';;;;;;i;;;"

_"r

the rungar cutinase

production during

t'lr.

pro..r,

oi

ittf..tiott

of

the

host-plant'

Vethankthe..AdministrationGénéraledelaCoopérationauDéveloppement,,(AGCD)'

Brussels, Belgium, for support to B' NasRaoul'

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y.

HrNrs, 1e85: Detection

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s

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_'ia

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"'-*';;od;;;J'uv

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47*75'

246

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