Enzymatic degradation of isolated plant cuticles and nectarine fruit epidermis by culture filtrates of phytopathogenic fungi

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Enzymatic degradation of isolated plant cuticles and

nectarine fruit epidermis by culture filtrates of

phytopathogenic fungi

Christophe Nguyen-The, A. Chamel

To cite this version:

Christophe Nguyen-The, A. Chamel. Enzymatic degradation of isolated plant cuticles and nectarine

fruit epidermis by culture filtrates of phytopathogenic fungi. Agronomie, EDP Sciences, 1991, 11 (2),

pp.101-108. �hal-02711409�

Plant

_pathology

Enzymatic degradation

of isolated

_plant

cuticles

and nectarine fruit

_{epidermis by}

culture filtrates

of

_{phytopathogenic fungi}

C

_Nguyen-The

A

Chamel

1

INRA, Station de Technologie des Produits Végétaux, Domaine St-Paul, 84140 Montfavet;

2_{CEA/Direction des Sciences du}

Vivant, DBMS/Laboratoire de

_Physiologie

Cellulaire

Végétale **,

CENG 85X, 38041 Grenoble Cédex, France

(Received 23

_April

1990; accepted 3 December 1990)

Summary —

In vitro

_degradation

of isolated cuticular membranes and in vivo

_degradation

of the

_epidermis

of

necta-rine fruit

_by

_Rhizopus

stolonifer and Monilia laxa were

_{investigated.}

Increases in the

_permeability

of cuticular

mem-branes to 86Rb and of

_epidermis

to 45Ca were used as a measure of

_degradation.

An isolate of Fusarium solani _{f sp}

pisi,

which

_produced

a

_large

amount of cutinase, was used as a reference. Culture filtrates of R stolonifer and M laxa

did not

_change

cuticular membrane

_{permeability. Conversely,}

both

_fungi

caused a marked deterioration in the

epider-mis of nectarine

fruit,

probably

caused

_{by pectinolytic}

_enzymes. The reaction is

_thought

to

_probably

take

_place

in the

cuticular microcracks present on the nectarine fruit surface. Penetration of nectarine fruit

_epidermis

_by

M laxa and R

stolonifer does not

_apparently

involve

_cutinolytic

enzymes.

Rhizopus

stolonifer / Monilia laxa / Fusarium solani / isolated cuticle / cutinase

Résumé —

_{Dégradation enzymatique}

de cuticules isolées de

_plantes

et de

_{l’épiderme}

de nectarines par des filtrats de culture de

_champignons

_{phytopathogènes.}

Dans ce

travail,

la

_dégradation

in vitro de membranes

cuti-culaires isolées et la

_dégradation

in vivo de

_{l’épiderme}

de nectarine par

Rhizopus

stolonifer et Monilia laxa ont été étu-diées. Cette

_dégradation

a été

mesurée,

d’une

_part

_par

_{l’augmentation}

de la

_{perméabilité}

de membranes cuticulaires

au

86

Rb, et

d’autre

_{part par}

_{l’augmentation}

de la

_{perméabilité}

de

_{l’épiderme}

au 45Ca. Un isolat de Fusarium solani

_{f sp}

pisi,

a servi de référence. Le traitement de cuticules isolées avec des filfrats de culture de ce

_champignon

_augmente

la

_{perméabilité}

cuticulaire au 86Rb

_(figs

2 et

_3);

_par contre, le transfert du 86Rb

_après

traitement des cuticules _{par des}

surnageants

de culture de R stolonifer et M

laxa,

dans des conditions

_comparables

d’activité

_enzymatique,

n’est pas

modifié

_(fig

_3).

Les filtrats de culture de Fusarium solani

_{f sp pisi}

causent aussi une

_perte

de

_poids

de cuticules isolées

décirées confirmant ainsi la

_présence

de cutinase dans les solutions de culture testées. Les 2

_champignons,

R

stolo-nifer et M laxa

_provoquent

une nette

_dégradation

de

_{l’épiderme}

de

nectarine,

visible

_après

coloration au bleu de

mé-thylène,

qui

se _{traduit par} une

_{pénétration}

_plus

élevée du 45Ca dans les fruits

_{(fig 4).}

Le traitement avec un filtrat de

culture de Fusarium solani

_{f sp pisi qui}

ne

_présente

_pas d’activité

_{pectinolytique,}

est sans effet. Les enzymes

pectino-lytiques produites

par R stolonifer et M laxa sont vraisemblablement à

l’origine

de l’altération observée avec ces

champignons,

qui

survient sans doute au niveau de microfissures cuticulaires

_présentes

à la surface des nectarines.

La

_{pénétration}

de

_{l’épiderme}

_{de nectarines par M laxa} et R stolonifer

_{n’implique probablement pas}

l’intervention

d’en-zymes

cutinolytiques.

Rhizopus

stolonifer /Monilia laxa /Fusarium solani / cuticule isolée / cutinase

*

Correspondence

and _reprints **

INTRODUCTION

Monilia

laxa

_(Aderh

and

_{Ruhl) Honey}

and

Rhizo-pus stolonifer

(Ehrenb

ex

_Fr)

Lind are the

princi-pal

fungal pathogens

in

_Europe

of

_peach

and nectarine

_during

_storage

_(Anderson,

_1925;

_Byrde

and

Willetts, 1977;

Bompeix

et

_al,

_1979).

Al-though

infection of fruit

_by

both

_{fungi frequently}

occurs

_through

wounds,

direct

_penetration

of

germinated

conidia of M laxa has been demon-strated

_(Wade,

_1956;

_Hall,

_1971).

Direct

penetra-tion of the

_plant

cuticle

_{by fungal pathogens}

(Rij-kenberg

et al, 1980;

Dickman

_{et al, 1982, 1983;}

Kolattukudy

and

Koller,

1983)

or bacterial

patho-gens

(Bashan

et

_al,

_{1985) requires}

the

produc-tion of

_{cutin-degrading}

_enzymes

_(cutinases).

Mi-crocracks on the surface of

nectarine fruit

_(Fogle

and

_{Faust, 1975,}

_{1976) might provide}

alternative sites for

_fungal

_penetration.

From electron

micro-scopic

observations,

microcracks can be the

main

sites of

_penetration

for the

_mycelium

of M laxa and R stolonifer

_(Nguyen-The

et

al,

1989).

The biochemical

_composition

and _{the presence}

or absence of

cutinized

material associated with these

microcracks

has not been determined. Therefore it seems

_important

to

_investigate

whether or not cuticular

_degradation

is involved

during

the

_penetration

_{processes of M} laxa and

R stolonifer in nectarine fruit. An isolate of Fusa-rium solani f sp

pisi

which

_produces

a

_large

amount of cutinase

_(Baker

et

al, 1982;

Kolattu-kudy, 1985)

was used as a reference. Increases

in the

_permeability

of nectarine fruit

epidermis

and of isolated pear cuticular membranes were

used to determine

the involvement

of extracellu-lar enzymes of both

fungi

upon cuticular

penetra-tion.

MATERIALS AND METHODS

Fungal

cultures

Strains of M laxa and R stolonifer were isolated from

decaying

fruits,

as for

_previous

_{investigations}

(Nguyen-The

et

_al,

1985a, b;

1989).

The isolate of F solani f sp

pisi

was

_purchased

from the

Centraalbu-reau voor Schimmelcultures

_(Baarn,

The

_Netherlands)

isolate CBS.188.35.

Fruits

Nectarine fruits of the

_Armking

cultivar were grown in

experimental

orchards

_{(INRA, Alenya, France).}

Fruits

were harvested at the

_stage

of commercial

_maturity.

Isolated cuticular disks

Cuticular disks

(1

cm

_diameter)

were isolated

_by

enzy-matic maceration

_{(2% pectinase;}

0.2%

cellulase,

Sig-ma

_USA)

from

_{fully developed}

_{pear leaves}

_(Pyrus

communis

_L,

cultivar Passe

_Crassane)

and from the skin of mature tomato fruit

_(Lypersicon

esculentum

Mill)

according

to the

_procedure

described in Chamel

and

_Bougie

_(1977).

The pear leaf cuticular disks used

were obtained from the astomatous _upper

surface;

the

absence of any visible hole was checked

_using

a

_light

microscope (x 150).

This material has

_already

been

used

_widely

as a model in cuticular

_permeability

stud-ies

_{(Chamel, 1986).}

Culture solutions

Two

_liquid

culture media were used for the

_production

of cutinase: a V8 medium

_supplemented

with 0.5%

_(w/

v)

of cutin

_{powder (Baker}

et

_al,

₁₉₈₂₎

and a

_synthetic

medium

_(Salinas

et

_al,

1986: 10 g

KNO

3

;

5 g

KH

2

PO

4

;

0.25 g

Mg

SO

4

,

7

_O;

₂

_H

0.4 mg

FeCl

3

;

3.6 _g

glu-cose; 250 _{mg cutin}

_powder;

_H

₂

_O

dist 1

_I).

To obtain

the cutin

_powder,

tomato fruits were

_dipped

in

_boiling

water and

_hand-peeled.

The skins were twice

macer-ated over a 1-d

_period

in a mixture of 1%

_pectinase

+

1 % cellulase

_{(Fluka, Switzerland)}

diluted in a 0.05

mol.l

-1

_pH

4.8 sodium acetate buffer. The skins were

rinsed in distilled water, then in methanol. The

cuticu-lar membranes obtained were dewaxed in a mixture of

chloroform/methanol

_(1/1)

for 1 h and in chloroform

twice for 1 h. The chloroform was decanted and the membranes were

_ground

in

methanol,

firstly

with an

Omni-mixer

_(Sorvall,

_Newtown,

_USA)

and

_secondly

with a

_{Polytron homogeniser (Kinematica,}

Switzer-land)

to obtain a very fine

_powder.

The methanol was

decanted

and

the

_powder

dewaxed in an identical

manner to that used for the cuticular membranes.

About 0.75 g of cutin

powder

were obtained from 2

_kg

of tomato fruits. The

_production

of

_pectinolytic

en-zymes was

_performed

in a

_{pectin liquid}

medium,

as

described

_{previously (Nguyen-The}

et al,

1984).

Fungi

were _grown at 25 °C on a shaker in 30 ml of culture solution

_placed

in an

_Erlenmeyer

flask. The

in-oculum consisted of _spores of R stolonifer

_(≈

10

5

spores/flask)

or 10 disks

₍₄

mm

_diameter)

of

_mycelium

of M laxa or F _{solani f sp}

_pisi

cut from a culture of the

fungus

on

_{potato dextrose agar. After}

5 d

_(R

stolonifer grown on

_{pectin medium),}

1 wk

_{(M laxa}

_grown on

pec-tin

_medium),

or more

_generally

3 wk

_(R

stolonifer,

M

laxa,

F solani f sp

pisi

grown on V8 + cutin solution or on

_{synthetic medium),}

the

_{respective liquid}

cultures

were

_centrifuged

for 20 min at 25 000 _{g and the}

fil-trates stored at -20 °C before use.

Enzyme

assays

Cutinase was

_{assayed by}

the

_degradation

of

_fatty

acid

(Sig-ma,

USA)

as the substrate. Release of

_{p-nitrophenol}

from

_{para-nitrophenol}

ester of

_butyric

acid is a

_rapid

method to _assay cutinase

_activity,

but it is not

_specific

since many

fatty

acid esterases do not

_hydrolyse

cutin

polymers (Kolattukudy, 1985).

The

_production

of

phe-nol was detected at 405 nm with a DMS-100

(Varian,

USA)

spectrophotometer.

Reactions were

_performed

at 30 °C in a Tris/HCl 0.1 M

_pH

8 buffer with 0.01%

Triton X-100

_(Fluka,

_Switzerland)

0.1% PNPB

_using

100

_μl

culture filtrate in 2.5 ml of the substrate solution.

Activity

was

_expressed

in absorbance units at 405 nm

per min

(Au/min).

Pectinolytic activity

was measured

_by

a viscosi-metric method or

_by

the release of a

_reducing

_group

and

_routinely

detected in culture filtrates with an _agar

diffusion assay

(Hornewer

et

_{al, 1987). Assays}

were

conducted at

_pH

5

_(sodium

acetate

buffer), pH

7

(sodium phosphate buffer)

and

_pH

8

_{(Tris/HCl buffer)}

in 0.05-mol.l-1 solutions.

Estimation of the

_permeability

of isolated cuticular membranes

The diffusion of 86Rb

_(a

185

_kBq/ml

solution which contained 0.1 mmol.l-1 Cl- ion as KCI +

_RbCl,

in a

10-mmol.l

-1

_pH

8

_Hepes

_buffer)

was measured

_by

the

following procedure.

A pear cuticular disk was

at-tached to the end of a thick-walled

_glass

tube

₍₇

mm

inner

_{diameter), dipped}

in a scintillation

_glass

vial

con-taining

10 ml of the non radioactive "receiver" solution

(Hepes

buffer, 10 mmol.l-1

_pH

_8).

The cuticular disk

was attached as

_previously

described

_(Chamel

and

Bougie,

1977;

Chamel, 1980)

using

RTV 111

_(silicone

adhesive

_substance,

_{Rhône-Poulenc),}

with its external

side

_(ie

waxy surface of the

_{cuticle) facing}

the inner

space of the tube.

_{System imperviousness}

was

checked

_{by replacing}

the cuticular disk

_by

a

micro-scope slide

(Chamel, 1980).

The solution of 86Rb

("donor" solution)

was

_placed

inside the

_glass

tube

_(0.5

ml),

and the tube was fixed so that the "donor" and

"re-ceiver" solutions were

_kept

at the same level

_during

diffusion

_{(fig 1). The permeability}

of the cuticular disk

was measured

_by

the amount of

_{radioactivity}

detected

in the "receiver" solution after 24 h at room

tempera-ture.

The

_degradation

of cuticular disks

_by

culture

fil-trates was assessed

by

the same method. Culture

fil-trates, diluted in

pH

8

_Hepes

buffer

_{(final molarity}

10

mmol.l

-)

supplemented

with 0.1% thimerosal

(Prola-bo, France)

to

_prevent

_growth

of

_{microorganisms,}

were

_placed

either inside the tube

_(to

attack the waxy side of the

_cuticle)

or both in the tube and in the

_glass

vial

_(to

attack both sides of the

_cuticle).

After a 24 h

incubation at room

_temperature,

the cuticular

mem-brane was rinsed several times with distilled water.

Diffusion of 86Rb

_through

the isolated cuticular

mem-brane was measured before and after the treatment

with culture filtrate and the results are

_{presented by}

the ratio R = diffusion after/diffusion before. In the case of low

_fatty

esterase

_activity,

the 24-h treatment

was

_repeated

3 times with

_freshly

thawed culture

fil-trate.

In situ

_degradation

of nectarine fruit

_epidermis

A

_{20-μl droplet}

of culture filtrate was

_deposited

on the

epidermis

in the

_equatorial

area of the nectarine fruit. Culture filtrate was _{either pure} or diluted

_{(1 : 1)}

in a

0.05-mol.l

-1 sodium acetate buffer

_pH

4 or a 0.05

mol.l

-1 sodium

_phosphate

buffer

_pH

7. The

_droplet

evaporated

within a few h at room

_temperature

and after 24 h the

permeability

of the

_epidermis

was esti-mated

_by

2 methods:

-

the fruit surface was stained with a water-ethanol solution of

_methylene

blue

_(Prolabo,

_France)

supple-mented with Triton

_X-100;

-

the

_penetration

of 45Ca inside the fruit was

meas-ured at the site

_previously

treated with the culture

fil-trate. In this latter case, 20

μl

of a 45Ca solution

₍₉₂₅

kBq/ml,

20 mmol.l-1

_CaCl

₂

in a 10 mmol.l-1

_pH

5 SADH

_buffer)

were

_deposited

on the fruit surface and after 24 h at room

_temperature,

the

radioactivity

which remained on the fruit surface was washed twice with 2

x 5 ml Tween 20 solution

_(0.1%

in a 10

mmol.l

-1

pH

5

SADH

_buffer).

As

_{radioactivity}

counted in the second

washing

solution was

_only

4.5% of the

_{radioactivity}

counted in the

first,

a third

_washing

was considered unnecessary.

Radioactivity

which had diffused into the skin of the fruit was measured in a 1-cm diameter disk

_peeled

with a razor blade and dissolved in 1 ml of

Lumasolve

_(Lumac).

Radioactivity

measurements

Radioactivity

of 86Rb was determined with a _gamma

counter

apparatus

(Intertechnique

CG

4000,

France).

Counts were

_performed

on 2-ml

_aliquots

removed

from the

"receiver"

solution and

_{20-μl aliquots}

re-moved from the "donor" solution. The latter were

made up to 2 ml with

_H

₂

_O

so that all the

_samples

had

Radioactivity

of 45Ca was determined in

_20-μl

ali-quots

of the 925

_kBq/ml

solution and in

_{100-μl aliquots}

of the

_washing

solution with a

liquid

scintillation coun-ter

_{(spectrometer}

Packard

_{4430). Aliquots}

were mixed with 10 ml of

_Lumagel

_(Lumac).

Skin

_samples

dis-solved in 1 ml Lumasolve were counted in 9 ml of

Li-poluma (Lumac).

Weight

loss of cuticular material

Dewaxed cuticular disks from tomato fruit were im-mersed for 24 h in the culture filtrate of F solani f _sp

pisi

(grown

in V8 + cutin

_solution)

mixed with a 20

mmol.l

-1

_pH

8

_Hepes

buffer

_(1:1).

Disks in heated

cul-ture filtrate

_{(10 min,}

100

_°C)

were used as control. The disks

_(≈

10

_mg)

were

_placed

in a scintillation vial with

3 ml of solution. The

_experiment

was

_performed

on 5

vials in each case

_(active

and inactive culture

_filtrate).

The 10 disks of a vial were

_weighed

before and after

treatment.

RESULTS

Permeability

of isolated Pear leaf cuticles Production of

_fatty

acid esterase

by

F

solani

f sp

pisi

and

_by

R stolonifer was

_higher

in V8 + cutin

solution

than in

the

_synthetic

+ cutin solution

(ta-ble

_I).

The isolate of M laxa that we used in this

investigation

did not

_produce

_{any detectable}

_fatty

acid esterase. The

_{following experiments}

were

conducted with

culture

filtrates of

_fungi

_grown on

V8 + cutin

solution,

the

fatty

acid esterase activi-ties of which are

_presented

in table I.

Incubation

of both sides of isolated cuticular membranes for 24 h with culture filtrates of F

so-lani f _sp

_pisi

led to an increase in the diffusion of

86

Rb.

The ratio

of increased diffusion

(R)

varied

greatly

among the

different

cuticular disks

treat-ed with the same culture filtrate

_{(fig 2)}

with R

ranging

from 1.3 to 5.2. The

_experiment

was

car-ried out on 2 different cuticle

_samples

and the

in-dividual variation was much lower in the second

sample.

It should be noted

that treatment of

the waxy side of the

cuticular disk

only

did not

cause

any increase in the diffusion of

86

Rb

(R

lower than

_1).

An increase in

86

Rb

diffusion was still

no-ticeable when the isolated cuticular disks were

in-cubated

with

F solani

_{f sp pisi culture}

filtrate

dilut-ed in

distilled

water

_(1/5,

_{1/10, 1/20;}

_{fig 3).}

The 24-h treatment was

_repeated

3 times before the

second diffusion of

86

Rb.

On the

_contrary

how-ever,

although

the 1/20

dilution of

F

solani

f

_sp

pisi culture

filtrate

and R stolonifer culture filtrate

(grown

on V8 + cutin

solution)

contained similar

fatty

acid esterase

_{activity (table}

_I),

the latter

had

no

_significant

_{effect upon the}

_permeability

of cuti-cular disks

_(compared

to a

_control).

Weight

of dewaxed tomato cuticles

Compared

to the increase in

_permeability

_{of pear} cuticular

disks,

the culture filtrate of F solani

_{f sp}

pisi

(grown

on

V8

+

_cutin)

caused a

_significant

loss of

_weight

in dewaxed

cuticular

membranes

(table II).

This confirms the presence of cutinase in the culture

solutions tested.

Degradation

of nectarine fruit

_epidermis

Culture

filtrate of R stolonifer grown on a

_pectin

medium

_deposited

on the

surface of

nectarine fruit caused a

marked alteration after

24 h at

room

_temperature.

It could be visualized

_by

staining

the surface with

_methylene

blue: after

rinsing

the

dye

with

_tap

water,

a network of blue-coloured cracks remained. No

such alteration

occurred when boiled

₍₁₀

min,

100 °C)

filtrate

was

_deposited

on the

_fruit,

but it could still be

ob-served when culture filtrate was mixed with 0.1

mol.l

-1

_pH

4 acetate buffer or

_pH

7

_phosphate

buffer

_(1:1).

Culture filtrate of M _{laxa grown in} the same

solution induced

a similar

_alteration,

but in a weaker and less reliable manner: altera-tion occurred in 80% of the fruits treated with R stolonifer and 40% of those treated with M laxa.

The

presence

of

pectinolytic

enzymes in

the

cul-ture filtrate was checked

_by

an

_agar

diffusion

as-say and M laxa culture filtrate contained

only

5% of the

_{enzymatic activity}

in the R

stolonifer

cul-ture filtrate.

Absorption

of

45

Ca

_by

the surface of nectarine

fruit was

_higher

in fruits

_previously

treated with culture filtrate

of

R

stolonifer

_(grown

on

_pectin

medium)

than with

fruits treated

with

the

same

culture filtrate heated 10 min at 100 °C. This could be demonstrated both

_by

the lesser

amount of

45

Ca

which

remained

on

the fruit

sur-face

_(ie

45

Ca

washed with

Tween

20

_solutions)

and

_by

the

_higher

amount detected in the skin

(fig 4).

The

addition

of the amounts of

45

Ca

measured in

_washing

solution

and

in the

skin

represented

74.6% ± 5.6 of

the

total amount

de-posited

on the fruit.

_{Approximately}

25% must

have

_{penetrated deeper}

into the

_{mesocarp and}

was not counted in our

_experiment.

Crude

cul-ture filtrate was used

_{(pH 4),}

the

_pectinolytic

ac-tivity

of which

_{(8 nkatal/ml)}

was

measured

and characterized in a

_previous

_{investigation}

(Hor-newer et

al,

1987).

However,

no

_fatty

acid

este-rase could be detected in this

_pectin

medium,

either at

_pH

_{4, 6,}

7 or 8. A

similar

_experiment

was conducted with crude culture filtrate of F _{solani f sp}

_{pisi (pH 7)}

_grown on V8 + cutin

me-dium,

the

_fatty

acid esterase

_activity

of which is

presented

in table I. No

significant

effect on the

penetration

of

45

Ca

could be noticed

_{(fig 4).}

It

should

be

noted that

no

_{pectinolytic activity}

could

be detected

in

this culture

filtrate,

at

_pH

5,

pH

7,

or

_pH

8.

DISCUSSION

In

_vitro,

in a

culture solution which contained

cutin,

F

solani

_{f sp}

_pisi

and

to a

lesser extent

R

stolonifer,

_produced

some

_fatty

acid

esterases.

When convenient dilutions of these culture

fil-trates were

tested,

so as to assess

the

same

fat-ty

acid esterase

_activity

in

_both,

F solani

_{f sp pisi}

caused a

marked increase

in the

_permeability

of

isolated

cuticular

membranes,

whereas R stoloni-fer did not. The considerable

_variability

observed within the same

_species

has

_already

been

report-ed in several studies relatreport-ed to _cuticular

pene-tration

_(Chamel,

1980;

1986).

The variations are

attributed

to

_{heterogeneity}

of

the

physicochemi-cal structure of the

cuticle. Baker

et al

₍₁₉₈₂₎

have demonstrated that cutinases

_purified

from a

culture solution of F _{solani f sp}

_{pisi increased}

the diffusion of

_glucose

and

_proteins

_through

isolated

cuticular

disks. The similar

results

we obtained with a culture filtrate

of

our isolate of

F solani

_{f sp}

pisi was probably

linked to the presence of a

cuti-nase.

The culture

filtrate of R

stolonifer did not

in-crease

the

permeability

of

isolated

cuticular

membranes;

the

_fatty

acid esterase

_{produced by}

the

_fungus

is therefore

_presumably

not a cuti-nase.

Our results are not sufficient to enable us to

state that R stolonifer could not

_produce

any

cuti-nase, since: -

only

2 culture media were tested. It

_might

be advisable to confirm the results of the

_present

in-vestigation by using

other

culture media without

a carbon source

_{(like glucose)}

which

could

re-press the cutinase

production.

The effect of

shaking

of the culture

should

also be

considered.

Lastly,

the

_permeability

_{assay with}

isolated cuti-cles should be tested at

_pH

values above

_8;

- the

technique

we used to _{detect cutinase may} not be as

sensitive

as

conventional methods with

radiolabelled

cutin

_powder

_{(Kolattukudy,}

_1985;

Salinas

et al,

1986).

Nevertheless,

this

_provides

a

_good

_argument

for

_considering

that cuticle

deg-radation is not of

_great

_importance

in the infec-tion of fruit

_by

R

_{stolonifer, contrary}

to F solani f sp

pisi (Koller

et

al,

1982).

This

_assumption

is

supported by

the

fact that

a

culture

filtrate of R stolonifer

_(grown

on

_{pectin medium)}

caused

a

marked increase in the

_permeability

of nectarine fruit

_epidermis,

whereas it did not contain

cutin-ase.

The

_{radioactivity}

of

45

Ca

measured

in

the

skin is

_mainly

attributable

to

Ca ions that have

penetrated

as

the

greatest

fraction of

Ca

retained

by

the cuticle

_(as

free ion and

_{exchangeable)}

was removed

_{by rinsing}

with

an

acid

solution

₍₁₀

mmol.l

-1

buffer,

pH 5),

as

_previously

shown

else-where

in

the

case of

isolated

_apple

fruit

cuticles

(Chamel, 1983).

Moreover,

it has

been shown

that Ca

can

_penetrate

into

apple

and

tomato

fruit

Whatever the

effect,

the results indicate a modifi-cation of the fruit surface

_following

its

treatment

by

the culture filtrate of R stolonifer. The results obtained with M laxa are less

consistent,

since

fatty

acid esterase

_production

was not detectable

and

_degradation

of

nectarine

fruit surface

(re-vealed

_by

the

_staining

with

_{methylene blue)}

was

not constant.

Nevertheless,

these results also

suggest

that cuticle

_degradation

is not involved

in the

infection

of nectarine

_by

M laxa.

Pectinolytic

enzymes

play

an

_important

role in

the

_{pathogenicity}

of M

laxa and

R

stolonifer

(Byrde

and

_{Willetts, 1977,}

_{Nguyen-The et}

_al,

1985a,

b),

especially

during post-penetration

pro-cesses.

From

the

_present

_{investigation,}

it can be

assumed

that

these enzymes are also

_important

during

the

_penetration

of both

_fungi

in nectarine fruit. This would

_imply

that non-cutinized

_(or

poor-ly

cutinized)

areas are

_present

on the surface of

nectarine

fruit. Microcracks that we observed on

the

cuticle

of nectarine fruit

_(Nguyen-The

et

_al,

1989)

are

_possibly

the sites of

_degradation

of the

epidermis by

R stolonifer and M laxa culture

fil-trates. Penetration of

_fungal

_pathogens

in fruits

through

microcracks has

_already

been observed in the case of

_apple

and

_Phytophthora

cactorum

(Mourichon

and

_{Bompeix, 1979)}

or in the case of grape and

Botrytis

cinerea

_(Bessis,

1972;

Pu-cheu-Plante

and

_Mercier,

_{1983), although}

a

di-rect

_penetration

of tomato

fruit

which involved

the

degradation

of cuticle has been demonstrated with B cinerea

_(Rijkenberg

et

_al,

_1980).

Research is in progress to

_investigate

the fine structure of cuticular microcracks on nectarine

fruit.

ACKNOWLEDGMENT

This work was

_{supported by}

a

_grant

from the Institut

National de la Recherche

Agronomique.

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