Activité peroxydasique de plantules de ray-grass anglais et de fétuque élevée inoculée avec des endophytes

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Peroxidase activity of perennial ryegrass and tall fescue

seedlings artificially infected with endophytes

W. Naffaa, C. Ravel, N. Boyer, J.J. Guillaumin

To cite this version:

W. Naffaa, C. Ravel, N. Boyer, J.J. Guillaumin. Peroxidase activity of perennial ryegrass and tall

fescue seedlings artificially infected with endophytes. Agronomie, EDP Sciences, 1999, 19 (7),

pp.611-619. �hal-02689494�

Note

Peroxidase

_activity

of

_perennial

_ryegrass

and tall fescue

seedlings

artificially

infected with

_endophytes

Walid Naffaa

a Catherine

Ravel

b

Nicole

_{Boyer’ Jean-Jacques}

Guillaumin

a

a

Unité de _{pathologie végétale} et _{mycologie, Inra, 234,} avenue du Brézet, 63039 Clermont-Ferrand cédex 02, France

b

Unité d’amélioration des _plantes, Inra, 234, avenue du Brézet, 63039 Clermont-Ferrand cédex 02, France c

Laboratoire de _{Phytomorphogenèse,} Université Blaise Pascal, Campus des Cézeaux, 24, avenue des Landais,

63177 _Aubière, France

(Received 11 _May 1999; accepted 15 _July 1999)

Abstract - An increase in

_{peroxidase activity}

is a common _response of

_plants

to various stresses,

especially

to infection

by

pathogens.

It seemed

_interesting

to

_study

the effects of

_{symbiotic fungi}

_{of fodder grasses} on the

_{peroxidase activity}

of their hosts. The

_{peroxidase activity}

of tall fescue cv. Clarine

_artificially

infected with

_{Neotyphodium coenophialum}

or

with

_e-endophytes

from other hosts _(N. lolii and

_LpTG-2

from _{Lolium perenne,}

_{Epicbloë festucae}

from Festuca

gigan-tea or Koeleria cristata,

Epichloë

bromicola from Bromus erectus) was

_generally

either lower or not

_{significantly}

dif-ferent from that of the non-infected control. Similar results were obtained with

perennial

_ryegrass cv.

_{Vigor artificially}

infected with different

_{e-endophytes.}

In _{contrast, artificial infection of both grasses with Gliocladium-like}

_fungi

belong-ing

to _{the group of}

_{p-endophytes provoked}

an increase in

_{peroxidase activity.}

These results _suggest that

_p-endophytes

can be considered as

_parasites

while

_{e-endophytes,}

which are not able to

_trigger

a

_non-specific

host defence _response,

are

_really

mutualistic. _(© 1999

Inra/Éditions

_{scientifiques}

et médicales Elsevier _SAS.)

clavicipitaceous endophytes

/ Gliocladium-like / Festuca arundinacea / Lolium perenne /

peroxidases

Résumé - Activité

_{peroxydasique}

de

_plantules

de _ray-grass

_anglais

et de

_fétuque

élevée inoculées avec des

endo-phytes.

Les

_plantes

stressées _(ou

_attaquées

_par un

_pathogène)

montrent

_{généralement}

une

_augmentation

de leur activité

peroxydasique.

Il a semblé intéressant d’observer l’effet de différents

_{champignons endophytes}

des

_graminées

sur

l’activité

_{peroxydasique}

de la

_fétuque

élevée _{(var. Clarine)} et du ray-grass

anglais

(var.

Vigor).

Clarine, artificiellement inoculée _par

_{Neotyphodium coenophialum}

ou _par des

_e-endophytes

isolés d’autres

_graminées

_(N. lolii et

_LpTG-2

de

Lolium _perenne,

_{Epichloë festucae}

de Festuca

_gigantea

et de Koeleria _cristata,

_Epichloë

bromicola de Bromus erectus) montre

_{généralement}

une activité

_{peroxydasique}

inférieure ou

_comparable

à celle du témoin. Des résultats similaires ont

été obtenus _pour

_Vigor.

En revanche, l’inoculation _par un

_champignon

_{Gliocladium-like,}

_{qui appartient}

au _{groupe des}

p-endophytes,

entraîne une

_{augmentation significative}

de l’activité

_{peroxydasique}

sur les deux

_{espèces-hôtes.}

Ces

résul-Communicated _by Hervé Thiellement (Geneva, Switzerland)

*

Correspondence and _reprints

tats

_suggèrent

le caractère

_{quasi-parasitaire}

des

_{p-endophytes,}

tandis _que les

_e-endophytes

se

_{comporteraient plutôt}

comme des

_symbiotes

dont la

_présence

ne déclenche pas

_{l’augmentation}

de l’activité

_{peroxydasique.}

_(© 1999

Inra/Édi-tions

_{scientifiques}

et médicales Elsevier _SAS.)

e-endophytes

/ Gliocladium-like / Festuca arundinacea / Lolium _perenne /

_peroxydases

1. Introduction

In recent _{years, the}

_importance

of

_symbiotic

interactions between grasses and

clavicipitaceous

fungal

endophytes

has been more

_{fully recognised.}

Knowledge

of their

_taxonomy

and

_{relationships}

with their hosts has

_{considerably progressed.}

It has been shown that these

_endophytes,

called

e-endo-phytes

because

_they

are related to

_species

of

Epichloë,

form a continuum from

_antagonism

to mutualism

_according

to their mode of transmission

[5].

Mutualism concerns

_symbionts

_only

able to

propagate

clonally

via the seeds of their hosts. These

_symbionts

are _{classified in the genus}

Neotyphodium, formerly

Acremonium section

Albo-lanosa. The main mutualistic effects of

clavicipitaceous endophytes

are the

_production

of

protective

anti-herbivore metabolites and their

ten-dency

to enhance host

_growth

and stress tolerance

[27, 29].

In

addition,

non-clavicipitaceous endophytes

from some

_species

of the genera Festuca and

Lolium,

referred to as

_p-endophytes

_[10]

or

a-endo-phytes

[22],

have also been described.

P-endo-phytes

include

_{Phialophora-like}

and Gliocladium-like

_{Deuteromycetes,}

which often occur

co-symbiotically

with

_e-endophytes

on Festuca

spp. and Lolium perenne,

respectively.

Although

some results

_suggest

that

_p-endophytes

are

com-mensal

_with,

or

_antagonistic

_towards,

their hosts

[26, 28],

the

_ecological

_importance

_{of p- and}

a-endophyte-grass

interactions has not

_yet

been determined.

It has been demonstrated that e- and

p-endo-phytes

can be introduced

_artificially

into different host grasses

[11].

Such

_experimental

inoculations

can

_help

to elucidate

_{compatibility}

_patterns

and the range of

endophyte

hosts.

Many

successful cross-infections of

_Epichloë

or

_Neotyphodium

endo-phytes

have been achieved within or between the

genera Festuca and Lolium

[3, 9, 13, 21].

However,

some of these novel associations showed

incompat-ibility

reactions such as the presence of stunted

tillers on the new host

[3]

or the

_degeneration

of

endophyte hyphae [9].

Little is known on the

_{physiological}

_processes

which could

_play

a role in

_{grass-endophyte}

rela-tionships.

No

_{specific pathogenicity}

or resistance mechanisms have been

_reported

_[12].

_However,

non-specific

reactions from the

_fungus

or the

_plant

have been

_described;

these reactions could

_explain

some cases of

_{incompatibility:}

_i)

_{Clavicipitaceous}

endophytes

can _express

_proteases

_homologous

to other

_proteases

involved in

_fungal

_{pathogenicity}

of insects or nematodes

_{[16, 23].}

Such

_proteases

_may facilitate colonisation of the host

_{by degrading}

the

plant

cell wall and/or the

_{apoplastic proteins}

pro-viding

a nutritional source for the

_fungus;

_ii)

Endophyte

infection can increase the

_expression

of

chitinase

_activity

_[24].

Chitinase is one of the

pathogenesis-related

(PR)

proteins

involved in

non-specific plant

defence responses.

The

_peroxidases

constitute a _{group of enzymes}

which

_play

the role of oxidoreductases and catal-yse the

cleavage

of the O-O link. In

_higher

_plants,

the

_peroxidases

are involved in several

physiologi-cal processes such as the oxidation of indole-3-acetic acid

_(IAA),

the oxidation of

_phenolic

com-pounds

and the

_assembly

of the cell wall. Peroxidases that

_{play a major}

role in the

_synthesis

of

_lignin

_[17]

are involved in the response of the

plant

to

_{pathogenic organisms}

_{[18, 19, 31]}

_by

strengthening

the cell wall. This

_phenomenon

can

delay

the

_penetration

of

_pathogens

into the

_plant

cells.

Therefore,

these

_peroxidases

are considered

as PR

_proteins

_[30].

Since Roberts et al.

_[24]

showed that chitinase

synthesis,

a

non-specific

_response to

_fungal

infec-tion,

can be modified

_by

_{the presence of} an

endo-phyte,

it has been assumed that

_peroxidase

_activity

could also be influenced

_{by endophytes.}

The

objec-tive of this

_study

was to examine the influence of several

_endophytes

on the

_{peroxidase activity}

of

tall fescue and

_perennial

_ryegrass.

2. Materials and methods

2.1. Materials

The artificial

_{host-endophyte}

associations used in this

_{study mostly corresponded}

to those created

_by

Naffaa et al. _[21].

_They

concerned tall fescue _(cultivar Clarine from which the natural

endophyte

was

_removed)

and

_perennial

_{ryegrass (cultivar}

_{Vigor, naturally}

endo-phyte-free)

inoculated with various

_endophytes

belong-ing

to _{different groups:} e-, p- and

a-endophytes

(table I).

2.2. Inoculation

Endophyte-free

seeds were surface sterilised. Seeds

were then

_placed

on water agar in Petri dishes and incu-bated in darkness at 23 °C ± 1 for 7

_days.

After

germi-nation of the seeds, a

_longitudinal

slit was cut

_through

the tissue of the young

seedlings

at the

_junction

of the

mesocotyl

and

_coleoptile

and a

_drop

of solution of

ground mycelium

in sterile water was

_placed

in the slit.

The

_mycelium

used for inoculation was taken at the

periphery

of the colonies.

For both cultivars, controls consisted of

_artificially

infected

_plants

where a

_drop

of sterile water was

intro-duced into a wound instead of

_mycelium.

After _inoculation, the

_seedlings

were incubated for 4

days

in darkness, then 3

_days

in

_light

_prior

to

_planting

in

pots. Then, the

_plants

were, after

adaptation,

grown in

the

_greenhouse.

The

_plants

used for this _assay were

_aged

3 months and had two to _{six young tillers.} The _assay was carried

out on tillers with three

_totally

_developed

leaves and in

addition, several internal _young leaves without

_flag

con-stituting

a

_pseudo-stem.

The

_fungus

was _present at least in the sheath of the three oldest leaves.

For most associations,

compatibility

or

incompatibili-ty reactions as well as the level of seed transmission, are

reported

in table II

_(from

Naffaa [20]).

The second leaf sheath of each studied tiller was used

for

_peroxidase

extraction. The first basal 5 cm of the

pseudo-stem

of these

_plants

was also used for other

extractions.

2.2. Biochemical methods 2.2.1. Extraction

_{of peroxidases}

Leaf sheaths and

_pseudo-stems

(600

mg fresh

pH

7, at a buffer to tissue ratio of 0.5 mL _{per g of fresh}

weight.

The

_homogenate

was

_centrifuged

at 10 000 _g for 15 min at 4 °C. The _supernatants were used as _enzyme

extracts. Each extraction was

_duplicated.

This

_procedure

allows extraction of soluble _enzymes,

but not of those _enzymes linked to the cell wall.

2.2.2. Proteins

_{quantification}

The

_proteins

were

_{quantified according}

to the method of Bradford [2]. Raw extract _{(5 mL)} was added to 1 mL

of Bradford reactant, then the whole was

_homogenised

mildly.

The

_{optical density}

was read after 5 min of

reac-tion at 595 nm _{per min with} a

_{spectrophotometre}

(Philips

Unicam).

2.2.3. Determination

_{of peroxidase}

_activity

Peroxidase

_activity

was determined

_according

to the

method

_{reported by Boyer}

et al. _[1] which is based on

the increase in

_absorption

of a substrate after its

oxida-tion

_by

_peroxidases.

Raw extract _{(5 mL)} was added to

1 000

_μL

of reaction medium

_containing

900

_μL

of Na-K

_phosphate

buffer _{(0.15 M,}

_pH

= _6.1) with _{0.1 %}

gua-iacol and 100

_μL

of 0.5 %

_H

₂

_O

₂

_.

Absorbance was read

at 470 nm after 1 min of reaction.

_{Specific peroxidase}

activity

was

_expressed

as the increase in absorbance at

470 nm _{per min and per mg} of

_proteins

_[6].

2.3. Statistical

_analysis

Specific

peroxidase

activity

was measured twice

(from two distinct _extractions)

_leading

to two

_replicates.

As Clarine and

_Vigor

were infected

_by

different sets of

endophytes,

the data sets from the two hosts were

analysed separately.

The influence of each

_endophyte

on

_specific

peroxi-dasic

_activity

in the leaf sheaths and

_pseudo-stems

of each host was

_{analysed by}

a variance

_analysis

with one

main effect

_(endophyte

_isolate). In the case of a

signifi-cant main _effect, means were

_{separated by}

the

two-tailed t-test _{of Dunnett which detects whether any} treat-ments are

_{significantly}

different from the control.

These

_analyses

were

_performed

_by

means of the

pro-cedures GLM of SAS _[25].

3. Results

Regardless

of the host

_plant,

the results showed that

_{specific peroxidase}

_activity

was

_remarkably

higher

in leaf sheaths than in

_pseudo-stems

_(tables

III and

_IV)

and the main

_’endophyte

isolate’ effect

was

_highly

_significant

_(P

<

_0.0001)

for each vari-able

_(specific

_{peroxidase activity}

in leaf sheaths

and

_{pseudo-stems).}

The

_comparison

of means _gave

different results

_according

to the associations

con-sidered.

3.1. Festuca arundinacea

In the association between Clarine and the Gliocladium-like

_{(Lp-80), specific}

_peroxidase

activity

in the second leaf sheath was

_slightly

high-er than that measured in the

_pseudo-stem

_(table

III).

In the other

associations,

the

_peroxidase

activi-ty

in the leaf sheath was

_{considerably higher}

than that measured in the

_pseudo-stem.

The Dunnett test shows that in associations with the

Gliocladium-like,

the

_{peroxidase activity}

in sheaths and

_pseudo-stems

was

significantly higher

than that observed in the

controls,

while these

vari-ables were not influenced

_{by LpTG-2}

and one

strain of

_{Epichloë festucae}

_(Kc-1).

Associations with N.

_coenophialum

_(Fa-3)

and E.

_festucae

from Festuca

_gigantea

_(Fg-1a)

exhibited a

significantly

weaker reaction in the leaf sheath than the

_controls,

whereas there was no difference in the

pseudo-stem. E. bromicola

_{significantly}

_{increased the} per-oxidase

_activity

in the

_pseudo-stem

of tall fescue while it did not influence that observed in the leaf

sheath.

3.1 Lolium _perenne

In most

associations,

_{peroxidase activity}

in the leaf sheath was

significantly

lower than that of the

control

_{(table IVa),}

with the

_exception

of E.

festu-cae

_(Fg-1a),

which did not alter the reaction

observed in the

_leaf,

and Gliocladium-like which

result was not observed in

_{pseudo-stems; only}

two associations

_(with

Gliocladium-like and E.

_festucae

from Koeleria

_cristata)

showed a

_peroxidase

activ-ity

significantly

different from that of the control

(table IVb).

In the

_pseudo-stem,

_following

_the gen-eral

_trend,

the Gliocladium-like increased the level of

_{peroxidase activity;}

_{in contrast, the strain of} E.

festucae

(Kc-1)

decreased this

_activity.

4.

Discussion

The infection _{of grasses}

_{by endophytic}

_fungi

is

significant

from both an

_ecological

and an

agro-nomical

_point

of

_view,

_{yet very little is known}

about interactions between the two

_organisms.

A better

_knowledge

of these interactions could be useful for

_generating

novel associations of

agricul-tural interest. Plants

_exposed

to various environ-mental stresses

_{respond by synthesising}

a set of

proteins, including peroxidases.

Moreover,

specific

lignin-forming peroxidases

are considered as PR

proteins

[30]

and classified as PR-6.

Therefore,

peroxidase activity

could be one out _{of many} markers

_indicating

how the host

_{plant responds}

to

endophyte

infection.

Our results show that soluble

_{peroxidase activity}

is influenced

_by

_{the age of the}

_plant

_{organs and}

_by

the presence of the

endophyte.

Peroxidase

_activity

was

_always

_higher

in the leaf

sheath than in the

_pseudo-stem.

Such a result is not

surprising

because

_{peroxidase activity generally}

increases with age

[1].

As the second leaf sheath

was older than the leaves

_forming

the

_pseudo-stem,

the age of each organ accounts for the results obtained.

As

_regards

the

_endophytes,

three different

reac-tions were observed: the

_{peroxydase activity}

of

each association was

_lower,

not

_{significantly}

differ-ent, or

_higher

than that of the control

_(table

III and

IV).

An enhanced

_{peroxidase activity}

was

_expected

because it is a common _{response of}

_plants

to stresses, such as

_pathogen

infection.

Moerschbacher et al.

_[18]

observed such a

response for resistant

near-isogenic

wheat lines

infected

_by

stem rust. Ye et al.

_[31]

also

_reported

increased

_{peroxidase activity}

in tobacco infected

by

the mosaic virus. This

_activity

was linked with

biochemical

_changes

in the cell wall and could be due to

_{specific peroxidases}

_(PR-6).

_Mouzeyar

_[19]

showed that the

_{global peroxidase activity}

of

sun-flower was increased

_by

infection

_by

the

_downy

mildew

_(Plasmopara

_halstedii).

In our

study,

infec-tion

_by

the Gliocladium-like

_{significantly}

increased the

_{peroxidase activity}

of the host. In the associa-tion

_involving

this

_{endophyte, peroxidase activity}

was

_high

even _{in young organs. This may} indicate

that the Gliocladium-like

_triggers

a

_systemic

defence mechanism thus

_confirming

that this

endo-phyte

is

_parasitic

rather than

_mutualistic,

as

postu-lated

_by

_{Siegel et al.}

_[28].

According

to Naffaa et al.

_[21],

_despite

the

delayed growth

of

_seedlings,

the association between

_perennial

_{ryegrass and Gliocladium-like is} stable

_(table

_II).

_Though

Gliocladium-like was not in

_co-symbiosis

with an

_e-endophyte,

the host

plants

looked

_healthy.

Such observations seem inconsistent with the enhanced

_peroxidase

_activity.

However,

the results were

_quite

different in F.

arundinacea. For tall fescue infected

_by

Gliocladium-like,

peroxidase activity

in leaf sheaths was almost

_equal

to that observed _in pseu-do-stems. In

addition,

the

_plants

with this

endo-phyte

showed

_{delayed growth}

and were able to

dis-card their novel

_{endophyte, resulting}

in a low rate of seed transmission

_{(table II).}

In this case, the

enhanced

_{peroxidase activity}

was associated with

morphological

responses. This confirms that Gliocladium-like is

_recognised

as

pathogenic by

tall fescue. As this

_endophyte

is not natural for this

species,

it cannot overcome the defence responses

of tall fescue.

Epichloë

bromicola

_(Be-1)

induced a

signifi-cantly

enhanced

_{peroxidase activity}

in the

pseudo-stem of tall fescue

_{(table IIIb).}

As this isolate from Bromus erectus can form stromata on its natural

host,

it is

_antagonistic

rather than mutualistic and it is not

_surprising

that it

_triggers

a defence response

from tall fescue. On the _contrary, it is more

surpris-ing

to observe that the

_{peroxidase activity}

_of peren-nial ryegrass in the leaf sheath can be

_{significantly}

decreased

_by

this

_fungus.

Leuchtmann and Schardl

[14]

reported

that E. bromicola contains both

stro-ma-forming

and non-stromal strains. Our results

suggest

that the same isolate of E. bromicola

might

have an

_antagonist

or mutualistic

effect,

depending

on the

_species

into which it is introduced.

Unfortunately, plants harbouring

E. bromicola died

as a result of technical

_problems,

and it was not

possible

to observe whether

_they

formed stromata

on the two hosts.

On the other

hand,

most

_e-endophytes

decreased

or did not

_{significantly}

alter

_{peroxidase activity}

of their novel hosts. In leaf sheaths of

_perennial

rye-grass, all the

e-endophytes

used

_(with

_the excep-tion of the strain

_Fg-1a

of E.

_festucae),

as well as

the isolate of

_{a-endophyte, Lpe-1,}

_{significantly}

decreased

_{peroxidase activity}

_{(table IIIa).}

The

same behaviour was also observed in tall fescue

inoculated with N.

_coenophialum

_(Fa-3)

and E.

fes-tucae

_(Fg-1a)

_{(table IVa).}

In the

_pseudo-stem

of

perennial

ryegrass and tall

fescue,

most associa-tions

_responded

as the control

(tables

IIIb and

IVb).

The _{different reactions of young and adult} leaves can be

explained by

the process of

plant

colonisation

_{by e-endophytes; endophytes}

are more

abundant in the first leaf sheaths than in the younger

tissues,

colonised later

[8],

and their meta-bolic

_activity

is

_higher

in mature tissues

_[7].

It is also worth

_noting

that different strains of a same

_species

can

_trigger

different

reactions,

as was

the case of

_Fg-1a

and

Kc-1,

both

_belonging

to E.

festucae.

This can be

_{explained by}

the considerable

genetic

differentiation of E.

_festucae

which can be

harboured

_by

several Festuca

_species

_[15].

Moreover,

Christensen et al.

_[4]

noted that different

isolates of E.

_{festucae provoked}

different

morpho-logical

reactions

(isolates

referred as

stunting

or

non-stunting)

when

_they

were introduced into

novel hosts. Neither of the two isolates of E.

festu-cae used in this

study provoked

_any

morphological

reaction in their novel

_host,

nor did

_they

enhance its

_{peroxidase activity.}

However,

there is no direct

_relationship

between

peroxidase activity

and

_{morphological}

reactions.

Despite

the

_{delayed growth}

of tall fescue

_seedlings

inoculated

_by

_Lp-1

_{(table II),}

their

_peroxidase

activity

was not

_{significantly higher}

than that of the control. This

_might

indicate that tall fescue

accept-ed this novel

_endophyte,

an

_hypothesis

confirmed

by

the

_high

rate of seed transmission of the

endo-phyte

in its novel host

_{(table II).}

_Conversely,

the association between

_Vigor

and N.

_coenophialum

did not _appear to be

_compatible

_{(table II)}

_though

the

_{peroxidase activity}

of the host was not altered

by

the presence of the

fungus.

Our results show that

_{e-endophytes,}

even intro-duced into novel

_hosts,

do not

_generally

enhance

peroxidase activity. Reddy

et al.

_[23]

_reported

that

mutualistic

_fungal

_endophytes

and some

Epichloë

species

express a

_protease

close to _proteases

sus-pected

to be virulent factors in

_pathogen

_systems.

Moreover,

Roberts et al.

_[24]

showed an increased level of chitinase in

_{endophyte-infected}

tall fescue.

Therefore,

in our

_experiment,

the host was

expect-ed to exhibit defence reactions. But this was not the case. The lack of enhanced

_{peroxidase activity}

by e-endophytes

may facilitate the colonisation of their host. This

_phenomenon

_{may indicate that} many

e-endophytes

are not

_{recognised by}

tall

fes-cue and

_perennial

_{ryegrass. These}

_fungi

_{may have}

developed

similar mechanisms to _escape

recogni-tion

_by

_grasses.

_However,

the

_peroxidases

are

_only

one _{group of enzymes involved in the defence}

reaction of

_higher

_plants

_{against micro-organisms.}

Study

of additional _systems

_(PAL,

_chitinases,

glu-canases,

etc.)

might

lead to

_slightly

different

con-clusions. In

_addition,

the

_procedure

used for

extrac-tion

_{yields only}

a fraction of the

_peroxisases

(solu-ble

_peroxidases)

_leaving

in the

_pellet

_{the enzymes} linked to the cell wall.

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