Dynamic conservation of variability responses of wheat populations to different selectives forces including powdery mildew

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Dynamic conservation of variability responses of wheat

populations to different selectives forces including

powdery mildew

V. Le Boulch, Jacques David, Philippe Brabant, Claude de Vallavieille-Pope

To cite this version:

Original

article

Dynamic

conservation of

_variability:

responses

of

wheat

populations

to

different selective forces

including

powdery

mildew

V Le Boulc’h

JL David

P Brabant

C de

_{Vallavieille-Pope}

1

Institut

National de la Recherche

_{Agronomique-UPS,}

Station de

_{Génétique Végétale,}

2

Ferme du

Moulon,

91190

_{Gif sur-Yvette;}

2

Institut

National de la Recherche

_Agronomique,

Laboratoire de

Pathologie Végétale,

Grignon,

78850

_{Thiverval-Grignon;}

3

Institut

National

_{Agronomique Paris-Grignon, 16,}

rue

_{Claude-Bernard,}

75005

Paris,

France

Summary - A programme of

dynamic

conservation of

_genetic

resources has been conduc-ted on winter wheat in France since 1984. One

_allogamous

and 2

_{preferential selfing}

popu-lations,

with a

_{large genetic base,}

made up the initial gene

pools.

Seed

_samples

of these

pools

were distributed

_throughout

a multi-site

_experimental

network. The

_populations

obtained were

multiplied

each _{year in} each site.

_{Populations developed}

_{significantly}

after 8 years of

multiplication: plant height

had

increased,

the

_proportion

of viable

_{pollen grains}

had increased in male-fertile

plants

of

allogamous populations

and a north-south

_gradient

for

_precocity

was observed for autogamous

populations.

Resistance to

_powdery

mildew had

_developed

in local

populations,

both for

_adult-plant

resistance and the

_frequency

of

specific

resistance genes to

Erysiphe

graminis f sp tritici. The differentiation

_depended

on the _gene

_pool

of the host

populations.

Virulence

_frequencies

of the parasite

popula-tions varied

_according

to the site and the year. There was no clear

_relationship

between virulence

_frequencies

and resistance gene

frequencies.

The

proportion

of

plants

without

specific

resistance genes seems to correlate with

adult-plant susceptibility

in local

auto-gamous

populations.

Most of the

_variability

was maintained

_{through propagation}

under various natural selection forces.

dynamic conservation

_/

lyiticum aestivum

_/

_{Erysiphe graminis}

_/

natural selection

_/

population

Résumé - Gestion dynamique de la variabilité : _réponses de _populations de blé tendre à différentes forces sélectives dont l’oïdium. Un _programme de

_gestion

_dynamique

des ressources

_génétiques

est

_développé

en France

_{depuis 1984}

sur le blé tendre. Deux

de

_grains

de ces

_pools

ont été distribués dans un réseau multilocal. Les

populations

ainsi

obtenues sont

multipliées

année

après

année dans

chaque

lieu du réseau.

_Après

8 ans de

_{multiplication,}

les

_populations

ont nettement évolué. On constate une

_augmentation

de la taille des

_{plantes adultes,}

une meilleure

production

de

_grains

de

_pollen

viable chez

les

_plantes

mâles

_fertiles

des

_{populations allogames}

et la mise en

_place

d’un

_gradient

de

précocité

Nord-Sud _pour les

_{populations autogames.}

La résistance à l’oïdium a évolué dans

les

_populations,

à la

fois

pour la résistance au stade adulte et pour les

_fréquences

de

_gènes

de résistance

_spécifique

vis-à-vis

_{d’Erysiphe graminis f sp}

tritici. La

_{différenciation dépend}

du

_{pool génétique auquel appartiennent}

les

_populations

hôtes. Les

_fréquences

de virulence

des

_populations

du parasite varient selon les sites et les années. Aucune relation claire n’a été mise en évidence entre les

_fréquences

de

_virulences

du

_parasite

et celles des

_gènes

de résistance

_spécifique

de l’hôte. La proportion de

_plantes

sans résistance

_spécifique

semble

corrélée à la sensibilité au stade adulte dans les

_populations

_autogames. La diversité des

milieux

formant

le réseau a

globalement permis

le maintien de la variabilité.

gestion dynamique

/

!iticum aestivum

_/

Erysiphe graminis

/

sélection naturelle

_/

population

INTRODUCTION

The conservation of the

_genetic

_diversity

of cultivated

_species

enables us to maintain

selected _genes involved in a

plant’s

_adaptation

to different environments. The

management

of

_genetic

resources

by

_storing

seed

_samples

ex situ is a

widely

used

method,

which answers an

_urgent

_problem

of

_conserving

the

_{genetic diversity}

of

a

_species

but

_only

represents

one

_aspect

of

_conserving

_genetic

resources. It

does,

however,

have limitations:

- the number of

samples

to be conserved _{is very}

_large

and continues to

_increase;

- evaluation of seed

samples

is

_costly

and can

_only

be carried out for a limited

number of traits and

_samples.

A

_major

_part

of the

_variability

stored is therefore

poorly known;

- the

variability

stored is isolated from the evolution of the natural environment.

The

_dynamic

_management

of

_populations

maintained in their natural

environ-ment has often been

_proposed

as

_{complementary}

to static

_management

_(Pernes,

1984;

Allard,

1990,

Henry

et

al,

1991).

Its

_objective

is to imitate nature in its

abi-lity

to maintain a wide

_variability

within numerous small diversified

_populations.

Humankind has

_played

an

_important

role in

diversifying

and

spreading

cultivated

species

over

_huge

_territories;

this

_activity

should therefore be

_envisaged

in a

dyna-mic conservation programme aimed at

_maintaining

resources which can be used in

plant breeding

(Gallais

et

_al,

_1992).

In order to establish a

_methodology

for

_dynamic

conservation and to evaluate

its

_potentials,

a programme was set _up on a national scale under the

_impetus

of

the Institut National

_Agronomique

of

_{Paris-Grignon}

_using

genetic

material selected

by

INRA. Since

_1984,

INRA and a number of

agricultural

universities and schools have

participated

in a network for the

_{multiplication}

of winter wheat

_populations

pyramidal

cross

_involving

16

_parents

_representing

a wide

_genetic

base. PSO was

obtained from a cross of 50 lines with the male-sterile

_variety

_Probus,

which is

_homozygous

_{for the recessive gene} mslb of the nucleus

_{(Trottet, 1988).}

Seed

samples

from these

_pools

were distributed

_throughout

sites

_representing

a varied range of environments. The

populations

obtained in this _way were

_multiplied

and resown year after year in the same site. To increase the

_diversity

of environmental

conditions, populations

were _{grown in} each site

_using

both an intensive

_farming

method

_(with

the same

_nitrogen

fertilizers and

_pesticides

as those used in the

region)

and an extensive

_farming

method

(one

third of the

_nitrogen

fertilizers used in the intensive method and fewer

_pesticide

_treatments).

This structure was chosen to enable the local differentiation of each

_population,

while

_maintaining

a

large

genetic

variability throughout

the network

_(David

et

_al,

_1992).

The

_populations

obtained

from PAO and PBO are

preferential selfing populations

and those obtained from PSO are outcrossed

_using

male

_sterility.

The

_objective

was to measure the

value of

_outcrossing

an

_autogamous

_species

to conserve its

_genetic

_diversity.

Precise

descriptions

of the 3

_populations

and how

_they

were obtained were

_{provided by}

Picard

_(1984),

Thomas et al

₍₁₉₉₁₎

and Pontis

_(1992).

The first results

_(David,

_{1992; Pontis,}

₁₉₉₂₎

showed that a _very distinct differen-tiation between

_{subpopulations}

of the same

_origin

_(PA,

PB or

_PS),

_appeared

after

only

a few years of

multiplication.

Genetic drift could not be ruled out to

_explain

this,

but such a

_rapid

differentiation would

_only

be

_expected

if the number of

re-productive

individuals was

_small,

which was not the case with the

experimental

protocols

used

_{(Pontis, 1992).}

Selection

_appeared

to be the most

_likely

cause of the

observed

_{developments,}

either

_directly

or

by hitch-hiking.

There are at least 3

_types

of selection _pressures in our

_experiment:

- intrinsic

pressures, which are the result of interactions between individuals within each

_{subpopulation;}

-

pressures

imposed

by

the external

physical

_{(pedoclimatic)}

conditions;

-

pressures

resulting

from the interaction of other biotic factors such as

pathogens.

After

_{briefly considering}

the evolution observed in traits

_subjected

to the first

2

_types

of selection pressures, we will

_present

in detail the results

_concerning

the evolution of resistance to

_powdery

mildew

_(Erysiphe

_graminis

DC ex Merat f sp tritici Em

_Marchal).

Selection forces intrinsic to

_populations:

the evolution

_{of plant}

size and

pollen production

In a

genetically heterogeneous population,

_competition

between individuals creates

selection forces which

_{change depending}

on the

_genetic

structure of the

_population

and the environmental conditions. The

_experiment

conducted on winter wheat

provided

2

_examples

of evolution under the influence of

_{inter-genotype competition.}

The first

_example

concerns the evolution of

_plant

size. The second concerns

pollen

production

in the PS

_{allogamous pool.}

A

_general

increase in adult

_{plant height}

was observed in all the

_populations

originating

from the PA and PB

_autogamous

_pools

_{(David, 1992).}

The tall

_plants

(1994)

showed in the PB

_population

that the tall lines were favoured in

competitive

situations with short lines. Their seed

_production

is

_higher

when in

_competition

than

in monoculture. For short

_lines,

the

_opposite

_phenomenon

has been observed:

_they

produce

more in monoculture than in a

_competitive

situation with taller

plants.

The

evolution observed in our

_experiment

was

_greater

and more

_rapid

than that observed in the United States in

_{barley populations}

under

_{comparable growing}

conditions

(Hockett

et

_al,

_1983;

_Allard,

_1988).

This difference in behaviour is due to the

genetic

composition

of the wheat

_populations:

the initial 1984

_populations

contained

2

_dwarfing

_genes

_(rhtl

and

_{rht2, McIntosh,}

₁₉₈₈₎

at

_{relatively high frequencies}

(Pontis, 1992).

The differences in size between

_genotypes

was therefore very distinct

and moreover _very heritable.

Figure

1 shows the

relationship

between the average

size of adult

_plants

within the

_populations

and the

_frequency

of individuals with

dwarfing

genes. This situation enabled a

_rapid

evolution of

plant

height

and a

_drop

in the

_frequency

of the rhtl and rht2 genes. The

rapidity

of the

phenomenon

can

also be

_{explained by}

the

_increasingly

_greater

_proportion

of tall

_{plants. Indeed,}

the

more tall

plants

there _are, the more the short

plants

are

disadvantaged.

depending

on environmental conditions:

_{rainfall, nitrogen dose,}

and soil

_depth.

For

_example,

_nitrogen

increases the

_intensity

of

competition,

first

_{by enabling}

dense

_plantations

to be established and second

_{by allowing}

substantial

_growth

in

height.

Therefore the

_autogamous

_populations

PA and PB _grown

_intensively

became

genetically

taller than the

_populations

grown

extensively.

At

_present,

it is not known

_just

how far _{the programme} can increase the

height

of wheat

_{populations. Very}

tall

_plants

_{(1.8 m)}

were observed which did not exist in

the initial

_populations

but which were obtained

_by

recombination. Plant

_height

of

adult wheat _{grown in} bulk is considered to be a trait

_subject

to

_stabilizing

selection

(Khalifa

and

_Qualset,

_1975),

the shortest

_being

eliminated

_by

_competition

and the tallest

_{by lodging. Lodging}

_may also favour tall

_plants

since

_they

remain above the

plant

cover, continue to

_capture

_light

and

stay

in a

satisfactory

state of health. This evolution in

_height

is a

problem

if we wish to maintain

_genetic

_diversity

for

subsequent exploitation.

Modern varieties are

_genetically

more

_productive

because

they produce

a

higher

_grain

weight

for the same aerial biomass

(Austin

et

al,

1989).

This evolution occurred because of the decrease in the

_length

of stems. Small size is also an

_adaptation

to the intensive

_growing

_practices

which involve

_greater

plantation

densities and

_high

levels of fertilization.

In addition to the

_problem

of time involved in

_converting

tall

_genotypes

to

cultivable

_genotypes,

_{losing dwarfing}

_{genes in}

_populations

would be an error, since

they

have numerous

pleiotropic

effects

(Pinthus

and

Gale,

1990).

It is therefore necessary that

populations

evolve with these _{genes in} order to select an

_adapted

gene

pool. Height

is also a

_physical

means of

escaping

from certain pressures

from

_parasites,

_particularly

on the

spike

(Septoria

nodorum,

Scott et

_al,

_1985).

This

phenomenon,

in

_uniformly

tall

_populations,

_prevents

_selecting

other

_genetic

resistance mechanisms which would function in short varieties.

A second

_example

of

_competition

between individual

_plants

is

_provided

_by

the evolution observed in

_{pollen production}

with the

_allogamous

PS gene

pool.

Wheat is

_naturally

_autogamous

and

_{produces relatively}

little

_{pollen. Introducing}

allogamy

in the

_species

creates a

major

disturbance. In

PS,

the

reproductive

value of

hermaphrodite plants depends solely

on their

_capacity

to fertilize females.

_They

are

used as males and these males

_compete

with each other in their

_ability

to

_pollinate.

Theoretically,

their viable

_{pollen production}

is

_expected

to

_improve

_(Charnov,

1982).

In

fact,

this

_improvement

was observed 6 _years after the

_beginning

of the

experiment

(David

and

_Pham,

_1993).

Selection _pressure on this trait is

_strong

and

can result in modifications of

_{morphological}

and

developmental

traits which also

have a role in

_adapting

the

_plant

to environmental conditions.

Competition phenomena

among

plants

are a

_major

factor in

_population

evolu-tion. When

_they

are

_present,

the

evolutionary

_response is

expected

to be

_directional,

but with differences in

_intensity

_depending

on the environments in which the

popu-lations are

_multiplied.

Difficulties arise in

_conserving

_diversity

for genes

subjected

to these selection forces.

Selection for

_adaptation

to the

_physical

environment

The

_evolutionary

_pressures exerted

_by

the environment can be considered as

Under these

_conditions,

_populations

are

expected

to differentiate in order to

adapt

to the characteristics of their environment.

_Contrary

to the

_example

of

inter-individual

competition,

the evolution may occur in directions

radiating

around the initial situation. This idea is at the basis of the

concept

of the centre of

origin

(Pernes, 1984);

the

_diversity

of environments creates and maintains

_genetic

diversity.

This also

_justifies

the

_dynamic

conservation of

_diversity.

This means that it is

_possible

to recreate or restructure a lost natural

variability by subjecting

a

genetically heterogeneous population

to a

particular

_environment;

the

adaptative

responses are

expected

to be

_repeatable.

For the

_autogamous

_populations

of the PA and PB

_pools,

we observed a

north/south

gradient

for

_precocity.

The

_{populations multiplied}

in the south became

genetically

more

_precocious

than those

multiplied

in the north

(fig

2).

This evolution was

presumably

an

adaptation

to the environment

firstly

because the trend occurred in numerous

independent populations

and

secondly

because of the

reproductive

cycle

of the

_plant.

In the

_south,

water stress _appears

_early

and late

_plants

do

not fill their seeds

_{satisfactorily.}

These seeds are scalded and are at a selective

disadvantage.

In the

_north,

this stress never appears or else _appears late. Late

plants

accumulate more

dry

matter and can be more

_productive.

More

_generally,

this

_phenomenon

can also be due to an

adaptation

to the

_length

of the

_day,

variable

on the

north/south

axis. A

gradient

for

north/south

precocity

has also been found for old wheat cultivars on a

regional

scale

_going

from the Middle East

through

to

Nepal

(Kato

and

_Yokoyama,

_1992).

In

_autogamous

_populations,

it seems that a

large

_part

of

inter-population

variability

is caused

_by

an

adaptation

to the environment and this

_applies

to

multiple

traits

_(Weir

et

_{al, 1974; Mitchell-Olds, 1992;}

Nevo and

Beiles, 1992;

Oosterom and

_Acevedo,

_1992).

It _may therefore be

_possible

to use environmental variations for

conserving genetic

resources.

An

_adaptative

response to the biotic environment

Selection _pressures due to

_parasites

are caused

by genetically

variable

living

organisms.

Parasites evolve in interaction with host

_plants

and exert variable selection _pressures which

depend

on the

location,

and evolve with

time,

as shown

by

the French

_populations

of

_Erysiphe

_graminis

f sp hordei

(Andrivon

and de

Vallavieille-Pope,

1993).

These pressures involve a

_system

of coevolution between

2

_types

of

_population,

host

_populations

and

pathogen

populations.

In

_barley

populations

in

California,

Allard

₍₁₉₉₀₎

observed a

_positive

correlation between an increase in the

_frequency

of resistance alleles and their selective

_advantage

towards the

_parasite

_{population Rhynchosporium}

secalis.

Resistance to

_powdery

mildew is a

_good

_marker,

since it is a _very

_widespread

disease

_causing

considerable reductions in

_yield.

_Moreover,

_powdery

mildew is

caused

_by

a

_biotrophic

_parasite

which has a _very

_specific

relationship

with its

host,

without a

_{saprophytic phase.}

Resistance to

_powdery

mildew in wheat has 2

_components:

_specific

resistance in accordance with the

_{gene-for-gene}

model

pressures due to

_parasites,

we characterized the virulence

_spectra

of these

popu-lations for some known genes.

_Second,

the

_frequency

of several _{resistance genes} was estimated in

_plant

_{populations multiplied}

on the same

sites,

harvested after 8 _years of evolution. In these

_populations,

each

_plant

was different and resistance genes were determined for each individual: this was an

_{original approach. Finally,}

resistance at the adult

_stage

of these

_populations

was estimated in the field.

MATERIALS AND METHODS

Virulence

_spectra

of the

_{pathogen populations}

Virulence

_frequencies

in E

_graminis

f _sp tritici

_populations

were estimated for 3 sites of the

_{multiplication network,}

Le Moulon

_{(Gif-sur-Yvette),}

Le Rheu

_{(near Rennes)}

and

Toulouse,

for 1991 and 1992. The

_frequencies

were determined

_using

_seedlings

of differential hosts

_{(table I),}

which were

placed

in the 3 sites to

_trap

_pathogen

population

spores.

PB for Le Moulon and

Toulouse,

and PB and PS for Le

_Rheu)

and were left for 1 d. For each differential set, the lines were

placed

at random. In order to have

2

_replications

for Le Moulon and Le

_Rheu,

2 differential host sets were

_placed

close to each

_population.

The

_experiment

was

_repeated

twice _{per season,} in the first

_days

of

_May

and June.

After a 10-d incubation

_period

at 20° C

_protected

from any outside

infection,

sporulating

lesions were counted on the

_primary

leaf of the

plants

in each differential host set. The

_frequency

of _{each virulence gene}

_(or

virulence gene

combination)

was

estimated from the infection rate on the

corresponding

differential

line,

which was

the ratio between the number of lesions on this line and the number of lesions on the most infected differential line.

ANOVA was used for statistical

analysis; preliminary

tests showed that

_’year’,

’site’ and ’differential line’ effects and interactions on infection rates were

_highly

significant.

The effects of the date of the

experiment

and the

population

in

_proximity

to the differential host set were not

_significant

_(P

>

_5%).

_{Consequently,}

we

_present

the results for each line per site and per year, the infection rates

being averaged

over the season.

Evolution of

_specific

_{resistance genes}

We studied _{12 resistance genes,} the Prra1 to Pm6

series,

Pm8,

Mli and the

_Michigan

cultivar gene

(Mia),

which are the most

_commonly

used in

_breeding

_programmes. In

order to detect these genes in

plants,

we selected a monocolonial isolate collection

with virulence

_patterns

_adapted

to

_identifying

the 12 _genes.

_Among

50 isolates

genes were indissociable:

Pml,a

and

Pm4b; Pm3c,

Pm5 and Mia. In

addition,

some tests were unable to

_distinguish

Mli from Pm8. These indeterminations will be referred to as

_Pm4a/Pm4b,

_Pm3c/Pm5/Mia

and

_Mli/PmB.

For each of the

_PA,

PB and PS

_{pools, analyses}

were conducted on the

populations

grown for 8 yr at Le

Moulon,

Le Rheu and Toulouse

(1992 harvest)

and on the

populations

PBO,

PSO and PAl. Because of the low

_{germination capacity}

of the initial

_{population PAO,}

we used the

_population

PAl _grown for _{1 yr at} Le Moulon

and harvested in 1985 as the reference for

_pool

A.

Between 40 and 75

_plants

were

analysed

_per

population.

To

identify

resistant genes in individual

plants,

each

_plant

was inoculated with the 9 isolates

_according

to the method described

_by

Limpert

(1985).

The first 2 leaves were cut into 9

segments

and each

_segment

was inoculated

_separately

with 1 isolate.

_Sporulation

was observed after a 10-d incubation

period

in a

mildew-proof growth

chamber at

16°C.

Evolution of adult

_plant

resistance

An

_experimental

_study

of adult

_plant

resistance to

_powdery

mildew was conducted at Le Moulon. It included the initial

_populations

PBO and PSO and

_populations

of the 3

_pools

_A,

_B,

and S _grown for _{6 yr} at Le

_Moulon,

Le Rheu and Toulouse

₍₁₉₉₀

harvest).

The

experimental

design

was a randomized block with 2

_replications

and 50

_plants

in each

_plot.

Plants were scored

_individually

twice

_(once

at the end of

_May,

and once at the end of

_June).

A 0-to-9 scale was used with 0 for a

plant

without

visible

_symptoms

and 9 for a

plant

with a

high density

of

_{sporulating pustules}

_up

to the last leaf.

RESULTS

Virulence

_spectra

of the

_pathogen

populations

The infection rates of each differential line for 1991 and 1992 at Le

Moulon,

Le Rheu and Toulouse were determined

_{(fig 3).}

At Le Moulon in 1991 _many virulence genes were

_present

in the

_pathogen

_population,

since

_only

Pm3b virulence and the Pm2 +

Mld +

Mlk virulence combination had low

_frequency.

From 1991 to

_1992,

_frequencies

of several virulence _genes decreased.

_Only

Pm3b

_{frequency clearly}

increased.

Numerous virulence genes were also

_present

at Le Rheu. There was _very little

difference between the _{2 yr.} In

_particular,

the

_frequency

of

Pml7, Pm3b, Pm3a,

Mlk and Pm2 +

Mld,

Pm2 + Mld + Mlk combinations remained very low.

Compared

with Le

_Moulon,

the

_{pathogen population}

of Le Rheu had more combinations of

several virulence _genes,

_except

those with Pm6 or Tal.

The results from Toulouse were

_considerably

different from those of the 2 other

sites,

because there were few virulence genes at

_{high frequency}

in the

_pathogen

population,

particularly

in 1992.

The correlation

_matrix,

established from common differential hosts for the 2 _yr

(table II),

shows the overall differences between sites and years. The 1991 and 1992

spectra

of the Le Rheu

_{pathogen population}

are

_clearly

similar

_(0.97

correlation

Moulon and 0.63 in

_Toulouse).

The correlations between

_spectra

of the Le Rheu

and Toulouse

_{pathogen populations}

are the lowest between-site correlations

_(0.45

for 1991 and 0.33 for

_1992).

For Le Moulon and

_Toulouse,

the differences between the _years are

comparable

to the differences between the sites: ’Within-site

between-year’

correlations are close to ’between-site

_{within-year’}

correlations.

Evolution of

_specific

resistance _genes

In the

_population

set

_{(table III),}

_Pml,

_Pm2,

_Mli,

Pm8 genes and

Pm4a/Pm4b

and

Mli/Pm8

were identified. Pm3b was considered to be

_present

_only

in

_pool

A and

Pm3c/Pm5/Mia

only

in

_pool

S. Other resistance _{genes may} have been

_present,

but our

_experiment

did not detect them.

In

_{pool B,}

the

_only

_significant

difference

_(at

P <

_10%)

between 1992

_populations

and the initial

_population

is the increase in the

_frequency

of

_{susceptible plants}

in

the

population

of Le Rheu. In

contrast,

populations

of

_pools

A and S had evolved.

For

_pool

_A,

_frequencies

increased for Pm2 and Mli in Le

_Rheu,

and for

_Mli/Pm8

in Toulouse. For

_{pool S,}

an increase of

_Pm4a/Pm4b

_frequency

was observed in the 3 sites.

_(P

<

5%

in Le Rheu and

_Toulouse,

but P <

10%

in Le

_Moulon).

_Overall,

the

populations

of Le Moulon evolved less than the

_populations

of Le Rheu and Toulouse.

To evaluate the total evolution of each

_pool,

we

_compared

the

_frequency

of

susceptible

plants

in the initial

_population

to the average in the 3 descendant

populations.

We noticed a

_{highly significant}

decrease

_(P

<

_1%)

in PS and a small

decrease

_(P

<

_10%)

in PA. The mean increase in PB was not

_significant.

Figure

4 shows the evolution of the gene combinations detected in the

experi-ment. In

_{pool A,}

combinations of _{3 genes}

_appeared

in Le Rheu and Toulouse. For

pool B,

combinations of

_2,

3 and _{4 genes} were identified whereas

_they

were

ab-sent in PBO. In

_{pool S,}

the Le Moulon and Toulouse

_populations

showed

_complex

combinations,

with

_probably

more than _{4 genes,} which were absent in PSO. On the other

_hand,

in the same

_pool,

the

_population

of Le Rheu

_possessed,

at

_most,

double

after 8 yr of

multiplication

showed an increase in the

_frequency

of

_plants

_carrying

a

combination of several

_specific

_{resistance genes. In 2}

_populations

of

_{pool S, complex}

combinations were selected.

Evolution of adult

_plant

resistance

ANOVA of

_powdery

mildew

_severity

in

_populations

harvested in 1990 showed a

highly significant ’population’

effect in both assessments. In the first one,

pool

A was

_{distinguishable}

from

pools

B and S because of its

_greater

resistance: a

Student-Newman-Keuls test

_(P

<

_5%)

_put

all the

_populations

of

_pool

A in one group, whereas

populations

of

pools

B and S were distributed into 2 _groups in which

_powdery

mildew was more severe. In the second

_assessment,

the same test

revealed 3

_overlapping

groups. Differences that had

appeared

at the first assessment were no

longer

distinct.

Nevertheless,

the most resistant group was

_composed

of 2

_populations

of

_pool

A

_(Le

Rheu and Le

_Moulon).

For

_pools

B and

S,

the resistance of the initial

_population

was

_compared

with

the mean resistance level of the 3

populations

harvested in 1990

using

the contrast

method. There was a

_significant

decrease in resistance

_(P

<

_5%)

in

_pool

B at the

first assessment. In

_pool

_S,

a mean increase in

_resistance,

(P

<

_5%)

_appeared

at

Relationship

between

_{specific susceptibility}

and adult

_plant

susceptibility

For each

_population,

the

_frequency

of

_{susceptible plants}

was

plotted

_against

the

2 assessments of

_powdery

mildew

_severity

observed at the adult

_stage

_(fig

_5).

For the first

assessment,

the

_populations

of the

_autogamous

_pools

A and B fitted

perfectly

on a line with a

_positive

_slope

(r

2

<

0.96).

The

_populations

of

pool

S were

_{clearly distinguishable.}

For

_these,

in

_spite

of the

_{high frequency}

of

_plants

carrying

specific

resistance genes, adult

_plant

resistance was poor,

except

for the Le Moulon

_population.

For the second field assessment of the

_populations

of the

2

_autogamous

_pools,

the linear

_relationship

between

_{specific susceptibility}

and

adult

_{plant susceptibility}

was

_maintained,

but the closeness of fit was not as

_good

DISCUSSION

The characteristics of the

_{pathogen populations}

in the 3 sites were different.

Selection _pressure due to

_parasites

was low in

Toulouse,

high

in Le Rheu and

intermediate in Le Moulon. The

_{pathogen population}

in Le Rheu was _very stable from one year to the next and the presence of numerous differential varieties

showing

a

_high

infection rate

_{(fig 3)}

indicates the

_{population’s}

abundance of virulence

genes, associated in multivirulent strains. In

contrast,

the Toulouse

pathogen

population

evolved

_rapidly.

It was in this

_population

that we found the

_greatest

interactions between years and varieties and between dates and varieties

(results

not

_shown).

_Furthermore,

the small number of

_strongly

infected differential varieties

(fig 3)

suggested

that the strains

_present

in Toulouse were mono- or

oligovirulent.

Selection _pressures due to

_parasites

in Le Rheu and Toulouse therefore

_represented

2 contrasted situations. Climatic factors were

probably

the cause of this situation.

Conditions in Le Rheu favoured the

_development

of this

_parasitic

_fungi

and thus the selection _pressure that it exerted each _year was considerable. The varieties most

cultivated in the

region

are multiresistant. In

_fact,

the

_Apollo

_variety

has the _{3 genes}

Pm2, Pml,b

and Pm8

_(McIntosh,

_1988).

Soissons

_probably

has a

_complex

resistance

but it does not seem to bear _genes which are

_currently

known

(Doussinault,

personal

communication).

The multivirulent

_fungi

strains for these _genes have a better

selective value than monovirulent strains and are in

competition

with the latter.

Each _year, these

_complex

strains must be

_rapidly

selected.

Therefore,

the

_pathogen

population

must be stable

_during

each season and from one _year to the next. In

contrast,

the climate in Toulouse does not allow

_major

_powdery

mildew attacks. The

_{pathogen population}

is not

_subjected

either to inter-strain

competition

or to

selection under the effect of very resistant varieties. Its

composition

in virulence genes remains

simple,

but drift and

migration

may

constantly modify

it.

Similarly,

differences in

_stability

of

_parasite

_populations

have been found between certain

regions

of

_Spain

_by

Molina-Cano et al

₍₁₉₉₂₎

for E

_graminis

f _sp hordei.

It is difficult to link the evolution of the

_frequencies

of

_specific

resistances in wheat

populations

to the

_spectrum

of virulence _genes of the

_pathogen

_populations

in the

different sites. Plants

_carrying

resistance _genes

_{corresponding}

to rare virulence _genes in the

_pathogen

are believed to have a better selection value.

However,

the

_types

of

evolution which we observed contradict this

_prediction.

Genes that have

_undergone

the

_greatest

increase in

_{frequency correspond}

to virulence genes which are very

frequent

in the

_pathogen.

Two models are

_possible

to

_explain

these

_findings.

Firstly,

the

_spectrum

of virulence _genes found in the

_pathogen

_population

is

not

_{representative}

of the

_region.

The _spores

_trapped

near a

_population

_(PA,

PB or

PS)

reflect the

_composition

of the

_{pathogen population}

which has succeeded in

developing.

Since the differential varieties were attacked with the same

_severity

no matter which host

_{population they}

were

placed

beside

(results

of the Anova are not

presented),

this

_hypothesis

alone cannot

_explain

the results.

Secondly,

if certain virulence _genes reduce the selection value of the

_fungi,

the

plants

with the

_{corresponding}

resistance _gene are attacked

_{only by}

strains which

are not _very

_aggressive

and the disease is

_delayed.

In this

_context,

the

_resistance,

even _overcome, is able to

_provide

a selective

_advantage.

In this

model,

certain

a situation has been described

_{by Kearney}

and Staskawicz

₍₁₉₉₀₎

for

_Capsicum

annuum

-Xanthomonas

cam

P

estris.

The

_relationship

between the

_frequencies

of

_specific

resistance _genes and viru-lence _genes also

_depends

on the differences in evolution of the

specific

resistance genes in the 3

populations

A,

B and S

multiplied

on each site. The selection forces

exerted

_by

the

_pathogen

did not lead to a

parallel

evolution of the

populations

multiplied

on the same site. In

_fact,

it seems that the evolution observed

depends

more on the initial

_genetic

context than on the selection pressures at stake.

The

_relationship

between

_specific

_{susceptibility}

and adult

_{susceptibility}

is _very

good

for

_populations

of the

_autogamous

_{pools. Allogamous pool}

S stands out

with a

_relatively

low

_frequency

of

_plants

without resistance genes and a

high

incidence of disease. Pool

_B,

with the

_{highest specific susceptibility level,}

had a better

_performance

in the field than

pool

S. General resistance

systems

are

_perhaps

present.

Pool A is _very resistant in the field and could combine both

mechanisms,

specific

resistance and

_general

resistance.

The distinctive behaviour of

_pool

S

_compared

with the 2

_autogamous

_pools

has

also been described for other traits

_{(David, 1992).}

It can be

supposed

that the

major

selection forces for this

_{allogamous pool}

are not the same as those

_acting

on the

_autogamous

_pools.

_Furthermore,

_harvesting

male sterile

_spikes

_requires

human intervention and thus a subconscious selection which interferes with natural

selection.

_{Heterozygosity}

and the recombination rate also differentiate

_pool

S from

the 2 others. The favourable

_epistatic

relations are less

_easily

maintained and selected. On the other

_hand,

the

_positive

effect of recombination is illustrated

_by

the creation of multiresistant

_plants

_{(fig 4).}

For

_{pool S,}

it seems that resistance in the field is linked more to the

_frequency

of multiresistant

_plants

_(fig

4 and

₅₎

than

to the

_frequency

of

_plants

without resistance _genes.

CONCLUSION

The selection pressure caused

by

the

pathogen

is variable

according

to time and

place. Populations

differ in terms of their

_composition

of

_specific

resistance _genes

and also show a variable level of adult resistance.

_Only

one _{resistance gene} seems to

have

_disappeared

_(Pm3b)

_during

the 8 yr of natural selection. The other genes have

kept

or increased in

_frequency.

With the creation of multiresistant

_plants,

overall

genetic

variability

in

_populations

for resistance to

_powdery

mildew is

_greater

in the

present

populations

than in the initial

_populations.

_{Only populations}

PB have not

undergone

any increase in their resistance to

_powdery

mildew. This could be due

to the low

_frequencies

of resistance in the initial

_population

or interaction with the

evolution of other traits

_subjected

to

_stronger

selection pressures. As a

_comparison,

the

_improvement

in resistance to

_{Rhynchosporium}

secalis in

_barley

_{(Allard, 1990)}

began only

about 15 _yr after the

_beginning

of the

_experiment.

_Generally,

and

especially

in

_autogamous

_pools,

the different traits do not evolve

_{independently}

of one another and

_{only global}

methods are

_appropriate

for

_managing

the

variability

of these

_populations.

In the

_present

_populations,

it seems that

variability

has been

correctly

maintained for _genes involved in

_adaptation

to the

_physical

environment

and for resistance to diseases

_(by

_{extrapolating}

the results obtained on

powdery

competition.

However it is necessary to maintain these genes for use in

breeding

programmes. Measures to

stop

the

disappearance

of

dwarfing

genes are in the process of

being

evaluated.

ACKNOWLEDGMENTS

This work was financed

by

the Direction G6n6rale de

1’Enseignement

et la Recherche

of the Ministbre de

_{t’Agriculture,}

the Bureau des Ressources

_G6n6tiques

and the Institut National de la Recherche

_Agronomique.

We would

_particularly

like to thank A Gallais and PH

_Gouyon

as well as JP

_Henry

for

_initiating

the

project

on the

_dynamic

conservation

of

_{genetic variability}

in winter wheat. We would also like to thank G Doussinault and

H

_Goyeau

for their

_help

and

advice,

and C

Young

for

_translating

the article into

_English.

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_autogame

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par croisements et

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_population

artificielle à 16

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Further

_analysis

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_{complex allozyme}

ANNEX I

Virulence _spectra of the 9 test isolates of

_{Erysiphe graminis}

f sp tritici for 12 genes.

The _spectra of the isolates were determined from numerous tests consisted of individual

infection of differential hosts. The tests used _segments of their

_primary

leaf.

To reveal the _presence of the _{12 resistance genes,} we _may have 2 isolates with the

following

spectra for each pair of genes: