Enhancement of yield and persistence of perennial ryegrass inoculated with one endophyte isolate in France

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Enhancement of yield and persistence of perennial

ryegrass inoculated with one endophyte isolate in France

C. Ravel, François Balfourier, J.J. Guillaumin

To cite this version:

C. Ravel, François Balfourier, J.J. Guillaumin. Enhancement of yield and persistence of perennial

ryegrass inoculated with one endophyte isolate in France. Agronomie, EDP Sciences, 1999, 19 (7),

pp.635-644. �hal-02698485�

Original

article

Enhancement

of

_yield

and

_persistence

of

_perennial

_ryegrass

inoculated

with

one

endophyte

isolate in France

Catherine Ravel

a

_François

Balfourier

a

Jean

_Jacques

Guillaumin

b

a

Unité d’amélioration des

_plantes,

Inra, 234 avenue du Brézet, 63039 Clermont-Ferrand cédex 02, France

b_{Unité de}

pathologie végétale

et

_mycologie,

Inra, 234 avenue du Brézet, 63039 Clermont-Ferrand cédex 02, France

(Received 13 March 1999;

accepted

6

_July

1999)

Abstract - The contribution of

_Neotyphodium

_endophytes

to the

_performance

of

_perennial

_{ryegrass in}

_Europe

is

_poorly

documented. The

_objective

of this

_study

was to _compare the

_yield

and

_persistence

of three

_{endophyte-infected}

_(E.I.)

and

endophyte-free

(E.F.)

perennial

ryegrass cultivars. The E.I. versions were obtained

_by

artificial inoculation of each

cul-tivar with one isolate of

_Neotyphodium

_belonging

to the second taxonomic _group

_(LpTG-2).

The field

_experiment

was

carried out for a

_{3-year period}

at five different locations in

France,

three of which could be considered

_climatically

stressful for

_perennial

_ryegrass. At all

locations,

the

_endophyte

enhanced the

_productivity

of each cultivar in the second

year after

sowing;

moreover, it

generally

improved

their

_persistence

in stressful environments. These results show that

endophyte

infection can increase the

_ecological

fitness of

_perennial

_ryegrass.

_Thus,

_plant

breeders can use

_endophytes

to

improve

_{perennial ryegrass, provided}

that the strains involved

_produce

no or low levels of toxins

_dangerous

for

mam-mals.

_(©

1999

Inra/Éditions

_{scientifiques}

et médicales Elsevier

_SAS.)

agronomic performance

/ environmental stress / Lolium perenne /

Neotyphodium

endophyte

/ trial

_plots

Résumé - Rendement et

_persistance

_{du ray-grass}

_anglais

inoculé avec un isolat

_{d’endophyte}

en France.

_L’impact

des

_endophytes

_{du genre}

_Neotyphodium

sur les

_{performances agronomiques}

_{du ray-grass}

_anglais

en

_Europe

est encore

mal connu. Une étude en

_plein

_champ

a été réalisée sur une

_période

de trois ans en

_cinq

lieux

_(dont

trois

_pouvant

être

considérés comme

_{climatiquement}

stressants _{pour le}

_ray-grass)

afin de _comparer trois

variétés,

chacune existant sous

deux formes:

_porteuse

_{d’endophyte}

_(E.I.)

et indemne

_(E.F.).

Les versions E.I. ont été _{réalisées par inoculation}

artifi-cielle des variétés avec une même souche de

_Neotyphodium

_appartenant

au _{second groupe}

_taxonomique

des

e-endo-phytes

du _ray

_{grass-anglais (LpTG-2).}

Les résultats montrent que la

présence

de 1’

_endophyte

_permet

_{d’augmenter}

le rendement en matière sèche de la deuxième année

_{d’exploitation}

de toutes les variétés et dans tous les lieux. La

péren-Communicated

_by

Max Rives

_{(Villeneuve-lès-Avignon,}

France)

*

Correspondence

and

_reprints

nité des variétés est

_{généralement}

_{améliorée par}

_{l’endophyte}

dans les environnements les

_plus

stressants. Il semble donc

possible

d’utiliser la

_symbiose

_{pour améliorer le ray-grass}

_anglais

à condition toutefois de

_disposer

de souches

hypotox-iques

pour les mammifères.

(©

1999

Inra/Éditions scientifiques

et médicales Elsevier

_SAS.)

performances

agronomiques

/ Lolium _perenne /

_Neotyphodium

/ essai en

_{plein champ}

/ stress environnemental

1.

Introduction

Perennial ryegrass is one of the _{many grass}

species

that can harbour

symptomless

endophytic

fungi belonging

to the

_{Neotyphodium species}

(for-merly

Acremouium

_).

Based on

_{morphology, ability}

to

_produce

_{spores in vitro and}

_isozyme

_patterns,

two _{distinct groups of}

_Neotyphodium

can infect

this host

_[8]:

the _{first taxonomic goup}

_(Lp-TG1)

correspond

to N.

lolii,

whereas the second taxo-nomic group

(Lp-TG2)

has not been

_given

a Latin name.

Such associations

_involving

_{grasses and}

Neotyphodium

endophytes

are

mutualistic

[9,

39].

The

_fungus

_grows

_{intercellularly}

in the shoot of its host

_excluding

leaf blades and

_{totally depends}

on

the

_plant

for

nutrition

and dissemination

_(seed

transmission).

From a

_general

_point

of

view,

Neotyphodium endophytes

are

_reported

to enhance the

_pest

and stress tolerance of its host as well as

its

_growth

_{[9, 17, 42].}

The

_protective

anti-herbivore effects of the

endophytes

are due to

_specific

metabolites

_[9]

such

as lolitrems and

ergopeptines acting particularly

against

mammals,

and

_peramine

and lolines

_acting

against

some insects.

_However,

the

production

of

mycotoxins

greatly depends

on the

_endophyte

genotype

[40].

Under controlled

conditions,

the tolerance of

symbiotic

plants

of tall fescue or

_perennial

_ryegrass

to abiotic stress has

_mostly

been studied in cases of

drought

and

_{nitrogen deficiency}

_[2,

43].

In tall

_{fescue/Neotyphodium endophyte}

associa-tions,

several mechanisms

_explain

the

_improved

drought

tolerance of

_{symbiotic plants}

such as their

enhanced root

_growth

_[5],

faster

_leaf-rolling

_[1],

enhanced osmotic

_adjustment

and the maintenance of

_turgor

levels under water stress

_[14].

The nitro-gen

deficiency

tolerance

of

symbiotic

tall

fescue

seems to be

_{improved by}

an increased

_activity

of

glutamine synthetase regardless

of

_nitrogen

level or

form

_favouring

the utilisation

of

_nitrogen

[27].

In

_perennial

_{ryegrass/Neotyphodium endophyte}

associations,

Ravel et al.

_[32]

showed that

endo-phyte

infection could also

_improve

the

_drought

or

nitrogen deficiency

tolerance of

_perennial

_ryegrass clones.

However,

according

to Lewis et al.

_[25],

endophyte

infection had few effects on Lolium

perenne

dry

matter

production

even in the case of

nitrogen deficiency.

These authors

_explained

their

negative

result

_by

a

_probably

too low N stress due

to the conditions of the

_{experiment performed}

with clones

_growing

in

_flowing

nutrient solution.

Under controlled

environments,

several studies have shown that

_endophyte

_{presence tends} to

increase

_growth

characteristics of tall fescue

_[1,

11],

perennial

ryegrass

[21 ] and

meadow fescue

[37].

However,

the effects of

_endophyte

on host

plant growth

are not consistent across host

geno-types.

It can also

_depend

on the age of the

_plants

and

_experimental

conditions such as the nutrient level

_[7].

In the absence of stress, the

higher

growth

of

_{symbiotic plants}

could be

_{explained by}

an increased

_efficiency

of

carbohydrate

utilisation

[18]

and

_by

a

_{phytohormone-mediated}

_response

[43].

For

_example,

auxin is a

_key

hormone in

regu-lating plant growth.

As indol acetic acid

_(I.A.A.)

production

was shown for N.

_coenophialum

_{in pure} culture

_[10],

the

_endophyte

_might

be involved in auxin metabolism

_leading

to an enhanced

_growth

of

_{symbiotic plants.}

Under field

conditions,

it is

_expected

that

endo-phyte-infected

(E.I.)

cultivars would show better

performance

than

_{endophyte-free}

_(E.F.)

cultivars.

However,

field

_comparisons

between E.I. and E.F.

cultivars gave

contrasting

results. For tall

_fescue,

Siegel

et al.

_[38]

observed no difference in

sur-vival,

herbage

and seed

_yield

between E.I. and E.F.

condi-tions,

whereas Read and

_Camp

_[33]

and Bouton et

al.

_[6]

_reported

that N.

_coenophialum

enhanced

growth

and

_persistence

in the same

_species

under

climatically

stressful conditions.

Similar

results

were

_reported

for

_perennial

_ryegrass. In a

cool,

moist

location

in New

_Zealand,

_Neotyphodium

endophyte

had no effect on

_perennial

_ryegrass

_[12].

In France

[31] and

Spain

[30],

the

effects

of

endo-phyte

on

the

_agronomic

traits

of

this

_species

depend

on environmental conditions: the drier

_they

are, the more

_important

are the beneficial effects of the

_endophytes.

However,

_frequent

interactions

between

endo-phyte

infection and

host

_plant

_genotypes,

_leading

to

_contrasting

_results,

are detected

_{[4- 6,}

_{11, 13,}

18, 23, 24,

34].

It seems that the beneficial effects

of

_endophytes

are the result of

_specific

interactions

between

_plant

_genotypes,

_endophyte

isolates and environmental conditions.

_Therefore,

_{extrapolating}

results obtained from clonal material under

con-trolled conditions to

_{heterogeneous}

_populations

in the field is difficult and field trials are

_required.

Moreover,

up to now, such interactions could be increased

_by

the

_great

_variability

of the material

tested;

in most

_experiments,

the

E.I./E.F.

_pairs

con-sist of a

_{genotype (or}

_cultivar)

with its native

endo-phyte

and the same

_genotype

_{(or cultivar)}

_deprived

of its

_{endophyte by}

selection or

_physical

treatment. Such material can make it

difficult

to

_separate

the

effects

of the

_genotypes

of the

_plant

and the fun-gus.

Although

the French

_perennial

_ryegrass

germplasms, especially

in hot and

_dry

_areas, were

found to be

_highly

infected

[26],

up to now, little is known about the

effects

of

_endophyte

infection upon

agronomic performances

of this host in French conditions.

_Moreover,

studies

_reporting

the effects

of

one

particular endophyte

strain in several

cultivars or of several

_endophyte

strains in

_only

one

_plant

_genotype

or cultivar are rare

_[22].

Therefore,

this

_study

was undertaken to

_investigate

the effects of

_only

one

_endophyte

isolate on the

growth

and

_persistence

of three

_perennial

_ryegrass cultivars at several French

locations,

whose climat-ic and soil conditions differed. This is the first time that the effects of one

_single

_endophyte

isolate were studied on several cultivars.

2. Materials and methods

2.1.

Materials

Three cultivars of

_perennial

_ryegrass were used in

this

_{study: Vigor,}

Parcour and

_Pacage.

One-hundred seeds of each cultivars were

_{microscopically}

examined

for

_endophyte

infection. As no

_endophyte

was

_found,

these cultivar were considered to be E.F.

_However,

such

a control did not allow the detection of a _very low infec-tion rate.

Thus,

to ensure that all the seeds to be used in

the

_experiment

were

_previously

_E.F.,

_they

were also

subjected

to a moderate heat treatment

_{[39, 35],}

which

kills the

_endophyte

without

_{affecting seedling vigour.}

2.1.1. E.I.

material

A total of 500 heat-treated seeds of each cultivar

were used to

_produce

axenic

_plantlets

which were

inoc-ulated with one strain of

_endophyte.

The

_endophyte

strain used in this

_experiment

was isolated from a

French wild

_population

of

_perennial

_{ryegrass. Based} on

morphology

and

_isozyme

studies,

it

_belonged

to

LpTG-2

_[8].

The inoculation was realised under sterile

condi-tions

_using

a method

_adapted

from that

reported by

Latch and Christensen

_[20]:

_plants

were inoculated

_by

placing

a

_drop

of sterile water with

_ground

_mycelium

in

a slit at the

_junction

of the

_mesocotyl

and the

_coleoptile

[29].

Two-month-old inoculated

_plants

were examined for

endophytes

and re-isolation was carried out from all the

plants

considered to be E.I. All of them contained the

LpTG-2 endophyte.

For each

cultivar,

about 50 E.I.

plants

were obtained. These

_plants

were then used in

three different

_polycrosses

(one

per

cultivar)

to

_produce

E.I. seeds. In these

_polycrosses,

E.F.

_plants

were

_planted

with the E.I.

_plants.

These E.F.

_plants

were not harvest-ed but

_only

used as

_paternal

_parents

to increase effective

population

size and thus avoid

_genetic

drift and to main-tain the allelic

_diversity

of each cultivar. As

_LpTG-2

was 100 % transmitted to the seeds of

_perennial

rye-grass

[29],

all the seeds collected from each of 50 E.I. mother

_plants

were E.I.

2.1.2. E.F. material

E.F. seeds consisted of heat-treated commercial

seed-lots.

_{Consequently,}

for each

cultivar,

the

_only

difference between E.F. and E.I. seedlots was the absence or pres-ence of a

_single

_endophyte

strain.

2.2. Trial

_{plot experiment (figure}

₁₎

Each

cultivar,

with and without

_endophyte,

was sown

at five locations in France:

in

_September

_1994,

at Les Alleuds

_(47°28’N,

0.33W),

Orchies

_{(50°28’N, 3°15’E)}

and Rodez

(44°21’N,

2°34’E);

in

_April

1995,

at Clermont-Ferrand

_(45°47’N,

3°05’E)

and

_{Verneuil l’Etang}

_{(48°32’N, 2°40’E).}

The environmental conditions at each location are

summarised in

_figure

1 . This

_figure

_gives

the climatic data and the index of Turc

[41]

for each location. This

index,

based on several data

_{characterising}

environmen-tal conditions

_(climatic

and

_edaphic

_data),

allows us to

estimate the

_potential

_forage

_production

at _every

loca-tion. Such an index also

_depends

on soil characteristics.

It was calculated

_{by taking}

into account the water reserve of different soils

_[28].

Each

_genotype

with and without

_endophyte

was sown

in

5-m

2

_plots,

at a rate _{of 2.5 g}

_seed·m

_-2

_,

in a

ran-domised block

_design

with five

_replicates.

Fertilisation

was

_applied

to the

_plots

at each location

_according

to

current

_{farming practices. Herbage yield}

was assessed at

4-week intervals

_using

a

_plot

harvester to cut and

_weigh

herbage

from each

_plot.

A

_sub-sample

of 1

_kg

of fresh

herbage

per

plot

was dried at 90 °C for 40 h and

reweighed

to determine the

_dry

matter content. Such data was accumulated to obtain three seasonal

_(spring,

summer and

_autumn)

_yields

and annual

_yields

for the first and _{second years}

_{following sowing.}

As summer

and autumn

_yields

were

_generally

low,

_they

were also

cumulated.

Moreover,

a visual estimate of the stand was

made on each

_plot

in the third _year

_following

_sowing.

This trait was scored on a scale from 1

_(ground

area

covered with

_perennial

ryegrass inferior to 10

_%;

_poor

persistence

of the

_plants

in the

_plot)

to 9

_(percentage

of

ground

area covered with

_perennial

_ryegrass

_superior

to

To check whether the level of

_endophyte

of each association fulfilled

_expectations

_(about

0 and 100 % in E.F. and E.I.

cultivars,

respectively),

at least 50 tillers

per cultivar

growing

in the field were examined for the

endophyte using

the method described

_by

Latch and Christensen

_[20]

in the first and third _years

_following

sowing

at each location _except at Les

Alleuds,

where

this control was carried out

_only

in the _{first year}

follow-ing sowfollow-ing.

No

_discrepancy

between

_expected

and observed results was noted. The levels of infection observed

_remaining

contrasted: 0 % for E.F. cultivars

and

_superior

to 90 % for E.I. cultivars.

2.3.

Statistical

_analysis

Data were

_subjected

to an

_analysis

of variance with

three main effects

_(location,

cultivar and

_endophyte

sta-tus)

and all the

_{interactions;}

the block effect was also

introduced in the model as

_being

nested to the location. Such an

_analysis

was

_{performed by}

the GLM

_procedure

of SAS [36].

In the absence of

_interaction,

as some data were

missing,

means for the main effect

_’endophyte

status’ were

_{compared by}

the method of Bonferroni.

3. Results and discussion

3.1.

_Endophyte

and

_yields

As no interaction was

significant

for

yield

(table

I),

we

_interpreted

the main effects. Location effects were

_{highly significant}

_(P

<

_0.001).

Cultivar effects were

_significant

for most traits

_(P

=

0.05)

with the

_exception

of the total

_yield

in the second year

following

sowing.

The

_endophyte

status

effect

was not

_significant

for

_yields

obtained in the first year

following sowing

(P

>

_0.05),

whereas

_they

were

_{generally highly significant}

_(P

=

0.001)

in the second year. The

comparison

of means

₍

table

II)

showed

_that,

in the case of

_significant

endo-phyte

status

effects,

E.I. cultivars

_outyielded

E.F. cultivars.

Endophyte

infection is

_reported

to

_{improve dry}

matter

_yield

and root

_growth

under controlled

con-ditions of tall fescue

_{[1] and}

_perennial

_ryegrass

[21].

Moreover,

although endophyte

effects on

agronomic

characters of

_field-grown

_plants

are less

con-trolled

_environment,

it is admitted that

Neotyphodium endophytes

of tall fescue and peren-nial ryegrass can

_improve

the

_yields

of their

_hosts,

especially

under

_{environmentally}

stressful field conditions

_[6,

30,

31]. Therefore,

from a

_general

point

of

_view,

our results confirmed that

_endophyte

presence

improves perennial

ryegrass

dry

matter

yield

in French conditions.

However,

in the

_present

_study,

E.F.

cultivars

out-performed

E.I. ones in the second year

_following

sowing,

whereas

_endophyte

did not

influence

the

productivity

of

_perennial

_{ryegrass in the first year}

following

sowing.

The absence of effect of

endo-phyte

infection on

_perennial

_ryegrass

_productivity

was

_{reported by}

Lewis

_[23],

Lewis and Clements

[24]

and Eerens et al.

_[12]

in cool and

moist

envi-ronments.

_According

to these

_authors,

insufficient-ly

stressful

conditions

could

_explain

their results. The lack of stress _{may also}

_explain

the results

obtained

_{the first year}

_following

_sowing

_{in the}

pre-sent

_study.

_Regardless

of the climatic

conditions

and the

_endophyte

_{status, the}

_drought

tolerance of

perennial

ryegrass decreases over time because its

roots become more

_superficial

_[15].

This

_implies

that,

in a similar

_environment,

older

_plants

can

undergo

more stress _{than younger}

_plants.

Therefore,

the

_productivity

_{of young}

_plots

is not

influenced

_by

_endophyte

infection because

_they

may not be

_subjected

to

_{sufficient stress,}

whereas

yield

may be

improved

by symbiosis

when

plants

become

older and more

_susceptible

to abiotic stresses.

3.2.

_Endophyte

and

_persistence

Table I shows that

_endophyte

status x location and

_endophyte

status x cultivar interactions were

significant

for

_persistence

_(P

< 0.01 and P &cong;

0.05,

respectively).

Therefore,

the effects of the

endo-phyte

on

persistence mainly depend

on

_plant

geno-type

and environmental

conditions.

As it was not

_possible

to

_interpret

the main

fac-tors,

the mean of

_persistence

_per

_genotype

_{and per}

location was

_plotted

in

_figure

2 .

This

_figure

illus-trates that the

_endophyte

did

not _{influence the} per-sistence of any cultivar at Orchies while

persis-tence was

_greatly

enhanced

_by

infection

at Les Alleuds. At the three other

_locations,

the effect of the

_endophyte

on

_persistence

_depended

on the

cul-tivar: the

_persistence

of

_Pacage

and Parcour was

enhanced

_{by endophyte,}

whereas that of

_Vigor

was

not influenced

_by

the

infection.

Perennial ryegrass is

susceptible

to

_drought

and hot

_temperature.

As Orchies is characterised

_by

the

lowest

maximal

_temperature

and

evapotranspira-tion

and the

_highest

Turc index of all the locations

(figure

1

_),

it can be assumed that the

environmen-tal conditions at this

location

are suitable

for

this

species.

The

four

other

locations

were

charac-terised

_by

more stressful climatic conditions

_(high

evapotranspiration)

which were modulated

_by

soil

characteristics

_(water

stress was increased at Rodez

by

rendzine

soils while

it was

decreased

at Clermont-Ferrand

_{by deep,}

black soils of

Limagne).

The low values of the Turc index

esti-mated for

these locations

confirm

that

_they

are

stressful

_(figure

_1).

This index was at its lowest at

Les Alleuds.

_Thus,

this location can be considered

to be the most stressful location of the network.

Therefore,

our results show that the

_Lp

TG-2

endophyte

could

_improve

the

_persistence

_of

peren-nial ryegrass when the survival of its host is

endan-gered

while it did not influence the

_persistence

of cultivars sown under more

_friendly

environmental

conditions. At locations characterised

_by

moderate-ly

stressful

conditions,

endophyte

could

_only

bene-fit certain cultivars while the

_persistence

of other cultivars was not influenced

_by

the infection.

Moreover,

it is worth

_noting

that the

_endophyte

never altered the

_persistence

of its host. Such a

result confirms the

_importance

of

_Neotyphodium

endophytes

in the survival of their host cultivated under stressful conditions

_[1,

_{6, 31,}

_44].

4. Conclusion

This

_study

shows the beneficial effects of one

strain of

_endophyte

on

_{field-performances}

of three

perennial

ryegrass cultivars. The commercial

seeds,

which were used for inoculation and

estab-lishment of E.F.

_plots,

were heat-treated to ensure

that seeds were

_{endophyte-free.}

This treatment was

gentle

and it was verified that it did alter neither seed

_germination

nor

_{seedling vigour.}

Such a

treat-ment was not

_applied

to E.I. seeds after

_harvesting

polycrosses

because,

in this _case, it was

_important

to conserve the

_endophyte

alive.

The

_population

size used to re-create the E.I. version of each

culti-var was considered to be sufficient to avoid

_genetic

drift and allowed the allelic

_diversity

to be main-tained

_{[3, 16, 19].}

It can thus be considered that the

E.I. seeds

_produced

were

genetically

_{very close} to

their initial cultivar

_although

not

_isogenic

sensu

stricto.

_Therefore,

it is

_possible

to conclude that the observed

differences

between E.I. and E.F.

ver-sions of a same cultivar were

_only

due to the

endo-phyte.

This

_study

_{shows that the presence of}

_endophyte

can

_improve

the

_productivity

and

_persistence

of

three

_perennial

_{ryegrass cultivars in French} envi-ronmental conditions. The increased

_dry

matter

yield

of E.I. cultivars may be linked to their enhanced

_persistence.

_However,

the

_yields

of E.I. cultivars in the second year

following sowing

were

increased

_by

the infection at all locations while their

_persistence

was

_improved

_only

in stressful

locations. This result may indicate a direct effect of

the

_endophyte

used on the

plant growth.

Such a

direct effect of

_endophytes

on the

_productivity

of

Nui

_perennial

_{ryegrass under field}

_conditions,

which were characterised

by

negligible

levels of stresses

_(insect

_predation

and

_drought),

has

_already

been

_hypothesised

_[22].

This

_study

confirms the beneficial effect of

endophyte productivity

of

_{field-grown perennial}

ryegrass even in French conditions. The beneficial

effect of the

_endophyte

on

persistence

is obvious

under stressful environmental conditions

_only.

Therefore,

the utilisation of

_endophyte

infection is

a means for

_plant

breeders to

_improve

the

adapta-tion of this

_species.

Some E.I. cultivars can be

developed

for turfs without risk to cattle.

However,

for

_forage

uses, the future of infected cultivars

depends

on the absence or the presence at low lev-els of anti-mammal

_mycotoxins.

In the

future,

such

a situation should

_change

the ways of

_improving

some _{grasses which harbour} an

_{Clavicipitaceous}

endophyte

because breeders would have to take into account the selection of both

_partners.

Acknowledgments:

The authors thank Dr Chosson

(R.A.G.T.),

Dr

_Bayle

_(Force

_Limagrain),

Dr Bourdon

(S.A.

Carneau

_Frères)

and Dr Baudouin

_(Verneuil

Recherches)

for

_providing

evaluation data and

_helpful

comments; and K.

_Lynch

for

_improving

the

_English

style

of this

_manuscript.

This work was

_{supported by}

a

grant from the French

_Ministry

of

_Agriculture.

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