Characterization and pathogenicity on seedlings of Pythium species isolated from soybean roots in the Toulouse area

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Characterization and pathogenicity on seedlings of

Pythium species isolated from soybean roots in the

Toulouse area

P. Davet, G.A. Forbes

To cite this version:

P. Davet, G.A. Forbes. Characterization and pathogenicity on seedlings of Pythium species isolated

from soybean roots in the Toulouse area. Agronomie, EDP Sciences, 1990, 10 (10), pp.825-830.

�hal-02713395�

Plant

_pathology

Characterization

and

_{pathogenicity}

on

seedlings

of

_Pythium

_species

isolated from

_soybean

roots

in

the Toulouse

area

GA Forbes

P

Davet

INRA, Laboratoire de _{Pathologie Végétale} de l’ENSA, 34060 _Montpellier, France

(Received

23

_July

1990;

accepted

9 October 1990)

Summary —

Isolation

_frequencies

and in vitro

_{pathogenicity}

are

_given

for several

species

of

_Pythium

isolated from

roots of

_{soybean plants growing}

near Toulouse

_{(SW France). Approximately}

half of all isolates were

_pathogenic

on

soybean seedlings (cv Kingsoy)

at _{20 °C. Some differences among isolates} could be

detected,

but the

_variability

among

species

was

_greater

in the isolates studied. The most

_{pathogenic species}

was P ultimum which reduced root

elongation

to

approximately

6% _{of the control but made up}

_only

10% of the total isolates in 2 _{years of}

_sampling,

slight-ly varying

from one _year to the next. P

_sylvaticum

and P

_irregulare

were isolated more

_{frequently (26}

and

13%,

re-spectively)

and were

_pathogenic

on

_{soybean, reducing}

root

_{elongation by}

30 to 50% of the control in 2 tests. The most

commonly

isolated group

(38%)

did not _{form oospores and} was classified as HS

_{(hyphal swelling).}

_{This group}

ap-peared

to have little effect on

_soybean

root

_growth

and caused no visible

_symptoms.

P ultimum was

_only

isolated from young

plants;

P

_irregulare

was isolated

_{primarily during}

the first two months of

_growth;

and P

_sylvaticum

and the HS group were isolated in a

_fairly

uniform manner

_throughout

the first 3 months of the season.

Pythium

ultimum /

_Pythium

_sylvaticum

/

_{Pythium irregulare}

/

_soybean

/

_Glycine

max

Résumé — Caractérisation et

_pouvoir

_pathogène

sur

_plantules

de

_Pythium

_{spp isolés de racines de}

_soja

dans la

_région

de Toulouse. Plusieurs

_espèces

de

_Pythium

ont été isolées de racines de

_soja

en 1986 et 1987 dans la

ré-gion

de

Toulouse,

à 3 stades de

_{développement}

des

_plantes.

Environ la moitié de l’ensemble des isolats se _révèle

pa-thogène

sur des

_germinations

de la variété

_Kingsoy

maintenues à 20 °C

_{(tableau I).}

La variabilité du

_pouvoir

patho-gène

à l’intérieur d’une même

_espèce

de

_Pythium

est

faible,

alors

_qu’il

existe des différences

_importantes

entre les

espèces

dans les échantillons observés

_{(tableau II).}

L’espèce

la

_{plus agressive}

est P ultimum

_qui

réduit

_{l’allongement}

des racines à

_près

de 6% de celui des témoins. Elle ne

_{représente cependant}

que 10% du total des isolements des 2

années,

avec une

_légère

variation d’une année à l’autre

_(fig

_1).

La

_fréquence

d’isolement de P

sylvaticum

et de P

irre-gulare est plus

élevée

(26

et 13%,

respectivement,

du total des

_isolements).

Ces 2

espèces

ont un

_pouvoir

_pathogène

important,

réduisant

_{l’allongement}

des racines à 30 à 50% de celui des témoins. Le _groupe le

_plus

_fréquent

_(38%

des

isolements),

dénommé HS

_{(hyphal swelling)}

ne _{forme pas}

_{d’oospores.}

II n’a _{pas d’effet}

_apparent

sur le

développe-ment des racines. P ultimum est isolé seulement sur les

_{plantes jeunes,}

P

_irregulare

est isolé au cours des

_{2 premiers}

mois;

on trouve P

_sylvaticum

et _{le groupe HS} au moins

_pendant

les 3

_premiers

mois de culture

_{(fig 2).}

Pythium

ultimum

_{/Pythium sylvaticum}

_{/Pythium irregulare}

_{/soja/Glycine}

max

*

Present address: International Potato Center

(CIP),

PO Box 5969, Lima, Peru.

**

INTRODUCTION

Several

_species

of

_Pythium

have been

estab-lished as

_pathogens

of

_soybean

seeds and

seed-lings,

and have been

shown

to cause

_major

damage periodically (Anonymous, 1975).

The

species

considered to be most

_important

are

P

_{aphanidermatum,}

P

_irregulare,

P ultimum and P

_myryotylum.

P

_debaryanum

has also been

fre-quently

associated,

but the

_legitimacy

of

this

species

has been

_questioned:

the

correct

de-nomination

_probably

_ought

to

have been P

syl-vaticum in most cases

_(Van

der

_{Plaats-Niterink,}

1981 ).

In

a

related

_study

_(Forbes

and

Davet,

1990)

we found that

_Pythium

_spp were isolated in the

growing

season

from

_{approximately}

20%

of

root

pieces sampled

in

the

_region

of SW France

near

the

_city

of Toulouse. An

_{earlier survey indicated}

that roots from

_{plants growing}

in this

_region

were

often more discoloured and/or had a

_greater

number of lesions than in other

parts

of France

(Davet

and

_Forbes,

_1988).

Several

_key

ques-tions arose:

_1),

what

_species

of

_Pythium

are

present

in the

region; 2),

do relative abundances

of

_{species change through}

the

season; and

3),

what

_proportion

of

isolates are

_pathogenic

on

soybean.

The

_{following study}

was

_designed

to

address these

_questions.

In

this

work,

pathoge-nicity

tests were limited to

_seedlings.

MATERIALS AND METHODS

Pythium

isolates used in this

_study

were collected

dur-ing

the 1986 and 1987

_growing

seasons. In

1986,

iso-lations were made from

_{randomly sampled plants}

(cul-tivars

_Kingsoy

and

_{Kador) originating}

from 12 fields in

an area of

_{approximately}

400 sq km delimited

_by

the

towns of

Toulouse,

Buzet sur

Tarn,

Lavaur,

and

Cara-man. This

_region

is

_{representative}

of one of the most

important

areas of

_{soybean production}

in France.

Twenty-four

isolates were

_kept

for characterization and 20 of these were

_compared

for

_{pathogenicity.}

In 1987 and as

_part

of a

_{larger study,}

10 fields

_(cv

Kador)

were

_{periodically sampled}

in the same

_region.

Sampling

and isolation

_procedures

have been de-scribed

_(Forbes

and

Davet,

1990).

Cultures of

Pythi-um were

_kept

from 3 of the

_sampling

dates: 28, 49

and 85 d after

_planting,

chosen to

_represent

the

_early,

middle and late

_growing

season. _{The isolation}

proce-dures described

by

Forbes and Davet

₍₁₉₉₀₎

were

also used for isolates taken in 1986.

Pythium

spp were maintained on

_potato

carrot _agar

(PCA)

(Van

der

_{Plaats-Niterink,}

₁₉₈₁₎

or

_{half-strength}

commercial corn _{meal agar}

_(CMA).

_Species

were

identified

_according

to the

_key

of Van der

Plaats-Niterink (1981).

Isolates were tested in the

_laboratory

for

pathoge-nicity

in a _{paper blotter}

_apparatus

as described

by

Sin-gleton

and Ziv

_(1981).

_{Surface-disinfected soybean}

seeds

_{(cv Kingsoy)}

were

_{pre-germinated}

at 24 °C for 48 h. Two

_seedlings,

selected for lack of contamina-tion and

_uniformity

of root

_growth,

were

_placed

in a

blotter

₍₁₀

cm wide

_by

15 cm

_long)

which was

sus-pended

in a

_plastic

_tray

such that the bottom of the

blotter was

_continuously

in water.

_Lighting

was

provid-ed

_by

fluorescent tubes at a distance of about 30 cm

from the

_top

of the

blotter,

adjusted

to a 14-h

photope-riod. After 24 h in the blotters at

_{20 °C,}

the

_seedlings

were inoculated

_{by placing}

a 0.5-cm agar disk

(CMA)

of a 1-wk culture

_adjacent

to the root

_tip.

At the time of

inoculation root

_length

varied between 3 and 5 cm.

Blotters,

each

_constituting

one

_experimental

_unit,

were

_{randomly arranged}

in the

_trays,

15 _{blotters per}

tray.

It was assumed that there was no effect of

_tray,

and data were

_{analyzed according}

to a

_completely

randomized

_design.

Twenty

of the 1986 isolates were

_compared

in an

experiment

with 3

_{replications.}

The effects of

inocula-tion were measured on

_{hypocotyl length}

and root

elon-gation beyond

the

_point

of inoculation. To aid

interpre-tation,

root

_elongation

was converted to a

_percentage

loss of the control in the

_following

manner: Y

_{= (A/B)}

x

100;

where Y = the

_{percentage value, A}

is the

meas-ured

_length

of an inoculated root, and B _{is the average}

length

of the

_replicated,

noninoculated roots.

In a second

_experiment,

53 isolates from 1987 were

tested with 2

_{replications.}

Root

_elongation

and a measure of

_severity

were used to assess isolates in

this second

_experiment.

The measure of

_severity

was

a scale based on the

_percentage

of the root

_system

af-fected where 0 = _no

lesion,

1 _{= a} small lesion

covering

up to 10% of the root

_system,

2 = lesion

spreading

to

50% of the root

_system,

and 3 = _more than 50% of the

root

system

affected.

RESULTS

Isolation

_{frequencies (IF’s)}

The isolates from 1986

and 1987

_primarily

com-prised

3

_species

and the HS

_{group which formed}

no _{oospores. This group} was

the

most common

type

isolated

making

up

approximately

38% of the 78

isolates characterized

_{(table I).}

P

_sylvaticum

followed

_{the HS group}

represent-ing

25.6% of the isolates. P

_irregulare

and P

ultimum followed with 12.8 and 10.3%

respec-tively.

These

3

_{species plus}

_{HS made up}

87%

of

all isolates. Three P

echinulatum and

one

each

of P

_{aphanidermatum}

and

P

violae

were

also

iso-lated. Five isolates

were not

identified

to

_species.

The

_general

_spectrum

of

_species

was the

types

considered

_together)

and HS

_having

the

greatest

IF’s

_{(fig 1).}

P ultimum and P

_irregulare

were

_{predominantly}

isolated

in

the

_early

_part

of

the

season

_{(fig 2).}

In

_contrast,

isolation date had

little effect

on P

_sylvaticum

and HS.

Species

characterization

Among

the isolates classified

as

HS there

were 2

types

that differed for several

characteristics.

Iso-lates of the dominant

_type

₍₂₄

of 30

_isolates)

on

PCA

were

_submerged

with a _very

distinct

"carna-tion"

_(Van

der

_{Plaats-Niterink,}

₁₉₈₁₎

_pattern.

Hy-phal

width reached

_{approximately}

5-7 μm and

colony

growth

was 14-15 mm _per

_day

at 24°C.

Hyphal swellings

were

abundant and

_averaged

20 μm in

diameter. There

was no aerial

_growth

on

CMA. Isolates of the second HS

type were

not

_homogeneous

for aerial

_growth

on

CMA

or

growth

pattern

on

_PCA,

but were

all

_{(6 isolates)}

characterized

_by

a

slow

_growth

rate

_(5-7

mm

per

day

at 24

_°C).

On

PCA,

P

_{irregulare appeared generally}

as

described

_by

Van der Plaats-Niterink

_(1981).

One

_exception,

which has been

reported

in

an-other

_{study (Chamswarng}

and

Cook,

1985),

was

hy-phal swellings

and

only

rarely produced large

globose hyphal swellings. Young

antheridia were

often

_highly

falcate

in water

culture,

but

ap-peared

to

swell with

maturation,

suggesting

that

this characteristic is less useful in

_diagnosis

than

has been

_{proposed (Vaartaja, 1967).}

P ultimum isolates did not

_{produce sporangia}

or zoospores and

therefore

belong

to the

_variety

ultimum. Colonies

produced

abundant

cottony

aerial

_growth

on

CMA but

no

discernible

_growth

pattern

on

PCA

or CMA.

Cultures of

P

_sylvaticum

were similar to P

ulti-mum

on

CMA and PCA. All

isolates

_except

one

produced

oospores when crossed with known

mating

types.

In

_1986,

isolates

were

_primarily

of the male

_mating

_type,

but in 1987 both

_mating

types

were

isolated

with

similar

_{frequency (fig 1).}

Pathogenicity

In the first

_{experiment involving}

the 1986

isolates

a

_significant

difference could

be established

among isolates for root

_{elongation (table II).}

Based

on

the level of

_significance

and R-

_square

values,

_{hypocotyl length}

did

not

_appear

to be a

good

indicator of differences among

fungal

iso-lates: the

_problems

associated with the measure

of

_{hypocotyl length}

in our

_{pathogenicity}

tests are

probably

due to

the

nature

of the

_apparatus.

The

uniform

_humidity

of the blotter

_supplies

sufficient moisture to the functional

part

of the root above the

_point

of inoculation.

Overall,

the

_analysis

of variance showed that

the

_majority

of

_variability

occurred

_among

spe-cies

_{rather than among isolates of}

a

_given

spe-cies. For

this reason we have

_reported

levels of

root

_elongation

and

_severity

for

_species

means

(table I).

P

_ultimum,

P

_sylvaticum

and P

irregu-lare

_{significantly}

reduced

root

_elongation

and caused severe root

necrosis.

Reduction in root

elongation

was

_generally

consistent

for both

tests

and

P

ultimum

had a

_greater

effect than the other 2

_{pathogenic species.}

The

_only

_other spe-cies which was

_{clearly pathogenic}

in this

_study

was P

_violae,

but it

was

_only

_represented

_by

one

isolate. The other

_species

and

_{HS group}

ap-peared

to have little or no effect on root

_growth.

DISCUSSION

In a

related

_study,

we

found that

_{approximately}

20% of all

root

_pieces

_sampled

in

the

_study

re-gion

were

infected

with

_Pythium

_spp

in a

_year

that was not

favourable

for

_seedling

disease

(Forbes

and

Davet,

1990).

IF’s of

_Pythium

_spp

decreased with

time,

and were more uniform in 10 fields

_sampled

than

were

IF’s for

most of the other

_{fungal species}

studied. Since about half of

the

_Pythium

isolates we

characterized

were

path-ogenic,

we

estimate that

_{approximately}

10%

of

all root

_{pieces sampled}

on 3 different

sampling

dates contained

_pathogenic

isolates.

This

esti-mate can be seen as

_{quantitative though}

indirect,

evidence for the

common

_assumption

that

Py-thium spp are

_{ubiquitous organisms}

that

cause

periodic damage

when

environmental conditions

are favourable

_(Hendrix

and

Campbell,

1973).

In both 1986 and 1987 we noticed a

_high

de-gree of

homogenicity

for

_{pathogenicity}

within

species.

Some differences among isolates could be

_detected,

but the

_variability

_among

_species

was much

_greater

_{(table II).}

This

_homogenicity

could result from the fact that all the

isolates

come

from

_only

1 or 2

cultivars of the

same host

and a limited

_{geographical region.}

If

_comparative

studies from other

_{geographical regions}

were to

support

this

_finding,

it would mean

that

simple

taxonomic determination of

_Pythium

_spp

on

soy-bean

would furnish

_important

information

con-cerning

the

_{pathogenicity}

of the

_Pythium

popula-tion

_{being sampled.}

The most

_{pathogenic species (under}

the

con-ditions of

our

_test)

was P

ultimum,

which was iso-lated at a

_relatively

low

_{frequency (10.3%).}

All P

ultimum

_isolates,

however,

were obtained

dur-ing

the

_early

_part

of the

season,

when

plants

are more

_susceptible.

The

greater

importance

of P ultimum in 1986

_probably

reflects the

wet

and

cool conditions which

_prevailed

after

_planting.

In the last 10

_days

of

_April,

24 mm

of rain fell and

soil

_temperature

at a

_depth

of 10 cm was

less

than 15°C.

In

the

same

_{post-planting period}

in

1987,

only

0.9 mm

of rain fell and soil

tempera-ture was

_consistently

above

15°C. If conditions

in

1987 had been conducive to this

_species,

which

is more

_pathogenic

in

cool,

wet soils

_(Lumsden

et al,

1976),

IF’s would

_probably

have been

high-er.

Nonetheless,

there is reason to

_suspect

that

the

_importance

of P ultimum has been

exagger-ated in the

_past.

It is one of the most common

soilborne

_Pythium

_spp

_(Van

der

_{Plaats-Niterink,}

1981)

and

recent

soil isolation studies do show

that

_high

levels of P

ultimum

occur in northern

European

soils

_(Messiaen

et

_al,

1977;

Bottcher

and

Behr,

1985).

However,

it may

not be valid to

based

on inoculum

_density

alone. In another

quantitative study

of root infection of a

legumi-nous

_plant,

P

ultimum

was

_only

isolated from 1 % of root

_segments

that

had been

_{plated (Hall,}

1983).

Conditions

at

the time

of

_sampling

the root

sys-tem

_appear

to

_greatly

influence the

_probability

of

finding

P ultimum. Our studies

indicate,

and

oth-ers

_(Schlub

and

Lockwood,

1981) directly

dem-onstrate

that P

ultimum

infection

_only

occurs in a narrow

_range

of soil

_temperatures

and

soil

mois-ture

levels. This

does not

_negate

the

_potential

for

severe attack

_by

P ultimum when these condi-tions exist. In

_fact,

_they

are

_frequently

met

with

at

emergence time.

Other

_{pathogenic species}

of

_Pythium

_appear

to be more common under a _{wider range of}

envi-ronmental conditions. P

_sylvaticum

and P

irregu-lare

_represented

_{approximately}

40.0%

of

our

iso-lates and

were

isolated

on

all 3

dates.

The most

_frequently

isolated

_pathogenic

spe-cies,

P

_sylvaticum,

was not mentioned in at least

one

review of

_soybean

diseases

_(Anonymous,

1975).

The lack of

_{importance given}

to P

sylvati-cum in earlier studies may be

due

to

the

_difficulty

of

_identifying

this

_species

without known

_mating

types.

Without

_mating

_types,

P

_sylvaticum

could

easily

be

_placed

in the HS group,

resulting

in an

unfortunate taxonomic mixture of

_pathogenic

and

_{nonpathogenic species.}

We found that iso-lates of the

_{HS group}

did not cause the

_typical

symptoms

of root

_{decay usually}

associated with

Pythium

spp and had little or no effect on root

growth.

Furthermore,

the

_greatest

number of HS

isolates

were

obtained from the last

_sampling

date

_{(fig 2),}

_{possibly indicating saprophytic}

activ-ity.

Overall,

these

_findings

confirm the

_high

inci-dence of

_{pathogenic Pythium}

_{spp in the} Tou-louse area. _{A further field survey in another}

_part

of SW France

_(Landes

and Adour

_valley)

re-vealed that the

same

_species

_occurred,

with

a

rather similar distribution. Field observations and

greenhouse

tests

_{(unpublished results)}

showed

that

_{pathogenic Pythium}

_{spp could}

cause

appre-ciable

_damping-off

under favourable conditions.

On

the other

hand,

we

lack

_precise

data

con-cerning

the

_practical

effects of

_pathogenic

Pythi-um

_spp

on

_{developed plants.}

Our results indicate the need for

some

investi-gation

into

_possible

varietal differences for

resis-tance.

_Very

few data

are

available

on

this

sub-ject.

Seed

treatment with

_{appropriate fungicides}

could lead

to

shorter-term results.

ACKNOWLEDGMENTS

Major

support

for the senior author was

_{given by}

the Chateaubriand

grant

for

_{post-graduate}

studies.

Signifi-cant

_financial

and

_physical

_support

was also

_provided

by

CETIOM

_{(Centre Technique Interprofessionnel}

des

Oléagineux Métropolitains).

P

_{sylvaticum mating}

_types

were

_{provided by}

J

_Mugnier,

Rhône Poulenc

Agrochi-mie, Lyon,

France.

REFERENCES

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Diseas-es

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JB;

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Am

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MN 55121

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LL,

Ziv O

₍₁₉₈₁₎

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21