Efficiencies of bacterial transmission from plants to seeds and … · 2018. 5. 14. ·...

Marie-Agnès Jacques

Efficiencies of bacterial transmission from plants to seeds and from seeds to

plantlets

Plant pathogenic MO

Pathogenic MO (E. coli STEC O104:H4)

PGPR

?

Seeds: vectors of a diversified microbiota

Strategic step to control diseases

Long-term inoculum survivalLong distance dissemination

Introduction to new area

Contaminated seed: source of primary inoculum

Critical step in plant pathogen ecology and

disease epidemiology

Seed transmission

Rely on a better understanding of mechanismsand pathways used for seed transmission

X. fuscans subsp. fuscansX. citri pv. fuscansX. axonopodis pv.phaseoli var. fuscans

Xap 1

Xap 2

Xap 3

Phaseolus vulgaris

The causal agents of CBB of bean: 4 genetic lineages

X. axonopodis sensu Vauterin et al., 2000; Rademaker et al., 2005

X. axonopodis pv. phaseoliX. fuscans subsp. fuscans

Role of look-alikes in transmission of Xff to bean seeds

Translocation of gfp-tagged strain of Xff to seeds using confocal scanning-laser microscopy

Efficiencies of transmissions of CBB agents from seeds to plantlets.

In vitro

antibiosis

and competition tests

to select strains among the 23 look-alikes that interact with CBB agents

Xff: 7767-R SNES 6V13

Xap GL1: 6546-R V12V13

Xap GL2: 6988-R SNES 27Xap GL3: 6996-R SNES 6

V13

Role of look-alikes in seed transmission of pathogens

4 CBB strains 7 look-alikesEach look-alike inhibitand/or outcompete one of the CBB agents

Pathogen alone

Look-alike alone

Mixtures Pathogen:look-alike (1:1)

5 plants / treatment

3 flower buds / plant

Flower bud inoculation(2 × 10 µL of a 1 × 106 cfu/mL)

Role of look-alikes in seed transmission of pathogenic strains

Xff: 7767-R SNES 6V13

Xap GL1: 6546-R V12V13

Xap GL2: 6988-R SNES 27Xap GL3: 6996-R SNES 6

V13

0

2

4

6

8

10

Popu

latio

n si

zes

(Log

10 C

FU/g

of

seed

)Population sizes of mature seeds

Pathogenic strain alone

Non-pathogenic strain alone

Pathogenic strain in mix

Non-pathogenic strain in mix

aa

aa

a a aa

a a

a a a a

b

b b b b b b b b b

b

b b b

Pathogen alone

Look-alike alone

Pathogen in mix

Look-alike in mix

Detection threshold

7767-RSNES 6

7767-RV13

6546-RV13

6546-RV12

6988-RSNES 27

6996-RSNES 6

6996-RV13


0

2

4

6

8

10

Popu

latio

n si

zes

(Log

10 C

FU/g

of

seed

)Population sizes of mature seeds

Pathogenic strain alone

Non-pathogenic strain alone

Pathogenic strain in mix

Non-pathogenic strain in mix

Detection threshold

aa

aa

a a aa

a a

a a a a

b

b b b b b b b b b

b

b b b

Pathogen alone

Look-alike alone

Pathogen in mix

Look-alike in mix

Detection threshold

7767-RSNES 6

7767-RV13

6546-RV13

6546-RV12

6988-RSNES 27

6996-RSNES 6

6996-RV13

No transmission of look-alikesNo effect of look-alikes on pathogen transmission


Xylem colonization by look-alikes and pathogenic strains

Cataphyll: 1st leaf

1st trifoliate

0 1 and 2 jpi

7767-R SNES 6V13

6546-R V12V13

6988-R SNES 276996-R SNES 6

V13

Pathogen alone

Look-alike alone

Pathogen in mix

Look-alike in mix

Popu

latio

n si

zes

(Log

10 C

FU/g

fres

hw

eigh

t)

7767-R / SNES6

01234567

Inoc Point > cata > cata

0 1 dpi 2 dpî

a a aab b bb

c cc

c

0

2

4

6

8


0 1 dpi 2 dpî

a a aab b b b

c c cc

7767-R / V13

01234567


0 1 dpi 2 dpî

a a aab b bb

c cc

c

6546-R / V13

012345678


0 1 dpi 2 dpî

a a aab b bb

c cc

c

6546-R / V12

01234567


0 1 dpi 2 dpî

a a aab b bb

c ccc

6996-R / V13

01234567


0 1 dpi 2 dpî

a a aab b bb

c cc

c

6996-R / SNES6

01234567


0 1 dpi 2 dpî

a a aab b bb

c c cc

6988-R / SNES27

No difference between pathogens and look-alikes

Pathogen alone

Look-alike alone

Pathogen in mix

Look-alike in mix

Xylem colonization by look-alikes and pathogenic strains

cataphyll

1st trifoliate

0 1 and 2 jpi

Look-alikes and transmission to seed of CBB agents

Plant pathogenic MO

Pathogenic MO

PGPR?

Not possible to monitor transmission to seeds for look-alikesBut look-alikes are isolated from seeds

Do not seem to interfere with pathogen transmission to seedNot good candidates for biocontrol

Mother plantSeed

Localization of Xff during seed formation on bean plants

Xff gfp-tagged cell following spray inoculation of bean plants at flower bud stage

seed

counterpalisade

funicule

Tracheid bar

parenchyma

hilar fissure

28 dai

Transversal cut at the hile

Mother plantSeed



seed

counterpalisade

funicule

Tracheid bar

parenchyma

hilar fissure

Aperture in the counterpalisade of the hile

Tracheid bar

funicule

seed



Mother plant

4 days after sowing in germ boxes 7 days after sowing in soil

Spray-inoculation of R5-beans

Harvest of seeds 6 wk later

Transmission rates of seed borne bacteria under optimal conditions


Large range of Contamination Rates of seed lotsAnd of associated population sizes

Popu

latio

n siz

es o

n se

eds

(Log

cfu

/g o

f see

ds)

Contamination rates of seed lots

0

1

2

3

4

5

6

7

8

0 20 40 60 80 100 120

Results from 29 bean seed lots harvested from plants inoculated with CBB agents


0

0,5

1

1,5

2

2,5

0 5 10 15 20 25 30 35

Tran

smiss

ion

rate

s

6546-R7430

6936-R

6937-R

6938-R

Xap GL1

6988-RXap GL2

6996-RXap GL3

7112-RXav4834-R

7767-R

4884-R

Xff

SNES22-R

0

0,5

1

1,5

2

2,5

0 5 10 15 20 25 30

6546-R7430

6936-R

6937-R

6938-R

Xap GL1

6988-RXap GL2

6996-RXap GL3

7112-RXav4834-R

7767-R

4884-R

Xff

SNES22-R

Tran

smiss

ion

rate

s

TR 4: CR plantlet lots 4 dai/ CR seed lots TR 7: CR plantlet lots 7 dai/ CR seed lots


High TR from seeds to plantlets: 0.6 to 1.5 (2) for CBB agents

Transmission efficiency varies for a same strain (high variability for Xav strain CFBP 7112)

TR ~ 1 when analyzed 4 dai, more variable when determined 7 dai

0

0,5

1

1,5

2

2,5

0 5 10 15 20 25 30 35

Tran

smiss

ion

rate

s

6546-R7430

6936-R

6937-R

6938-R

Xap GL1

6988-RXap GL2

6996-RXap GL3

7112-RXav4834-R

7767-R

4884-R

Xff

SNES22-R

0

0,5

1

1,5

2

2,5

0 5 10 15 20 25 30

6546-R7430

6936-R

6937-R

6938-R

Xap GL1

6988-RXap GL2

6996-RXap GL3

7112-RXav4834-R

7767-R

4884-R

Xff

SNES22-R

Tran

smiss

ion

rate

s

TR 4: CR plantlet lots 4 dai/ CR seed lots TR 7: CR plantlet lots 7 dai/ CR seed lots


All seed contaminations lead to plantlet contamination if analyzed 4 daiContamination rates of plantlets lots are more or less predictable from mean population sizes in seedsPopulation threshold (1 × 103 cfu/g of seed) on seeds for an efficient contamination of plantlets (4 dai)

If analyses are made on leaves of 7 days-old plantlets, contaminations are highly variable and not predictable from populations sizes determined on seeds.

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

Population sizes on seeds (Log cfu/g of seeds)

Cont

amin

atio

n ra

tes o

f pla

ntes

0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

Plantlets 4 d after imbibition Leaves of 7 days-old plants

How do look-alikes transmit to seeds?Are they stopped by plant defenses in flower buds?

Bean bud inoculation lead to vascular transmission of Xff to seeds, and not only to ingress via the micropyle.

Analysis of plantlets 4 dai in germination boxes allows a good estimation of seed contaminations,BUT tend to over-estimate efficient transmission to plants as determined by contamination of the cataphylls

Conclusions

Acknowledgments

Armelle DarrasseJean-François Guimbaud

Anne Préveaux

INEM

Thank you for your attention

Efficiencies of bacterial transmission from plants to seeds and … · 2018. 5. 14. ·...

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