study of plant resistance inducers (pri) on apple: …

Colloque Elicitra - Natural Products & Biocontrol 26 Septembre 2018

STUDY OF PLANT RESISTANCE INDUCERS (PRI) ON APPLE:

FROM THE LAB TO THE FIELD

MATTHIEU GAUCHER / MARIE-NOËLLE BRISSET

Angers, France

Production in France

1,5 - 2 millions of tonnes

3rd european producer

Source : Ecophyto R&D., 2009

37 300 cultivated ha 7 600 farms Mean Nb of treaments / year = 35,1 (Mainly toward apple scab)

-30% in 11 years

Apple farming in France

+20% in 15 years

Current situation regarding PRIs

In controlled conditions In the field

Control of disease rarely significant, with poor result reproducibility

Applicable to all crops

Global objective of the team: find answers for apple crop

Significant and reproducible control of disease

Applied research Basic research

Screening methods

Study of factors influencing effectiveness

Integration into pest managment programs

lab

field

Mode of action

Research on Plant Resistance Inducers

Screening of PRI candidates

PRI candidates

Defense activation

Protection efficacy

and/or

pathosystem ?

PRI candidates

Defense activation

Protection efficacy

qPFD

Screening of PRI candidates on apple

3 pathosystems Fire blight

(Erwinia amylovora)

Apple scab (Venturia inaequalis)

Rosy aphid (Dysaphis plantaginea)

a ready-to-use molecular tool for the profiling of the expression of 28 defense genes by quantitative real-time PCR

1 plate = 1 cDNA sample

qPFD = Puce Faible Densité quantitative

SA JA ET

Barrièreschimique&physique



Antagonisme Addi vité

28 defense genes

(quantitative Low Density Array)

Pulv. des produits à J0

(± H202 à J1)

Prélèvements de disques

foliaires à J0, J1, J2, J3 Extraction d’ARN qPFD

Plant material

Apple seedlings - population - sustained production

Method

Material and methods


(± H202 à J1)


foliaires à J0, J1, J2, J3 Extraction d’ARN qPFD


(± H202 à J1)


foliaires à J0, J1, J2, J3 Extraction d’ARN qPFD Pulv. of products at J0 Foliar disks prelevement

at J0, J1, J2, J3 RNA extraction + RT qPFD

Molecular analysis using the qPFD tool (RT-qPCR on 28 defense genes)

A PR1

PR2

PR4

PR5

PR8

PR14

PR15

PAL

ANS

CHS

DFR

PPO

HMGR

FPPS

FAR

CSL

APOX

GST

POX

CAD

CalS

PECT

EDS1

WRKY

LOX

JAR

ACCO

EIN3

B

Induction Répression

28 defense genes

Products

PR proteins

Secondary metabolism

Cell-wall

Oxidative burst

Signalling pathways

Material and methods

Calculation of a global rate of induced defense per product

Ranking of products against each other

Example of screening results

-4 -2 0 2 4 6

Kendal cops

VG+

Isomag

Boralgine

Sérénade max

BasFoliar SI

Vitalgue

Fytosave

Altex

Sarriette

Vacciplant

Eau

Aliette

H2O2

Defender

Régalis

Alexin

Fructose

Chitoplant

Sucrose

Optiplant petit pois

Nectar Duo

Trainer

PRM12

Armicarb

Kendal

LBG01F34

Trafos Mg-Ca-Si

Bion

Sreening of 25 commercial products

Market autorization as phytopharmaceutical

4 synthetic products / 5

PEPS

Bion 50 WG functionnal analog of SA (ASM)

Trafos Mg-Ca-Si P, Mg, Ca, Si

LBG01F34 potassium phosphonate (KHP)

Kendal organic matter, glutathion

Armicarb potassium bicarbonate

Biocontrol product (NS 11/2016)


Screening methods



lab

field

Mode of action


-8

-6

-4

-2

0

2

4

6

8

ELCal

ELEau

ELEau+S

ELBion

ELBion+S

FUCal

FUEau

FUEau+S

FUBion

FUBion+S

GACal

GAEau

GAEau+S

GABion

GABion+S

GOCal

GOEau

GOEau+S

GOBion

GOBion+S

PICal

PIEau

PIEau+S

PIBion

PIBion+SElstar

(++) Fuji (+)

Gala (+++)

Golden (+++)

Pink Lady (++++)

Genotypes (susceptibility to apple scab)

Glo

bal

rat

e o

f d

efen

ses

J0

Water

Bion J3

Results

DEFISTIM

Time (days) 0

Method

Defense analysis (qPFD)

3

Grafted genotypes

Durability of the induced resistance

Quality of the application

Interaction with other

inputs

Persistance of action

Abiotic stress Genotype

PRI Water

PREDIRE

0

1

2

3

4

5

6

7

8

seve

rity

of

app

le s

cab

génotypes

Water

Bion

Protection test against apple scab




inputs



Results

PREDIRE

NT

PRI




inputs



Hypothesis on genotypes exhibiting the same susceptibitliy level

0

2

4

6

8

10

12

+

++

+++ = constitutive level of defenses ≠ response to PRI

≠ constitutive level of defenses = response to PRI

0

2

4

6

8

10

12

=

=

=

No protection with the PRI

Slight response to PRI

Too low defense level

Def

ense

leve

l

Def

ense

leve

l

Mobilization of apple geneticists at Angers (C.E. Durel, IRHS) PhD

PRI efficacy

Partial resistance (apple scab and fire blight)

Constitutive expression of defenses

400 génotypes

PREDIRE




inputs



genetic determinism explaining the difference of PRI effectiveness?

Question

Individual stress or stress combination?

Complexe experiment designs !

PRI treatment

Initial φ state Elicited state

PRI

Time




input



Time (days) 0 21 2 -2

Bion Inoculation FB Symptoms assessment 1

2 3 4 5 6 7

Day 25°C

Night 17°C

Day 35°C

Night 5°C

Water

Example of experiment (∆Temp. day/night)

PEPS

Conditions to be diversified to answer the question!

Rel

ativ

e in

fect

ion

(%

)

1 2 3 4 5 6 7 0

50

100

150

Result

Water

Bion




inputs



PRI treatment

Initial φ state Elicited state

Question

Correlations

Receptivity level to PRI

Find a way to measure this initial φ state




inputs



Measure of the receptivity level: 2 projects

1 - Expression of abiotic stress-responsive genes

Retrotransposon

DNA

Retrotranscription

Insertion Transcription

Stress

ESTIM

2 – Transposable element activation (retrotransposons)

CARE




inputs



Soon available !

Time

PRI

Inputs Inputs Question Inputs

+ PEPS




inputs



Some results with fungicides, insecticides, thinning agents,…

qPFD analysis with inputs applied alone

Individus (axe F1 et F3 : 40,06 %)

Axe F1 (28,56%)

Axe

F3

(12

,04

%)

Bion

Rhodofix

Brevis 0

25

50

75

100

125

0

25

50

75

100

125

Fire blight

Apple scab

a

b b

c

a

b

b

c

Water

Bion

Rhodofix

Water

Rel

ativ

e in

fect

ion

(%

)

PRI or water ± foliar fertilizers

PRI or water

≠ fertilization regimes

Defense analysis + protection tests

• Soil fertilization (nitrogen)

• Foliar fertilizers (oligoelements)

Defense analysis + protection tests




inputs



Tavinnov

• Foliar fertilizers (oligoelements)

6 products tested in combination or applied 6 days before treatment : no identified interactions

• Soil fertilization (nitrogen)

0

20

40

60

80

100

7 days before PRI

14 days before PRI

bcd b bcd

efg fg

def cde

b a

g

ef

ab Water N1 Water N4 Water N16

Bion N1 Bion N4 Bion N16

Inci

den

ce (

%)

Fire blight

N supply:




inputs



Tavinnov

PRI

days 0 3 7 10 14

Defense analysis

Water

Method

-2

-1

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14

-2

-1

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14

Glo

bal

rat

e o

f d

efen

ses

Glo

bal

rat

e o

f d

efen

ses

Time (days) Time (days)

treated leaf untreated leaf Result

µPRI Water

treated leaf treated leaf

untreated leaf

PEPS

Weekly applications of PRIs can be recommended in orchard, especially during the growing season




inputs



Controlled-conditions Orchard (spray gun) (Traditionnal sprayer)

Purchase of an automated and adjustable spray

chamber (2019)

Discrepancy between controlled conditions / Orchard

0

20

40

60

80

100

120

140

160

180

Facesup Faceinf Facesup Faceinf

Quan

tédeproduit(µg)/cm2defeuille

Orchard Greenhouse

Quantification

Am

ou

nts

of

pro

du

cts

(µg)

/cm

2 o

f le

af

lower side upper side lower side upper side




inputs



Mobilization of population geneticists of Venturia inaequalis (B. Le Cam, IRHS)

2) Orchard

Sampling of strains on trees that have received PRI-treatments for several consecutive years (100 strains)

Experimental evolution

1) Controlled conditions

Comparison of strain

aggressivness Inoculation 20 strains

Elicited plants

Control plants

… x10

… x10




inputs



Tavinnov

SNP analysis


Screening methods



lab

field

Mode of action


National network for the fiel study

Angers

Lanxade

Sées

Montauban

Marsillargues

St Yrieix

Prayssas

St Epain

Malemort

Coordination

8 experimental sites

in 3 production areas PEPS

Control for apple Scab (spring) and postharvest diseases (autumn)

Integration of PRI into pest managment programs (toward apple scab)

Strategy consensual protocol between partners

July..

Secondary contamination

March

Classical IPM

Classical IPM

15 days after the last lesions

Classical IPM

Classical IPM Classical IPM

Classical IPM

Classical IPM

Light IPM

Light IPM + PRI every week

Primary contamination

Fungicide treatments managed with an enhanced acting threshold compared to the IPM (RIMpro apple scab prediction model)

0

100

200

300

400

500

600

700

800

900

1000

Apple scab risk (RimPro values)

Classical IPM

fungicide

Light IPM

IPM = Integrated Pest Management

0

25

50

75

100

Expected result

Untreated control

Light IPM

IPM

Light IPM + PRI

Dis

ease

inci

den

ce (

%)



systematic preventive fungicides (any risk) Strategy in 2015 curative fungicides if RIM value > 800 (severe risks)

Light IPM =

Results

0

25

50

75

100

Dis

ease

inci

den

ce

NT IPM Light IPM Light IPM + PRI

Results in most experimental units

Too low acting threshold in light IPM

95,2

4,2

61,3

25,2

5,4

0

20

40

60

80

100

a

d

b

c

d

Golden 98

22,5

83

64,5

6,5

0

20

40

60

80

100

a

d

b b

d

Pink Lady

55,5

8,5

23,5

7,53

0

20

40

60

80

100

a

c b

c c

Braeburn

48,5

9,5 10,55

10

0

20

40

60

80

100

a

b b b b

Gala

100

7075

53

3 5

69

0

20

40

60

80

100

a

b

b

c c

b b

Pink Lady 100,0

5,0

33,8

7,8 9,5

0

20

40

60

80

100

a

c

b

c c

Golden

19

1,7 2,3 1,7 0,30

20

40

60

80

100

a

b b b b

Frequin rouge

68,8

9,5

34,0

11,6

30,2

17,5

0

20

40

60

80

100

Gala

a

c

b

c

b c

Results

NT IPM Light IPM LBG Bion Trafos Armicarb Kendal Light IPM +

X X


no fungicide for the 1st risk Strategy in 2016 preventive and curative fungicides for severe risks

Light IPM =

++

+

- -

In 2017: focus on 3 PRI to increase the number of experimental sites

40% of fungicides during the periode (Nb treatment = 12) But 8,5 PRI applications

51,33

9,005,33

21,00

9,33

0,00

20,00

40,00

60,00

80,00

100,00

94,50

45,00

93,00

22,00

34,00

12,00

0

20

40

60

80

10091,50

5,83

20,4215,00

2,50 1,67

11,67

0

20

40

60

80

100 a

c bc

c bc

100,0

6,0

41,5

5,3 7,8

0

20

40

60

80

100a

c

b

c c

95,0

2,5

67,5

50,8 48,3

0

20

40

60

80

100

a

d

b

c c

31,3 31,7 30,4

17,520,4

34,6

0

20

40

60

80

100

X

a a a b

a

85,00

21,00

30,50

14,50 15,33 17,50

0

20

40

60

80

100 a

b b

b b b

74,5

12

28,523,5

6

20

0

20

40

60

80

100

Golden a

b

a

b b

Pink Lady

Braeburn

a

bc

c c bc

Gala

b

Pink Lady Golden

a

bc c b

bc

Frequin rouge Gala

Results

b c

c

b

X X X


no fungicide for the 1st risk Strategy in 2017 preventive and curative fungicides for severe risks

Light IPM =

New modalities = NT (no fungicides) + PRI

LBG 1/2 LBG Bion Kendal Light IPM + NT IPM Light IPM

LBG Kendal NT + LBG 1/2

PRI effectiveness in fungicides-free orchard

50% of fungicides during the periode (Nb treatment = 12) With 8 PRI applications

Non-reproductible results with some PRI

No treatment

PRI every week Classical pgm

Classical pgm

2 months before harvest

Classical pgm

1 month before harvest Harvest

Integration of PRI into pest managment programs (toward postharvest diseases)

Application of PRI 6-8 weeks before harvest (depending on the rain)

Strategy consensual protocol between partners

Storage of the harvest in a cold room for a period of > 3 months

0

20

40

60

80

100100

80

60

40

20

0

Infe

cted

fru

its

(%)

NT Bion LBG Trafos Kendal Ref

Gloesporioses (summary on 3 years)

a a a

b b b b

Integration of PRI into pest managment programs

Unintended effect of PRI application?

Quantification of major allergenic proteins (PR5 and PR10) in fruits when PRI have been applied in spring or before harvest

Fruit flesh

Fruit skin Controls Bion LBG Kendal

0

50

100

150

200

250

0 25 50 75 100 125

PR

10

PR5

Traitements Printemps

0

50

100

150

200

250

0 25 50 75 100 125

PR

10

PR5

Traitements AutomneSpring (apple scab)

Before harvest (postharvest diseases)

Results (ELISA)

PR

10

(O

D/m

g p

rot.

/min

)

PR

10

(O

D/m

g p

rot.

/min

)

PR5 (OD/mg prot./min)

PR5 (OD/mg prot./min)

No impact of the PRI use on the fruit allergenicity

Results summary

LBG (K-phosphonate)

Field effectiveness

Other products

+++ +/-

0%

40%

80%

120%

160%

200%

240%

Eau Captan LBGWater LBGCaptan(fungicide)

0

240

200

160

120

80

40

Co

nid

ia g

erm

inat

ion

(%

)

400000

600000

800000

1000000

1200000

1400000

Conidia germination Fungal growth

Water

LBG

Plant resistance induction, and antimicrobial activity?

Too good to be true !

Factors influencing effectiveness

Much work remains to be done !

Outlooks on PRI

In controlled conditions

genotype fertilization abiotic stress

Methods to measure :

the physiological state the elicited state

Effect on the PRI efficacy :


In the field

PRI positioning

Protocol for integration of PRI:

resistant genotypes (partial)

Crossover with other alternatives?

Improvement of prediction models in taking into account:

physical technics (rain cover)


the plant's receptivity to disease and PRI according to abiotic constraints

Rain cover

Colloque Elicitra - Natural Products & Biocontrol 26 Septembre 2018

Registered PRI on apple (France)

Biocontrol product

Regalis® (prohexadione-Ca) -BASF-

(apple scab / fire blight / aphids / powdery mildew)

Vacciplant® (laminarine) -Goëmar-

Sérénade® Max (Bacillus subtilis) -BAYER-

Aliette® Flash (fosetyl-Al) -BAYER-

(fire blight)

(apple scab / fire blight)


Bion® 50WG (Acibenzolar-S-methyl) -SYNGENTA-


Delan® PRO (Dithianon + K-phosphonate) -BASF-

(apple scab)

4 PRI

1 PRI + fungicide

1 PRI soon registered

study of plant resistance inducers (pri) on apple: …

Documents