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Assessing brown rot resistance in peach fruitsIgor Pacheco, Bénédicte Quilot-Turion, Daniele Bassi

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BR-resistance assessment is hindered by its high environment-associated variability

Disease Incidenc

e

Fungal virulence and density

- Occurrence of cold and humidity in late spring

- Orchard hygienic conditions

- Skin cracking- Phenolics content at

maturity- Pathogenesis-related (PR) protein activities

- Nitrogen applications- Rainfalls near harvest

Seasonal conditions/Agronomical practices

Pathogen factors

Fruit factors- maturity variability inside

the plant- fruit size variability inside a

seedling

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Developed methods in Fruit Breedomics:

• Orchard spray-based (high-throughput)

• Laboratory drop-based (detailed parameters)

0 50 100 150 200

020

4060

80

E36

temps_heure

diam

_inf

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Paper-protected fruit clusters+3fruit clusters containing 3-6

fruits each

Fruit cluster inoculation of Monilinia laxa

(105 spores/ml until runoff)

incubation time (e.g. 7 days in dry environment; 3 days in moist environment)

Methods for BR-resistance phenotypingOrchard spray test

Register number of infected and healthy fruits

Infection probability - Drop 2014 (lab)

Infection probability

Num

ber o

f gen

otyp

es

0.0 0.2 0.4 0.6 0.8 1.0

010

2030

4050

Infection probability - Spray 2014 (orchard)

Infection probability

Num

ber o

f gen

otyp

es0.0 0.2 0.4 0.6 0.8 1.0

010

2030

40• BC2 (Zephir x [(Summergrand x P.davidiana) x Summergrand]; 98 to 118 individuals in 2013 and 2014. INRA-Av

• Bolinha-self. Around 90 individuals in 2013 and 2014. INRA-Av

• Contender x Elegant Lady F2. 120 individuals in 2012 and 2013. UMIL

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2. Hot water disinfection (40s at 55ºC).

Control Drop Spray (e.g. nCi = 10 nSKi = 20 nFLi = 20)

4. Disease incubation(90-100% RH, 25ºC)

5. Susceptibility scoring

spore suspension

1. Harvest at physiological ripening (e.g. 60 fruits, IAD < 0,6)

3.Fruit inoculation

Seedling i

105 sp/ml

each 24 hours for rot kinetics exp.(preferred 72 and 120 hpi)

- ∆Ameter-based maturity evaluation- Fruit size registration

72 h 96 h 120 h

Methods for BR-resistance phenotypingLaboratory “drop” protocol (1)

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time after infection (h)

Rot d

iam

eter

(mm

)Rot progression curve parameters (resistance values):

• % infected fruits (infection probability after n hours)

• rot diameter (after n hours)

•Infection delay (penetration time)

V1 V2

D1 D2

•rot speed (progression slope)

Methods for BR-resistance phenotyping -Laboratory “drop” protocol (2)

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Phenotyped material (Drop-lab)

• BC2 (Zephir x [(Summergrand x P.davidiana) x Summergrand]; 98 to 118 individuals in 2013 and 2014.

• Bolero x OroA. 80-120 individuals in 2012 and 2014.

• Contender x Elegant Lady. 50 individuals in 2013-2014

Infection minimal lead time

(hour)

Num

ber o

f gen

otyp

es

50 100 150 200

05

1015

2025

Infection maximal lead time

(hour)

Num

ber o

f gen

otyp

es

50 100 150 200

05

1015

20

Maximal speed of progress of infection diameter

(mm/h)

Num

ber o

f gen

otyp

es

0.0 0.5 1.0 1.5 2.0 2.5 3.0

05

1015

2025

3035

Infection diameter at 120 hours after drop deposit

(mm/h)

Num

ber o

f gen

otyp

es

0 10 20 30 40 50 60 70

05

1015

2025

r2 = 0,28 p = 0,00842013

2014

2013

2014

r2 = 0,32 p = 0,0011

Spray - orchard Drop - Lab

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Phenotypic dissection of BR-resistance trait:Analysis of infection variables (drop-lab)

- concentration of spores in inoculum drop- drop surface- total number of inoculated spores- density of inoculated spores

AIM: estimate effect of infection surface and number of spores in the genotype-specific infections

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Rot d

iam

eter

(mm

)

Time after inoculation (h) * ranks inside cultivar

Phenotypic dissection of BR-resistance trait:Analysis of fruit variables (drop-lab)

commercial maturity

physiological maturity

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A

50 µm

Light microscopy (LM) in young fruit surface

julian days

Infe

ctio

n pr

obab

ility

100 150 200 250

0.0

0.2

0.4

0.6

0.8

1.0

2012 no wound2013 no wound2013 wounded

Summergrand

tabCinet$jul

tabC

inet

$pro

bafin

100 150 200 250

Zephyr

Infe

ction

pro

babi

lity

Julian days

julian days

cutic

ular

con

duct

ance

(cm

/h)

100 150 200 250

050

010

0015

0020

00

Summergrand

100 150 200 250

Zephyr

Julian days

Cutic

ular

con

duct

ance

(c

m/h

)

Stage I: young fruits High infection probability High conductance at early stage High stomatal density

Phenotypic dissection of BR-resistance trait:Analysis of fruit variables (drop-lab) – (1)

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cutic

ular

con

duct

ance

(cm

/h)

100 150 200 250

050

010

0015

0020

00

Summergrand

100 150 200 250

Zephyr

julian days

Infe

ctio

n pr

obab

ility

100 150 200 250

0.0

0.2

0.4

0.6

0.8

1.0

2012 no wound2013 no wound2013 wounded

Summergrand

tabCinet$jul

tabC

inet

$pro

bafin

100 150 200 250

Zephyr

Infe

ction

pro

babi

lity

Julian days

Stage I: young fruits High infection probability High conductance at early stage High stomatal density Stage II: pit hardening Low infection probability Low cuticular conductance Max. wax layer

40 60 80 100 120 140 160

05

1015

20

total cuticular wax quantities (mg/dm²)

DAB

wax

acc

umul

atio

n m

g/dm

²

SGZE

Wax

(mg/

dm²)

Days after bloom

Cutic

ular

con

duct

ance

(c

m/h

)

Phenotypic dissection of BR-resistance trait:Analysis of fruit variables (drop-lab) – (2)

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cutic

ular

con

duct

ance

(cm

/h)

100 150 200 250

050

010

0015

0020

00

Summergrand

100 150 200 250

Zephyr

julian days

Infe

ctio

n pr

obab

ility

100 150 200 250

0.0

0.2

0.4

0.6

0.8

1.0

2012 no wound2013 no wound2013 wounded

Summergrand

tabCinet$jul

tabC

inet

$pro

bafin

100 150 200 250

Zephyr

Infe

ction

pro

babi

lity

Julian days

B

Scan electron microscopy (SEM) of fruit surface nectarine at maturity

Stage II: pit hardening Low infection probability Low cuticular conductance Max. wax layer Stage III: maturity High infection probability Increase of cuticular conductance Increase of microcracks incidence

Cutic

ular

con

duct

ance

(c

m/h

)Phenotypic dissection of BR-resistance trait:Analysis of fruit variables (drop-lab) – (3)

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Phenotypic dissection of BR-resistance trait:Effect of fruit size on cracking (…and opening doors)

Probability of infection value is affected by fruit size

Summergrand

Zéphir

SMALL FRUIT LARGE FRUIT

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• Qualitative relationship BR resistance and fungal colonization on fruit tissue

• Effects of pathogen colonization on fruit tissue of cultivars with contrasting BR-resistance

• Morphology of physical fruit barriers (e.g., µcracks, cuticle, epidermis)

Phenotypic dissection of BR-resistance trait: Microscopy features of BR infection in fruits

Bolinha - 48 hpi Zéphir - 48 hpi

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• Fungal inhibition proportional to compound concentration• Inhibitor effect (in function of concentration): FA > PCA > CA• Related with rot progression speed??

7

9

11

13

15

17

19

21

*

*

*

eau stérile

1% éthanol

CA

PCA

FA

Colo

ny d

iam

eter

(mm

)

0,1 0,5 1 2 Concentration (mM)

72 hours after inoculation

Caffeic acid [CA]

p-coumaric acid [PCA]

Ferulic acid [AF]

Phenotypic dissection of BR-resistance trait: Antifungal activity of polyphenolic compounds

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0

1

2

3

4

5

6

Proba infection _ Spray _ carte P.davidiana

Chromosome

lod

1 2 3 4 5 6 7 8

IMCIMMQM_1Covar

0

1

2

3

4

Proba infection _ Drop _ carte P.davidiana

Chromosome

lod

1 2 3 4 5 6 7 8

IMCIMcovainteractiveCovar

0.0

0.5

1.0

1.5

2.0

Proba infection _ Spray _ carte Zephir

Chromosome

lod

1 2 3 4 5 6 7 8

IMCIMMQM_1Covar

0.0

0.5

1.0

1.5

2.0

Proba infection _ drop _ carte Ze_corr

Chromosome

lod

1 2 3 4 5 6 7 8

IMCIMMQM_auto

0

1

2

3

4

Proba infection _ Vitmax _ carte P.davidiana

Chromosome

lod

1 2 3 4 5 6 7 8

IMCIMinteractiveCovarMQM_1Covar

0

1

2

3

4

Proba infection _ Vitmax _ carte P.Zephir

Chromosome

lod

1 2 3 4 5 6 7 8

IMCIMinteractiveCovarMQM_1Covar

Spray – infection probability – P. davidiana

Drop – infection probability – P. davidiana

Max. rot speed – P. davidiana

Spray – infection probability – Zéphir

Drop – infection probability – Zéphir

Max. rot speed – Zéphir

4. Towards WP3: application of phenotyping in genetic analyses – BC2 progeny (INRA-Avignon)

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4. Towards WP3: application of phenotyping in genetic analyses – BxOa progeny (just lab drop test; U.Milan)

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Conclusions and Perspectives

•Two methods have been developed for BR resistance scoring.

•Four populations have been phenotyped for two years and phenotypic data is under analysis

•Different “sub-traits” have been associated to the BR impact:•“open doors” (cuticular conductance and stomata number) are

associated to infection probability•sample factors (maturity index and fruit size) affect the extent of

open doors•rot progression speed can be affected by polyphenol composition in

cuticle, epidermis and flesh

•different sub-traits could be pyramided to generate more BR-resistant cultivars