06 pacheco
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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
$pro
bafin
100 150 200 250
Zephyr
Infe
ction
pro
babi
lity
Julian days
Stage I: young fruits High infection probability High conductance at early stage High stomatal density Stage II: pit hardening Low infection probability Low cuticular conductance Max. wax layer
40 60 80 100 120 140 160
05
1015
20
total cuticular wax quantities (mg/dm²)
DAB
wax
acc
umul
atio
n m
g/dm
²
SGZE
Wax
(mg/
dm²)
Days after bloom
Cutic
ular
con
duct
ance
(c
m/h
)
Phenotypic dissection of BR-resistance trait:Analysis of fruit variables (drop-lab) – (2)
YOUR LOGOjulian days
cutic
ular
con
duct
ance
(cm
/h)
100 150 200 250
050
010
0015
0020
00
Summergrand
100 150 200 250
Zephyr
julian days
Infe
ctio
n pr
obab
ility
100 150 200 250
0.0
0.2
0.4
0.6
0.8
1.0
2012 no wound2013 no wound2013 wounded
Summergrand
tabCinet$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
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