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06 pacheco
1. YOUR LOGO
Assessing brown rot resistance in peach fruits
Igor Pacheco, Bénédicte Quilot-Turion, Daniele Bassi
2. YOUR LOGO
BR-resistance assessment is hindered by its
high environment-associated variability
Seasonal conditions/
Agronomical practices
Pathogen factors
Fruit factors
- maturity variability inside
the plant
- fruit size variability inside a
seedling
3. YOUR LOGO
Developed methods in Fruit Breedomics:
• Orchard spray-based (high-throughput)
• Laboratory drop-based (detailed parameters)
0 50 100 150 200
020406080
E36
temps_heure
diam_inf
4. YOUR LOGO
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 phenotyping
Orchard spray test
Register number of infected and
healthy fruits
Infection probability - Drop 2014 (lab)
Infection probability
Numberofgenotypes
0.0 0.2 0.4 0.6 0.8 1.0
01020304050
Infection probability - Spray 2014 (orchard)
Infection probability
Numberofgenotypes
0.0 0.2 0.4 0.6 0.8 1.0
010203040
• 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
5. YOUR LOGO
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 phenotyping
Laboratory “drop” protocol (1)
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time after infection (h)
Rotdiameter(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)
8. YOUR LOGO
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
9. YOUR LOGO
Rotdiameter(mm)
Time after inoculation (h)
* ranks inside cultivar
Phenotypic dissection of BR-resistance trait:
Analysis of fruit variables (drop-lab)
commercial maturity
physiological maturity
10. YOUR LOGO
A
50 µm
Light microscopy (LM) in young fruit surface
julian days
Infectionprobability
100 150 200 250
0.00.20.40.60.81.0
2012 no wound
2013 no wound
2013 wounded
Summergrand
tabCinet$jul
tabCinet$probafin 100 150 200 250
Zephyr
Infectionprobability
Julian days
julian days
cuticularconductance(cm/h)
100 150 200 250
0500100015002000
Summergrand
100 150 200 250
Zephyr
Julian days
Cuticularconductance
(cm/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)
11. YOUR LOGO
julian days
cuticularconductance(cm/h)
100 150 200 250
0500100015002000
Summergrand
100 150 200 250
Zephyr
julian days
Infectionprobability
100 150 200 250
0.00.20.40.60.81.0
2012 no wound
2013 no wound
2013 wounded
Summergrand
tabCinet$jul
tabCinet$probafin
100 150 200 250
Zephyr
Infectionprobability
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
05101520
total cuticular wax quantities (mg/dm²)
DAB
waxaccumulationmg/dm²
SG
ZE
Wax(mg/dm²)
Days after bloom
Cuticularconductance
(cm/h)
Phenotypic dissection of BR-resistance trait:
Analysis of fruit variables (drop-lab) – (2)
12. YOUR LOGOjulian days
cuticularconductance(cm/h)
100 150 200 250
0500100015002000
Summergrand
100 150 200 250
Zephyrjulian days
Infectionprobability
100 150 200 250
0.00.20.40.60.81.0
2012 no wound
2013 no wound
2013 wounded
Summergrand
tabCinet$jul
tabCinet$probafin
100 150 200 250
Zephyr
Infectionprobability
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
Cuticularconductance
(cm/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
14. YOUR LOGO
• 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
15. YOUR LOGO
• 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
Colonydiameter(mm)
eau stérile
1% éthanol
CA
PCA
FA
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
16. YOUR LOGO
0
1
2
3
4
5
6
Proba infection _ Spray _ carte P.davidiana
Chromosome
lod
1 2 3 4 5 6 7 8
IM
CIM
MQM_1Covar
0
1
2
3
4
Proba infection _ Drop _ carte P.davidiana
Chromosome
lod
1 2 3 4 5 6 7 8
IM
CIM
cova
interactiveCovar
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
IM
CIM
MQM_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
IM
CIM
MQM_auto
0
1
2
3
4
Proba infection _ Vitmax _ carte P.davidiana
Chromosome
lod
1 2 3 4 5 6 7 8
IM
CIM
interactiveCovar
MQM_1Covar
0
1
2
3
4
Proba infection _ Vitmax _ carte P.Zephir
Chromosome
lod
1 2 3 4 5 6 7 8
IM
CIM
interactiveCovar
MQM_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
Editor's Notes
Hello, I’m Igor Pacheco, and I am here to present to you the main results in the research for methods to phenotype and characterize resistance to Monilinia fungi (namely brown rot) on peach.
This task is developed by the collaboration of the Unit of Genetics and Improvement of Fruits and Vegetables of the INRA Avignon, and the Department of Agricultural and Environmental Sciences of the University of Milan.
- is the major cause of fungicide applications in stone fruits
- threat to sustainable production of these crops
- interest in develop cultivars more resistant to this disease
…that is strongly influenced by the environment
by one hand the v and d of the f
- in the other hand , rainfalls and agronomical practices affect different aspects influencing the occurrence of constitutive barriers in the fruit such
given this great environmental influence, in this work we aimed to develop phenotyping tools for the BR resistance assessment in cultivars and crosses
Orchard test was designed as an more high-throughput alternative to lab test.
To setup this approach, we used 3 levels of humidity maintenance, in order to enhance pathogen activity.
After inoculating fruit clusters with a spore suspension, fruit clusters were enclosed with paper and plastic.
after a period of incubation, we determined the proportion of infected fruits.
Paper protection presented practical advantages at the moment of identify specifically Monilinia-rotten fruits
MICROSCOPY ANALYSIS
Ferulic acid easy to test, complex to terpenoids??
Difficulty to perform experiments in hydrophobic compounds