Download Etude de l`adaptation du mildiou de la vigne à la pression fongicide

May, 12th 2007
Magistère of Biotechnologies, University of Orsay
Pierre ABADIE
INRA Bordeaux-Aquitaine, UMR Santé Végétale
DOWNY MILDEW ADAPTATION TO
FUNGICIDE PRESSURE: FITNESS STUDY
BIOLOGICAL CYCLE OF PLASMOPARA VITICOLA AND
ASSOCIATED SYMPTOMS
Oomycota family (brown algae)
Biotrophic parasite, introduced in France in 1878
SCIENTIST CONTEXT AND GOALS
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Since 1996, massive use of the fungicide famoxadone
Consequence: resistant pathogen strains of Plasmopara
viticola quickly emerged
Resistance to famoxadone is due to a punctual mutation
(G143A) in the mitochondrial gene coding the cytochrome b
General goal: acquire data on spreading and maintainance of
P. viticola resistant strains, in the optic of improving fungicides
application management.
My training period goal: studying fitness of resistant and
sensitive strains to famoxadone
-> Is there a cost of resistance?
THE COMPETITION TEST
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Goal: following the evolution of sensitive/resistant strains proportions during 8
cycles
Cycle 0
Reinoculation on leaves
(1 week)
5 couples R/S
3 initial mixes
- 20%R 80%S
- 50%R 50%S
- 80%R 20%S
Cycle 1
5 couples R/S
3 mixes
- ?%R ?%S
- ?%R ?%S
- ?%R ?%S
FUNGICIDE SCREENING QUANTITATIVE PCR
(1 week)
To estimate
Visual notation to estimate
resistant and
resistant and sensitive
sensitive
percentages
percentages
Cycle 8
STRAINS COUPLES CARACTERISTICS
INOCULATION ON LEAVES
Inoculation of 24 drips of
15µL, adjusted to 40,000
sporangias/mL
One week at
22°C
Sporulation
FUNGICIDE SCREENING
Inoculation on leaves disks
sprayed with famoxadone
(100mg/mL)
Visual notation
BIOLOGICAL RESULTS
BIOLOGICAL MEASURES STANDARDIZATION
- Good correlation between the percentage of resistant and the
notation scale (linear correlation)
- At 40,000 sporangias/mL, notation extended from 0 to 5
BIOLOGICAL COMPETITION TEST (first assay)
No significant variation detected
Statistical work required
BIOLOGICAL COMPETITION TEST (second assay)
Statistical work required but general tendencies observed
Diminution of resistant proportion in 3 couples
QUANTITATIVE PCR MEASURES
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Goal: determination of the resistant and sensitive strains rates
in the biological competition test mixes (to improve fungicide
screening measures)
Experiment progress: the protocol is set up, first results coming
soon…
Protocol:
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« Sybr green » fluorescent probe is used
P. viticola DNA is extracted from the leaves used in the competition test
2 primers couples: one that is specific to P. viticola cytochrome b gene,
and another one specific to resistant P. viticola strains cytochrome b
gene allele
30 cycles of PCR amplification
QUANTITATIVE PCR MEASURES
(5’)
1021 TTATGCGTGATGTAAATAACGGTTGGTTAATTCGATATATACATGCGAATGGTGCATCTT
1081 TTTTTTTTATTGTTGTATATATACATATTTTTAGGGGTTTGTATTACGGATCTTATATTA
1141 CACCTAGAGAAGCTTTATGGTGTTCAGGGGTAATTATTTTTATTTTAATGATGGCGACTG
1201 CATTTATGGGTTATGTTTTGCCTTGGGGACAAATGAGTTTTTGGGGTGCAACAGTTATTA
1261 CAAATTTATTCTCGGCTATCCCATTAATTGGAAAAGAAGTTGTTGACTGGTTATGGGGTG
1321 GATTCGCCGTTGATAATCCAACATTAAATCGTTTTTTTAGTTTACATTTCACCTTTCCAT
1381 TTGTAATTGTAGGGGCTGTACTAATACATTTAATTTTATTACATGAGGTAGGTTCAAATA
(3’)
G
: SNP G143A leading to resistant phenotype
: unspecific primers amplifying R and S
: resistant-specific primers
DISCUSSION AND PERSPECTIVES
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Previous data on fitness didn’t show any significant global
differences between resistant and sensitive strains
In this study, the competition test seems to corroborate
previous fitness data: low-fitness strains are less competitive
Costs of resistance may have been detected
But: statistical work is required
Waiting for Q-PCR measures to improve the results
Realize a model of evolution of resistant and sensitive strains
mixes including fitness data