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

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
SCIENTIFICAL CONTEXT
BIOLOGICAL CYCLE OF PLASMOPARA VITICOLA AND
ASSOCIATED SYMPTOMS
Oomycota family (brown algae)
Biotrophic parasite, introduced in France in 1878
SCIENTIFIC 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
Cycle 8
5 couples R/S
3 mixes
- ?%R ?%S
- ?%R ?%S
- ?%R ?%S
FUNGICIDE SCREENING
(1 week)
Visual notation to estimate
resistant and sensitive
percentages
QUANTITATIVE PCR
To estimate resistant
and sensitive
percentages
STRAINS 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
COMPETITION TEST (SECOND ASSAY)
Statistical work required but general tendencies observed
Diminution of resistant proportion in 3 couples
MOLECULAR EXPERIMENTS
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