may, 12th 2007 magistère of biotechnologies, university of orsay pierre abadie inra...
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May, 12th 2007Magistère of Biotechnologies, University of OrsayPierre ABADIE INRA Bordeaux-Aquitaine, UMR Santé Végétale
Oomycota family (brown algae)Biotrophic parasite, introduced in France in 1878
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?
Goal: following the evolution of sensitive/resistant strains proportions during 8 cycles
5 couples R/S3 initial mixes- 20%R 80%S- 50%R 50%S- 80%R 20%S
Cycle 0
Cycle 1
5 couples R/S3 mixes - ?%R ?%S- ?%R ?%S- ?%R ?%S
Reinoculation on leaves(1 week)
FUNGICIDE SCREENING(1 week)
Visual notation to estimate resistant and sensitive percentages
QUANTITATIVE PCR
To estimate resistant and
sensitive 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
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: « 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’)
: SNP G143A leading to resistant phenotype
: unspecific primers amplifying R and S
: resistant-specific primers
G
DISCUSSION AND PERSPECTIVES
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
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