May, 12th 2007 Magistère of Biotechnologies, University of Orsay Pierre 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/S 3 initial mixes - 20%R 80%S - 50%R 50%S - 80%R 20%S Cycle 0Cycle 1 5 couples R/S 3 mixes - ?%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 CATTTATGGGTTATGTTTTGCCTTGGGGACAAATGAGTTTTTGGG G TGCAACAGTTATTA 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 didnt 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