May 12 th 2007 Magistre of Biotechnologies University

May, 12 th 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 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 (G 143 A) 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 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 FUNGICIDE SCREENING QUANTITATIVE (1 week) PCR Visual notation to estimate To estimate resistant and sensitive resistant and percentages sensitive percentages Cycle 8

STRAINS COUPLES CARACTERISTICS

INOCULATION ON LEAVES Inoculation of 24 drips of 15µL, adjusted to 40, 000 sporangias/m. L One week at 22°C Sporulation

FUNGICIDE SCREENING Inoculation on leaves disks sprayed with famoxadone (100 mg/m. L) Visual notation

BIOLOGICAL RESULTS

BIOLOGICAL MEASURES STANDARDIZATION - Good correlation between the percentage of resistant and the notation scale (linear correlation) - At 40, 000 sporangias/m. L, 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 TTATGCGTGATGTAAATAACGGTTAATTCGATATATACATGCGAATGGTGCATCTT 1081 TTTTATTGTTGTATACATATTTTTAGGGGTTTGTATTACGGATCTTATATTA 1141 CACCTAGAGAAGCTTTATGGTGTTCAGGGGTAATTATTTTAATGATGGCGACTG 1201 CATTTATGGGTTATGTTTTGCCTTGGGGACAAATGAGTTTTTGGGGTGCAACAGTTATTA 1261 CAAATTTATTCTCGGCTATCCCATTAATTGGAAAAGAAGTTGTTGACTGGTTATGGGGTG 1321 GATTCGCCGTTGATAATCCAACATTAAATCGTTTTTTTAGTTTACATTTCACCTTTCCAT 1381 TTGTAATTGTAGGGGCTGTACTAATACATTTAATTTTATTACATGAGGTTCAAATA (3’) G : SNP G 143 A leading to resistant phenotype : unspecific primers amplifying R and S : resistant-specific primers

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