May, 12th 2007 Magistère of Biotechnologies, University of Orsay Pierre ABADIE INRA...

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