Jeanne Tourvieille

Single nucleotide polymorphisms reveal multiple introductions into France of Plasmopara halstedii, the plant pathogen causing sunflower downy mildew.

Plasmopara halstedii, the causal agent of sunflower downy mildew, displays a gene-for-gene interaction with its host plant, Helianthus annuus and other species of the genus. Monitoring of the evolution of virulent races in France over a 19-year period led to the identification of 14 different races (or pathotypes). Twelve expressed sequence tag (EST)-derived markers displaying SNPs and insertion-deletion variations have recently been identified in P. halstedii. We used these markers to study the genetic structure and the evolution of sunflower downy mildew races. Bayesian assignment analysis identified three genetically differentiated groups of isolates organized around the first three races described in France. Strong genetic substructuring according to geographic origin of races was observed, confirming that these three groups corresponded to three separate introductions into France of isolates with different genetic and phenotypic backgrounds. Our results suggest that multiple introductions of P. halstedii isolates may have provided the raw material for more complex processes in the evolution of races, such as recombination between races or clonal evolution through mitotic instability.

Resistance to metalaxyl in isolates of the sunflower pathogen Plasmopara halstedii.

Plasmopara halstedii isolates showing an atypical reaction to metalaxyl were collected in France, in 1995 and 1996, and tested in the laboratory for their level of sensitivity to this fungicide. Primary and secondary infections caused by one of these isolates were not controlled by the metalaxyl concentration registered for seed treatment. The EC50 of this isolate was 12 800 mg a.i. kg-1 compared with 22 mg a.i. kg-1 for sensitive isolate, indicating a 582-fold decrease in sensitivity to the compound. There was no reduction in the agressiveness of the resistant isolate. Using other anti-oomycete fungicides, it appeared that propamocarb, contact fungicides (fluazinam, folpet, mancozeb) and the mixed formulations dimethomorph + mancozeb, cymoxanil + mancozeb and ofurace + folpet were effective against primary infections made with metalaxyl resistant and sensitive isolates, but not against secondary infections. Metalaxyl mixed with fluazinam, folpet or mancozeb was more effective against primary infections with the resistant isolate than metalaxyl alone. The EC50 of five other isolates ranged from 5 800 to 32 900 mg a.i. kg-1, indicating a variability in metalaxyl sensitivity of resistant sunflower downy mildew isolates. This is the first report of physiological resistance to metalaxyl in Plasmopara halstedii.

Molecular Variability Within Diaporthe/Phomopsis helianthi from France.

ABSTRACT Diaporthe/Phomopsis helianthi causes brown stem canker of sunflower (Helianthus annuus) and is responsible for considerable yield loss. This species shows considerable variation for morphological characters, growth, and pathogenicity. Molecular variability of two sample groups was assessed with amplified fragment length polymorphism (AFLP) markers. Isolates of the first sample were collected from infected sunflower tissues from the main regions in France where the crop is grown, whereas isolates from the second sample came from stems within a single field of sunflower. A soybean strain was taken as an outgroup for AFLP analyses. Within sample one, the greatest genetic distance among isolates was 0.97, whereas it was 0.44 within sample two isolates. For the whole of France, the average genetic distance was 0.68, whereas in the one field it was 0.12. Neis genetic diversity indices were 0.20 and 0.06 for France and for one field, respectively. The greatest genetic distance was found between isolates from the most northern crops. The greatest genetic distance between D. helianthi isolates and the strain isolated from soybean was similar to that observed for D. helianthi isolates from different geographical areas. The problems in defining the genus Phomopsis are discussed. It is shown that internal transcribed spacer sequencing could be a useful criteria for Diaporthe/Phomopsis species determination. The considerable genetic variability of the pathogen could lead to the occurrence of new strains that could be more aggressive or more resistant to chemical control.

Variation in form and size of Plasmopara halstedii (sunflower downy mildew) zoosporangia

Zoosporangia form and size were studied on a collection of 94 strains of Plasmopara halstedii (sunflower downy mildew). Both oval and round forms were present in all strains analysed. The proportion of two forms varied significantly according to strain and plant age but more especially to host plant genotype. Whatever the strain or host genotype, oval zoosporangia were larger than round ones, but there was no relation between the proportion of the oval form and mean zoosporangia size. There was no relation between zoosporangia form or size and race virulence profiles or aggressiveness criteria, with the possible exception of zoosporangia size and sporulation density. It is concluded that, for this obligate parasite, although form and size of zoosporangia depend on pathogen strain, these characters also vary according to growth conditions of Plasmopara halstedii, in particular to the genotype of the plant host.

Development of a polymerase chain reaction diagnostic test for the detection of the biotrophic pathogen Plasmopara halstedii in sunflower

The obligate parasitic fungus-like organism Plasmopara halstedii (Farl.) Berl. et De Toni, is the causal agent of downy mildew disease in sunflower (Helianthus annuus). New races of this economically important parasite are regularly detected throughout the world. In addition, fungicide-resistant isolates have been reported in Europe and North America. These observations of parasite evolution, as well as the risk of propagation of the disease by infected seeds, means that it is necessary to guarantee the absence of Plasmopara halstedii in seed shipments. We report here the development of a rapid assay that can be used to detect infection by Plasmopara halstedii in plant tissues. Based on the nucleotide sequence information obtained from one cloned random amplified polymorphic DNA fragment, specific oligonucleotides were designed and used as primers for in vitro DNA amplification by polymerase chain reaction. An amplification product was detected on agarose gel stained with ethidium bromide when DNA from various Plasmopara halstedii races was tested, whereas no amplified DNA was detected when DNA from other origins was tested, including DNA from the host plant. The sensitivity of the technique was evaluated. The assay successfully reveals the presence of Plasmopara halstedii in infected sunflower plants prior to sporulation.