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Changes in virulence of Plasmopara halstedii populations under different major gene (Pl) management strategies were studied over 5 years continuous cropping of one sunflower hybrid under netting cages. Strategies were monoculture of forms of the hybrid with 1 gene or with combinations of 2 genes, alternation of different genes, and mixtures of several different forms of the hybrid. Monoculture with single resistance genes led to loss of efficient resistance after 3 years, with high levels of disease and increased variability of the pathogen, whatever the Pl gene used. Combinations of genes, alternation and mixtures gave longer term control of downy mildew. In particular, combinations of resistance genes coming from both female and male parents of the hybrid (such that even impurities had a resistance gene) gave the best control and least variation in pathogen virulence. Results are discussed with the object of durable control of downy mildew by all methods available.

Plasmopara halstedii, the causal agent of downy mildew of sunflower, is an obligate parasite but viable sporangia and oospores of the pathogen may be found in a quiescent state in seeds of sunflower and therefore may be transported with sunflower seeds in international commercial exchanges. In order to prevent the spread of this pathogen, especially the introduction of potentially new races, an efficient method to analyse sunflower seed samples is required. In this study, a P. halstedii-specific polymerase chain reaction (PCR) test was developed based on the ribosomal large sub unit (LSU) DNA. The forward (PHAL-F) and reverse (PHAL-R) PCR primers were designed from two polymorphic regions of LSU. After screening 22 isolates of P. halstedii corresponding to different races and countries and 32 other oomycete, deuteromycete and ascomycete isolates, the PHAL-F/R primers amplified only a single PCR band of c. 310 bp from P. halstedii. The PHAL-F/R PCR test could detect as little as 3 pg of P. halstedii genomic DNA per 20 mu L reaction volume and enabled the direct detection of P. halstedii in 35 g sunflower seed samples without the need for any prior biological baiting step. An internal amplification control (IAC) was developed to help discriminate against false negative samples due to the potential presence of inhibitory compounds in DNA extracts. The test was successfully used on samples of naturally contaminated seeds. These new molecular tools should be of great interest for quarantine seed testing purposes

Downy mildew of sunflower (Helianthus annuus L.), caused by the pathogen Plasmopara halstedii, is a potentially devastating disease. Seventy-seven isolates of P. halstedii collected in twelve countries from four continents were investigated for RAPD polymorphism with 21 primers. The study led to a binary matrix, which was subjected to various complementary analyses. This is the first report on the international genetic diversity of the pathogen. Similarity indices ranged from 89% to 100%. Neither a consensus unweighted pair group method with arithmetic means (UPGMA) tree constructed after bootstrap resampling of markers nor a principal component analysis based on distance matrix revealed very consistent clusterings of the isolates, and groups did not fit race or geographical origins. Phylogenies were probably obscured by limited diversity. Analysis of molecular variance (AMOVA) and Nei's genetic diversity statistics gave similar conclusions. Most of the genetic diversity was attributable to individual differences. The most differentiated races also had the lowest within-diversity indices, which suggest that they appeared recently with strong bottleneck effects. Our analyses suggest that this pathogen is probably homothallic or has an asexual mode of reproduction and that gene flow among countries can occur through commercial exchanges. Knowledge of the downy mildew populations' structure at the international level will help to devise strategies for controlling this potentially devastating disease.

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