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.) Be rl. et De Toni, is the causal agent of downy mildew disease in sunflower (H elianthus annuus). New races of this economically important parasite are re gularly detected throughout the world. In addition, fungicide-resistant iso lates have been reported in Europe and North America. These observations of parasite evolution, as well as the risk of propagation of the disease by i nfected seeds, means that it is necessary to guarantee the absence of Plasm opara halstedii in seed shipments. We report here the development of a rapi d assay that can be used to detect infection by Plasmopara halstedii in pla nt 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 polyme rase chain reaction. An amplification product was detected on agarose gel s tained with ethidium bromide when DNA from various Plasmopara halstedii rac es was tested, whereas no amplified DNA was detected when DNA from other or igins was tested, including DNA from the host plant. The sensitivity of the technique was evaluated. The assay successfully reveals the presence of Pl asmopara halstedii in infected sunflower plants prior to sporulation.