Phylogenetic Analysis of Downy Mildew Pathogens of Opium Poppy and PCR-Based In Planta and Seed Detection of Peronospora arborescens

Abstract

Severe downy mildew diseases of opium poppy (Papaver somniferum) can be caused by Peronospora arborescens and P. cristata, but differentiating between the two pathogens is difficult because they share morphological features and a similar host range. In Spain, where severe epidemics of downy mildew of opium poppy have occurred recently, the pathogen was identified as P. arborescens on the basis of morphological traits. In this current study, sequence homology and phylogenetic analyses of the internal transcribed spacer regions (ITS) of the ribosomal DNA (rDNA) were carried out with DNA from P. arborescens and P. cristata from diverse geographic origins, which suggested that only P. arborescens occurs in cultivated Papaver somniferum in Spain. Moreover, analyses of the rDNA ITS region from 27 samples of downy-mildew-affected tissues from all opium-poppy-growing regions in Spain showed that genetic diversity exists within P. arborescens populations in Spain and that these are phylogenetically distinct from P. cristata. P. cristata instead shares a more recent, common ancestor with a range of Peronospora species that includes those found on host plants that are not members of the Papaveraceae. Species-specific primers and a PCR assay protocol were developed that differentiated P. arborescens and P. cristata and proved useful for the detection of P. arborescens in symptomatic and asymptomatic opium poppy plant parts. Use of these primers demonstrated that P. arborescens can be transmitted in seeds and that commercial seed stocks collected from crops with high incidence of the disease were frequently infected. Field experiments conducted in microplots free from P. arborescens using seed stocks harvested from infected capsules further demonstrated that transmission from seedborne P. arborescens to opium poppy plants can occur. Therefore, the specific-PCR detection protocol developed in this study can be of use for epidemiological studies and diagnosing the pathogen in commercial seed stocks; thus facilitating the sanitary control of the disease and avoidance of the pathogen distribution in seeds.