3.3.44
MOLECULAR PHYLOGENY OF THE PERONOSPORALES AND PYTHIALES BASED ON REGIONS OF rDNA

DEL COOKE, NA WILLIAMS and JM DUNCAN

Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK

Background and objectives
The Peronosporaceae are a diverse family (consisting of seven genera) of obligate biotrophic plant pathogens, collectively known as the downy mildews. Due to the paucity of morphological features and their biotrophic nature, distinguishing between species within a genus has proved difficult and, inevitably, has been based on host association. Such a classification has resulted in inconsistent, and often inadequate, species definitions and it is widely accepted that a more rigorous analysis of the downy mildews and related genera is required [1]. A phylogenetic analysis of RDNA sequence data would allow such a re-examination, but molecular analysis of these fungi is often hampered by the intimate association of host and pathogen. Sequence analysis of the Internal Transcribed Spacer (ITS) regions of ribosomal DNA has proved a useful for interspecific comparisons and phylogenetic analysis of Phytophthora species [2]. The objectives of this work were to a) develop PCR primers which facilitate the molecular characterisation of downy mildew species even in the presence of plant material b) assess intra and interspecific relationships within the Peronosporales and c) re-examine the current classification of the Peronosporales and Pythiales.

Materials and methods
Sequences of the 18S RDNA gene from a range of organisms (oomycetes, true fungi, plants and bacteria) were extracted from the EMBL database, aligned and compared. A PCR primer (DC6) was designed to allow amplification of Phytophthora species even in the presence of plant DNA. The specificity of this primer was tested against a range of oomycetes before being utilised for specific amplification of the ITS regions of 17 isolates (11 species) of Peronospora and 2 Albugo species from infected plant material. After direct sequencing and alignment with published sequences of Pythium and Phytophthora species (plus our own unpublished sequences of over 40 Phytophthora species) phylogenetic analysis was performed using the PHYLIP computer package.

Results and conclusions
In a screen of a number of species in the phylum oomycota, the primer DC6 only amplified members of the Peronosporales and Pythiales. Re-amplification and direct sequencing of the strong 1.2Kb product confirmed it contained the ITS regions. Conventional DNA extraction from lesions and DNA released from freeze-thawed sporangia provided suitable templates for PCR. Sequences of Peronospora and Phytophthora species were broadly similar and could be accurately aligned whereas those of Pythium, Achlya and Albugo proved more difficult to align. Within the Peronospora genus, P. sparsa (from Rosa spp.) and P. rubi (from Rubus spp.) sequences were identical which, alongside evidence of cross infection suggests they may be conspecific. Contrary to this, ITS diversity amongst isolates of P. parasitica from different Brassica hosts was suggestive of reproductive isolation. Phylogenetic analysis indicated that the 11 species of Peronospora examined were monophyletic and this single clade formed a part of the tree containing papillate species of Phytophthora. The genetic distances between Peronospora species and the nearest Phytophthora species were less than those between some non-papillate and papillate Phytophthora species (e.g. P. cinnamomi and P. infestans). The two blister rusts, Albugo spp., were not closely related but formed an outgroup along with Achlya and Pythium. There are clearly many more species from other genera yet to be examined but this study is the first to suggest that the genus Peronospora consists of a group of biotrophic Phytophthora-like species and a re-examination of the taxonomy of these groups will need to be considered.

References
1. Hall GS, 1996. Plant Pathology 45, 1009-1026.
2. Cooke DEL, Duncan JM, 1997. Mycological Research 101, 667-677.