The genus Phytophthora, one of the most devastating plant pathogens, currently consists of more than 50 recognized species, which can be divided into six main groups based on morphological features of the sexual structures and sporangia. Other criteria widely used to distinguish species are cardinal growth temperature and mating behavior. Identification of Phytophthora species based on morphology is difficult due to intraspecific variation and overlap of morphological characters between species. Specialists capable of identifying Phytophthora to the species level are less common and available. Since the last decade a number of biochemical techniques have been developed to aid in identification: Isozyme-, RAPD- and mtDNA analysis. For Phytophthora, internal transcribed spacer (ITS) sequence analysis of rDNA has been shown to be very useful for species identification. Although some species have exactly the same ITS sequence (e.g. P. infestans and P. mirabilis), most species have a unique sequence. In this way new species have been identified: e.g. P. multivesiculata, P. ipomoeae, P. pistaciae, P. brassicae, P. psychrophila, P. europaea, P. uliginosa and P. ramorum. These sequence differences can be used to develop very sensitive diagnostic methods using PCR. In this way we developed several tools for diagnosis and detection of P. fragariae.
Another important technology is AFLP (amplified fragment length polymorphism) in which a DNA fingerprint is generated to study genetic differences within a species. Although designed for construction of genetic maps, we used AFLP to study more than 400 isolates comprising many Phytophthora species. Clear differences between species of Phytophthora have been observed. In this way an AFLP database has been constructed.
Also gene sequencing can be used for identification of species. Sequence analysis of house-keeping genes with structural and metabolic functions was used for phylogenetic analysis, and unknown isolates could be placed in the right species, based on sequence homology.
Since the discovery of Phytophthora ramorum on Rhododendron in the Netherlands and on oak in the USA, several of the techniques mentioned above have been used for identification and detection of this new species of Phytophthora. Minor differences between USA* and EU# isolates of P. ramorum exist and can be shown with the techniques at hand. Isozyme profiles, gene sequences and minor variation in AFLP fingerprint patterns support that USA and EU populations belong to the same species (P. ramorum), despite the difference in mating type (A2 in the USA, A1 in Europe) and unsuccessful attempts to cross European and American isolates.
*USA isolates used in this study were obtained from Dave Rizzo, Matteo Garbelotto and Kelly Ivors (University of California, USA).
#EU isolates from Germany used in this study were obtained from Sabine Werres (BBA, Braunschweig, Germany).
Their collaboration is greatly appreciated.
