3.3.26
IDENTIFICATION AND DETECTION OF PYTHIUM SPECIES BY REVERSE DOT BLOT HYBRIDIZATION

AWAM DE COCK1, A QUAIL2, R DESCALZO3, C MAYES2 and CA LEVESQUE2

1 Centraalbureau voor Schimmelcultures, Yeast Division, Julianalaan 67, 2628 BC Delft, Netherlands; 2 Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, Summerland, B. C., Canada, V0H 1Z0; 3 IG Micromed Environmental Inc., 190-12860 Clarke Place, Richmond, B.C., Canada, V6V 2H1

Background and objectives
Species identification in Pythium is often difficult due to variable and overlapping characters. A relatively novel technique called reverse dot blot hybridization has been successfully utilized for the detection in a single assay of a range of single point mutations related to human genetic disorders. Utility of this method to identify some Pythium species was recently reported [1]. The objectives of this study were to show the feasibility of this technique for the identification of a wide range of pathogenic Pythium species and to demonstrate that the technique can detect these species directly from infected roots.

Materials and methods
Ribosomal ITS regions of all available (neo-)type isolates of Pythium species were sequenced. Sequences were aligned using GCG 7.0; and the software Oligo 5.0 was used to design oligonucleotides for more than 30 important plant pathogenic species. Oligonucleotides were synthesized, coupled with a reactive amine group and covalently bound to nylon membranes. DNA was extracted from pure cultures and from infected roots of turfgrass. The DNA was then simultaneously amplified and labelled [2] using a universal primer located at the 3' end of the small ribosomal subunit and an Oomycete specific primer located at 350 bases from the 5' end of the large ribosomal subunit. Hybridization and detection were performed as previously described [1].

Results and conclusions
The reverse dot blot technique was used to successfully identify several Pythium species and was able to differentiate closely related species such as P. aphanidermatum and P. deliense. Cross-hybridization reactions were encountered but after designing a series of oligonucleotides for the problematic species, an oligonucleotide array was developed with minimal and weak cross hybridization reactions with pure cultures of all available (neo-)type isolates. The technique was then expanded to detect Pythium species directly from infected turfgrass roots. Turfgrass roots often contained more than one Pythium species. In most cases(~90%), direct processing of root tissue detected the same species that was isolated from segments of roots plated on culture medium. However, traditional plating methods detected only approx. one quarter of the species that were detected by reverse dot blot hybrization using amplified root DNA. Some species probably grow better than others on agar and surface sterilization required for culturing may kill several species still present after thorough washing before DNA isolations. Root sampling for DNA extraction may also have given a more representative sample of the Pythium species present than fragments selected for plating. The reverse dot blot technique is not only faster than traditional methods for Pythium species identification, but it eliminates problems with detection of species in a mixed population and/or species that are difficult to culture.

References
1. Levesque CA, Harlton CE, de Cock AWAM, 1998. Phytopathology 88, (in press).
2. Kawasaki ES, Saiki R, Erlich H, 1993. Methods in Enzymology 218, 369-381.