PCR Amplification of Ribosomal DNA for Species Identification in the Plant Pathogen Genus Phytophthora

Phytophthora species are responsible for economically important diseases of a wide range of agronomic and ornamental crops. Species identification for Phytophthora has traditionally been based upon microscopic examination of morphological characters and growth characteristics of the pathogen on specific media (27, 35). Variations in the morphological characters of both the sexual and asexual stages of this group of pathogens exist, leading to difficulties in accurate identification by traditional methods. In addition, identification based on pathogenicity assays or growth characteristics are time-consuming. Accurate and rapid identification of Phytophthora species in plant material is important for several reasons. First, in many hosts, such as citrus, walnut, strawberry, raspberry, potato, and tomato, multiple species of Phytophthora can infect the plant, and the relative severity of disease and the plant part infected can vary among pathogen species (6, 25, 37, 43). Preplant identification of Phytophthora species can be important for quarantine purposes and is important for restricting the spread of pathogens in plant material (23). In addition, accurate diagnosis of the species of Phytophthora is important in disease management and control. Molecular tools including isozyme analysis, restriction fragment length polymorphisms in nuclear and mitochondrial DNA, randomly amplified polymorphic DNA PCRs, serological assays, DNA probes, and PCR of internal transcribed spacer (ITS) regions and nuclear small- and large-subunit ribosomal DNA (rDNA) have been used to evaluate intraspecific and interspecific variation in Phytophthora species (1, 5, 8, 11, 13, 14, 22, 28). Molecular techniques have also been used to study genetic diversity and evolutionary origins in populations of many different fungal genera (2). Nucleotide sequences of rRNA genes have been used in studies of phylogenetic relationships over a wide range of taxonomic levels with many organisms (2, 9, 31, 41). The nuclear small-subunit rDNA sequences evolve relatively slowly and are useful for studying distantly related organisms, whereas the ITS regions and intergenic region of the nuclear rRNA repeat units evolve the fastest and may vary among species and populations (41). Mitochondrial rRNA genes also evolve rapidly and can be useful at the ordinal or family level (41). The evolutionary lineage of the oomycetes has been elucidated by sequencing studies with small-subunit rRNA sequences (9). We have adopted a quick extraction procedure for DNA and a reliable PCR technique for amplification of DNA from Phytophthora species. This method is based on procedures developed by Lee and Taylor (21) and Lee et al. (22) for Phytophthora species and involves amplification of the ITS and 5.8S rDNA. We used ITS primers 5 and 4 and PCR to amplify the entire 5.8S rDNA gene, both ITS regions I and II, and a portion of the 18S nuclear small-subunit rDNA gene. The amplified DNA was then cut with a series of restriction enzymes to develop species-specific restriction fragment patterns for rapid identification of the important plant-pathogenic Phytophthora species from all the different taxonomic groups in the genus that infect economically important hosts (35). In addition, we devised a PCR primer to specifically amplify P. capsici, an important pathogen of pepper, and used this primer (PCAP) to compare PCR to traditional isolation methods for identification of the pathogen in infected pepper tissue from the field.