USE OF POLYMERASE CHAIN REACTION-AMPLIFIED RIBOSOMAL INTERGENIC SEQUENCES FOR THE DIAGNOSIS OF VERTICILLIUM-TRICORPUS

Verticillium dahliae, V. albo-atrum, and V. tricorpus are common pathogens of potato. Currently, polymerase chain reaction (PCR) assays based on sequence differences in their ribosomal RNA genes are available for the specific diagnosis of the first two species. Using the same principles, we developed an analogous assay to detect V. tricorpus. The 18-28S rDNA intragenic region of V. tricorpus was obtained by PCR amplification of genomic DNA from the fungus. Sequence analyses indicated that the 5.8S rDNA sequences were conserved among all three Verticillium species but the internal transcribed spacer regions of V. tricorpus clearly were divergent. These sequence differences were used to synthesize a specific primer set for the diagnosis of V. tricorpus. The same primers (VT primers) also were used to prepare a heterologous internal control DNA template, which can be added to assays to standardize the quantification of fungal biomass. VT primers amplified a specific 337-bp fragment from DNA extracted from V. tricorpus cultures isolated from various host species, including potato, or from infected potato stems, but no amplification occurred with DNA from V. dahliae, V. albo-atrum, or from uninfected potato stems. The addition of V. tricorpus internal control DNA template allowed the quantification of the pathogen in diseased field plants. The development of an assay that is specific for V. tricorpus completes the diagnostic set necessary for the investigation and monitoring of the Verticillium-potato pathosystem.