An Economic and Reliable PCR Approach to Distinguish the Primary Pathogens Armillaria mellea and A. ostoyae From Other European Armillaria Species

Armillaria is a soil-borne genus of basidiomycetes whose species can cause stem and root rot in woody plants. The effects of plant-pathogenic Armillaria species are well known in forests, but are underestimated in urban areas, where cases causing damage to trees and shrubs in green spaces have been steadily increasing in Switzerland since the 1980s. In this study, we present a simple, rapid, and cost-effective protocol for high-throughput diagnostics of the two primary pathogens A. mellea and A. ostoyae based on partial PCR amplification of the RPB2 gene. The specificity and sensitivity of the presented duplex PCR-I and single-plex PCR-II were evaluated using different methods: (i) testing both PCRs on tree pathogenic or soil-borne fungi of genera other than Armillaria, (ii) using dilution series of Armillaria-DNA to determine a minimum detection limit, and (iii) sequencing the selected RPB2 region to verify the primer sequences and positions. The utility of PCR-I and PCR-II as a high-throughput method was successfully tested on 65 DNA samples of Armillaria from Switzerland. Finally, an uninvolved person compared both classical methods, pairing test and sequencing, with PCR-I and PCR-II in a blind test. This study provides a reliable and alternative protocol for the rapid diagnosis of A. mellea and A. ostoyae causing root rot of woody plants.