Special Interest Group Meeting

Rapid detection and differentiation of forest pathogenic fungi associated with mountain pine beetles by padlock probes and rolling circle amplification

Clement Tsui¹, Bin Wang², Lily Khadempour³, Brent Murray⁴, Jörg Bohlmann⁵, Richard Hamelin¹, ¹Department of Forest Science, University of British Columbia, Vancouver, Canada, ²Centre for Virus Research, Westmead Millennium Institute, The University of Sydney, Australia, ³Department of Wood Science, The University of British Columbia, Vancouver, Canada, ⁴Ecosystem Science and Management Program, University of Northern British Columbia, Prince George, Canada, ⁵Michael Smith Laboratory, The University of British Columbia, Vancouver, Canada

Fifteen million ha of pine forests in Canada have been attacked by the mountain pine beetle (MPB), leading to economic losses. Grosmannia clavigera and Leptographium longiclavatum, are fungi associated with MPB and important components of the epidemics. To discriminate these related pathogens, we utilized a method based on ligase-mediated nucleotide discrimination with padlock probe technology, and signal amplification by hyperbranched rolling circle amplification (HRCA). Padlock probes were designed to target species-specific single nucleotide polymorphisms (SNPs), located at the rRNA, that allow discrimination between the species. Multiple strains of G. clavigera and L. longiclavatum were tested with this assay. The HRCA results were largely in agreement with the identification based on morphology or molecular methods. Both probes also efficiently distinguished the two fungi from other related fungi in the Ophiostomatales. We tested this diagnostic method for the direct detection from the DNA of MPB. A nested PCR approach was used to enrich amplicons for signal detection. The results confirmed the presence of these two fungi in MPB. Thus, the padlock probe assay coupled with HRCA is an accurate and reproducible method for rapid identification and detection of these Ophiostomatoid fungi.