New Taxon-Specific Heterobasidion PCR Primers Detect and Differentiate North American Heterobasidion spp. in Various Substrates and Led to the Discovery of Heterobasidion irregulare in British Columbia, Canada

Abstract

Heterobasidion annosum sensu lato is a species complex of pathogenic white-rot wood decay fungi which cause root and butt rot in conifer and hardwood species across the Northern hemisphere. Annual losses to forest managers are valued in the billions of dollars, due to tree mortality, reduction in timber yield, and wood decay. In North America, H. irregulare and H. occidentale have a partially overlapping host and geographic range, cause similar disease symptoms and produce similar fruiting bodies, making discrimination between the two of them often difficult. We developed two sets of primers that bind specifically to conserved, but species-specific portions of glyceraldehyde 3-phosphate dehydrogenase and elongation factor 1α alleles. The method is sensitive enough to detect either species from infected wood. Analysis of North American isolates has further clarified the distribution of both species on this continent, including the detection of H. irregulare for the first time on ponderosa pine (Pinus ponderosa) and eastern white pine (Pinus strobus) in British Columbia. This method has the potential to be a valuable tool for the detection of the pathogen in exported/imported wood products, as well as for the further identification and assessment of the distribution of North American Heterobasidion species.

Rapid detection of Heterobasidion annosum using a loop-mediated isothermal amplification assay

“…In general, most PCR amplifications are carried out with a DNA concentration of 20 ng/mL. Conventional PCR amplifications used to detect Heterobasidion species are carried out with a DNA concentration of 20 pg/mL (Shamoun et al, 2019). However, the LAMP assay tested in our study was found to detect H. annosum with a DNA concentration of 100 pg/mL.…”

“…Several fragments of genes have been described for detection of species of Heterobasidion (Fabritius and Karjalainen, 1993;Johannesson and Stenlid, 2003;Linzer et al, 2008;Chen et al, 2014, Chen, 2015Shamoun et al, 2019;Pellicciaro et al, 2021;Yuan et al, 2021). Although the use of PCR techniques is more successful as a method of detection, it still requires specialized equipment and highly trained personnel, and it is difficult and time-consuming to implement the technique in remote areas and ports.…”

Diversity and molecular diagnostics of fungi and oomycetes in plants

“…Several fragments of genes have been described for detection of species of Heterobasidion (Fabritius and Karjalainen, 1993;Johannesson and Stenlid, 2003;Linzer et al, 2008;Chen et al, 2014, Chen, 2015Shamoun et al, 2019;Pellicciaro et al, 2021;. Although the use of PCR techniques is more successful as a method of detection, it still requires specialized equipment and highly trained personnel, and it is difficult and time-consuming to implement the technique in remote areas and ports.…”

Efficacy of Chemical and Biological Stump Treatments for the Control of Heterobasidion occidentale Infection of California Abies concolor

“…Colonies were marked and counted, and three colonies per disc were sub-cultured on 30% malt extract agar (MEA) plates using a sterile scalpel. We used DNA that was collected from each colony as an identification method for Heterobasidion species via a PCR analysis which employed Heterobasidion-specific primers and an established laboratory protocol [44].…”

“…The area of the disc that was covered by these colonies was delineated with a marker at each examination time. A sample of each colony per wood disc was obtained with a sterilized surgical needle to confirm the presence of H. occidentale establishment via direct culturing followed by PCR analysis performed on DNA that was extracted directly from fungal colonies growing on 30% MEA [44]. On the final examination (day 12), each wood disc with its delineated and confirmed colonies was photographed with a 30 cm long ruler as a reference.…”