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

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.


Introduction
Heterobasidion annosum (Fr.) Bref. sensu lato (s.l.) is a significant forest pathogen that causes root and butt rot primarily in conifers across the Northern Hemisphere. The tree disease caused by Heterobasidion spp. is referred to as Annosus root and butt rot, and the mortality it causes in old growth forests is regarded as an important driver of forest turnover and natural biodiversity. However, damages to managed forests and plantation forestry include wood quality loss, timber yield reduction and tree mortality, and have been valued up to 790 million annually in the EU alone [1], making it

Results
Alignments of EFA and GPD DNA sequences of 18 isolates of H. irregulare and 10 isolates of H. occidentale were analyzed for the presence of interspecific polymorphisms. Variable blocks between the two species were identified for both genes and are shown, together with primer locations and sequences, in Figure 1. The two differentiation primer sets were tested on several North American pure cultures (Table 1), herbarium samples and on infected wood samples (Figures 2 and 3). The Irr-1 For and Irr-1 Rev primer set was highly specific and only amplified a 165 base pair (bp) amplicon from H. irregulare. DNA from all H. irregulare isolates/samples tested amplified successfully. The Occ-0 For and Occ-0 Rev primer set produced a 365 bp amplicon for the H. occidentale samples tested, and never amplified H. irregulare samples. There were no instances in which both amplicons were present, nor were amplicons of unexpected length ever produced. The primers were tested with a serial dilution of template DNA to determine the limit of detection of the primers. Using conventional PCR, the limit of detection was 20 pg/µL. In general, most PCR amplifications were carried with a DNA concentration of 20 ng/µL.         The primer sets were tested against other species, including the Eurasian species: H. annosum, H. abietinum, and H. parviporum ( Figure 4, Table 2). The Irr-1 For and Irr-1 Rev primers did amplify a 165 bp band when tested on H. annosum isolates, and the Occ-0 For and Occ-0 Rev primers amplified the expected 365 bp amplicon from H. parviporum and H. abietinum isolates, although when amplicons were visualized through agarose gel electrophoresis, the bands from H. abietinum isolates were significantly "weaker". Heterobasidion ecrustosum and H. orientale of the H. insulare complex collected in Japan, as well as H. araucariae from New Zealand were tested and failed to produce a PCR product ( Figure 4, Table 2). The primer sets were tested against other species, including the Eurasian species: H. annosum, H. abietinum, and H. parviporum ( Figure 4, Table 2). The Irr-1 For and Irr-1 Rev primers did amplify a 165 bp band when tested on H. annosum isolates, and the Occ-0 For and Occ-0 Rev primers amplified the expected 365 bp amplicon from H. parviporum and H. abietinum isolates, although when amplicons were visualized through agarose gel electrophoresis, the bands from H. abietinum isolates were significantly "weaker". Heterobasidion ecrustosum and H. orientale of the H. insulare complex collected in Japan, as well as H. araucariae from New Zealand were tested and failed to produce a PCR product ( Figure 4, Table 2).
In addition to DNA extracted from mycelia of pure Heterobasidion cultures, the diagnostic primers were successful in amplifying DNA from basidiocarp herbarium samples that were collected in British Columbia, Canada, more than 20 years ago (Table 1  In addition to DNA extracted from mycelia of pure Heterobasidion cultures, the diagnostic primers were successful in amplifying DNA from basidiocarp herbarium samples that were collected in British Columbia, Canada, more than 20 years ago (Table 1,  The specificity of the primers was tested against other forest rot species commonly found in North America. The root rot fungi Armillaria ostoyae, the tomentosus root rot fungus (Onnia (Inonotus) tomentosa), and the laminated root rot fungus (Phellinus weirii) were not amplified by either set of primers ( Figure 4). The red ring rot fungus (Porodaedalae pini), the white mottled rot fungus (Ganoderma applanatum), Trametes versicolor, as well as the polypore brown crumbly rot fungus (Fomitopsis pinicola) were not amplified by either primer set. All these root rot fungi were collected from coastal British Columbia forests, except for Onnia tomentosa was collected from northern British Columbia forests near the Prince George region. Thus, there was no cross-reactivity with any of the non-Heterobasidion species tested (Figure 4), however DNA was available and amplifiable from all samples tested as demonstrated by the successful amplification of the internal transcribed spacer  (ITS) of all control species using primers ITS-1Fand ITS4 (Table 3) and PCR conditions described by [24,25]. Validation of the primers using an additional internal universal plant primer PUC-UPC7 For and PUC-UPC7 Rev (Table 3) confirmed that DNA of both fungi and plant were amplifiable and showed no cross-amplification when done in single, duplex or triplex PCR (Figures 5-7). Finally, when combining red alder or red pine wood with DNA of the two Heterobasidion species in the same sample, the three expected specific amplicons were amplified by running in multiplex the three primer sets Irr-1, Occ-0 and PC-UPC7 (Figure 8).      The specificity of the primers was tested against other forest rot species commonly found in North America. The root rot fungi Armillaria ostoyae, the tomentosus root rot fungus (Onnia (Inonotus) tomentosa), and the laminated root rot fungus (Phellinus weirii) were not amplified by either set of primers ( Figure 4). The red ring rot fungus (Porodaedalae pini), the white mottled rot fungus (Ganoderma applanatum), Trametes versicolor, as well as the polypore brown crumbly rot fungus (Fomitopsis pinicola) were not amplified by either primer set. All these root rot fungi were collected from coastal British Columbia forests, except for Onnia tomentosa was collected from northern British Columbia forests near the Prince George region. Thus, there was no cross-reactivity with any of the non-Heterobasidion species tested (Figure 4), however DNA was available and amplifiable from all samples tested as demonstrated by the successful amplification of the internal transcribed spacer (ITS) of all control species using primers ITS-1Fand ITS4 (Table 3) and PCR conditions described by [24,25]. Validation of the primers using an additional internal universal plant primer PUC-UPC7 For and PUC-UPC7 Rev (Table 3) confirmed that DNA of both fungi and plant were amplifiable and showed no cross-amplification when done in single, duplex or triplex PCR (Figures 5-7). Finally, when combining red alder or red pine wood with DNA of the two Heterobasidion species in the same sample, the three expected specific amplicons were amplified by running in multiplex the three primer sets Irr-1, Occ-0 and PC-UPC7 (Figure 8).

Discussion
The identification of North American Heterobasidion to the species level using the simple PCR protocol described in this paper has allowed us to diagnose both North American species directly from infected woody tissue, saving time and costs compared to culture-based methods. When performed in duplex or triplex, these two species-specific primers do not cross-amplify plant DNA and can be reliably used on environmental samples. Further, the small product size also allowed for the testing of preserved herbarium collections, where DNA integrity is low and conventional PCR

Discussion
The identification of North American Heterobasidion to the species level using the simple PCR protocol described in this paper has allowed us to diagnose both North American species directly from infected woody tissue, saving time and costs compared to culture-based methods. When performed in duplex or triplex, these two species-specific primers do not cross-amplify plant DNA and can be reliably used on environmental samples. Further, the small product size also allowed for the testing of preserved herbarium collections, where DNA integrity is low and conventional PCR methods are troublesome.
The two primer sets differentiate the two North American species, but they do cross-react with all three Eurasian species of the Heterobasidion annosum complex, thus, unfortunately, these primers would not be able to differentiate H. annosum s.s. from H. irregulare, nor H. parviporum from H. abietinum in those parts of Europe where multiple species exist.
Using this novel method, we have identified H. irregulare for the first time on ponderosa pine and eastern white pine in the Okanagan Valley of British Columbia. The first detection of H. irregulare on British Columbia's pine species is specifically significant as H. irregulare is considered the most aggressive pathogen in the entire species complex [10]. It is unknown whether: (1) BC isolates may represent a historical Northern boundary of the Western US population, and may have existed there a significant time period without our knowledge; (2) BC isolates represent may be a recent natural Northward expansion of the Western US population, maybe associated with climate change; or (3) BC isolates may be the result of human transport, likely via the planting of infected saplings, and may, thus, represent a geographically and genetically disjunct population from the Western US one.
Although we acknowledge that the significance and rate of occurrence of H. irregulare on ponderosa pine and eastern white pine is unknown and needs further investigation, forest management in this area may have to adjust to the presence of this pathogen as done in the USA and Eastern Canada. Additionally, pine species are important export species for Canada. The potential presence of H. irregulare in British Columbia pines is of high phytosanitary concern now that H. irregulare is on the list of regulated organisms by the European and Mediterranean Plan Protection Organization (EPPO) A2 list of pests recommended for regulation as quarantine pests in September 2015 (http: //www.eppo.int/QUARANTINE/Pest_Risk_Analysis/PRA_intro.htm).
The primers and PCR assays described in this study could be used by the Canadian Food Inspection Agency (CFIA) to certify timber and plants as Heterobasidion-free, thus, facilitating Canadian export of pines, as EPPO national members will start, including H. irregulare, as a fully regulated pathogen in their trade policies. This will also assist the provincial forestry agencies to take suitable control measures to prevent threats to timber production and sustainability of forest production. Furthermore, the assay here described is a cost-effective, fast, and reliable method for detecting and differentiating the two North American Heterobasidion species. Thanks to this assay, it was also possible to identify H. irregulare for the first time in British Columbia, Canada. There are multiple advantages provided by this new diagnostic method: (1) Current distribution of Heterobasidion in North America can be mapped to the species level; (2) wood for export can be tested specifically for the presence of Heterobasidion; and (3) species-level diagnosis of Heterobasidion may help formulate better disease management strategies.

Study Sites and Isolates
A representative selection of North American Heterobasidion isolates in pure culture was used in this study (Table 1). Isolates were chosen to best represent diverse regions and host species within Canada and the United States. Herbarium samples and infected wood samples from British Columbia (Canada) were also included in the study. DNA from herbarium specimens was obtained by excising 50-100 mg from dry basidiocarps with a sterile blade and carefully excluding tissue from the outside surface of the sample to minimize contamination. Samples of wood infected by H. occidentale were collected from wind-thrown or from standing western hemlocks in Mt. Doug and Sandcut Beach municipal forests (Victoria, British Columbia). A hatchet was used to remove the bark, and diseased wood was collected for DNA extraction. Roots were drilled, and the drill shavings were used for DNA extraction. Samples of H. irregulare infected wood were collected from two trees a red pine plantation in Lacrosse County, Wisconsin as follows. Infected wood was collected by drilling 5 cm into infected trees near the root collar and collecting the drill shavings generated for DNA extraction [28,29]. Finally, roots of diseased trees were excavated, and a cross-section from each was collected for DNA extraction.

DNA Extraction
DNA was extracted from isolates grown on 2% Malt Extract (Difco) and grown at room temperature for 5-7 days. Approximately 50-100 mg of mycelia was harvested, centrifuged to remove excess liquid and transferred to a sterile 2-mL lysing matrix A tube (MP Biomedical; Solon, OH). The tissues were frozen in liquid nitrogen and homogenized for 10 seconds at 4 m/s using a FastPrep-24 5 G benchtop homogenizer (MP Biomedicals; Solon, OH). Wood samples were similarly treated, frozen in liquid nitrogen, but homogenized twice to pulverize the tissue completely. Conversely, for herbarium samples, homogenization speed and time was reduced to avoid shearing of DNA. DNA of samples of H. irregulare infected red pine tissues from Wisconsin was extracted at the Wisconsin Department of Natural Resources in Fitchburg, WI. DNA was extracted using a modified. CTAB extraction protocol with choloroform and ethanol washes [30].

PCR, Sequencing and Primer Design
Two loci, namely the elongation factor 1 alpha and the glyceraldehyde 3-phosphate dehydrogenase, were sequenced from a selection of North American Heterobasidion cultures (Table 1). Sanger sequencing was done directly from PCR products using big dye terminators on the ABI 5730xl Data Analyzer at Centre hospitalier de l'Université Laval, Quebec, Canada. Sequences were aligned with the ClustalW extension of BioEdit and edited manually. All sequences used in this study are available in GenBank, while alignments are available from TreeBase (submission ID 17241; www.treebase.org). Allelic blocks that were highly divergent between the two species were identified in the final alignments and selected for primer design. Two sets of candidate species-specific primers, namely, Irr-1 For and Irr-1 Rev and Occ-0 For and Occ-0 Rev (Table 3) were designed manually from these regions focusing on indels and high variation at the 3' end of the primer. To ascertain that DNA extractions from both pure cultures and environmental samples were successful, a pair of universal plant-specific primers (Table 3) derived from the chloroplast genome (http://bfw.ac.at/200/2043.html) was used as an internal control during the validation step.

Primer Testing and Validation of the Assay
The two differentiation primer sets (Table 3), namely Irr-1 For and Irr-1 Rev and Occ-0 For and Occ-0 Rev, were tested for their specificity using DNA extracted from pure cultures and herbarium samples of North American Heterobasidion species (Table 1) Table 3), extension at 72.0 • C for 55 s; and a final extension at 72.0 • C for 5 min. The differentiation primer sets Irr-1 For and Irr-1 Rev and Occ-0 For and Occ-0 Rev were always used together in the same reaction, each at 0.25 µM. PCR products were visualized using electrophoresis with 1.5% agarose gels stained with ethidium bromide.
Further validation of the differentiation primers sets Irr-1 For, Irr-1 Rev, Occ-0 For and Occ-0 Rev were done on DNA extracted from pine and hemlock wood infected by H. irregulare and H. occidentale, respectively; on DNA from pure cultures of closely-related Heterobasidion species; as well as on DNAs of common North American heartwood and root rot fungi. Amplification was conducted using identical PCR conditions as stated above using the primer sets Irr-1 For, Irr-1 Rev, Occ-0 For and Occ-0 Rev, in duplex PCR reaction.
A third validation assay using the two Heterobasidion differentiation primers and the internal plant-specific primer (Table 3) was performed to confirm that DNA extractions were successful, and the differentiation primers did not cross-amplify plant DNA. We used DNA from pure cultures and herbarium samples of North American Heterobasidion (Table 1), as well as DNA from representative conifer species. The PCR was run using the three sets of primers in single-, duplex and triplex.
A fourth validation assay, using three primer tools, was also tested on samples containing DNA from H. occidentale, H. irregulare, and wood shavings from red alder (Alnus rubra) and red pine (Pinus resinosa) in all possible combinations.