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Pathogens 2019, 8(3), 156; https://doi.org/10.3390/pathogens8030156

Article
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
1
Pacific Forestry Centre, Canadian Forest Service, Natural Resources Canada, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada
2
Department of Forest and Conservation Sciences, University of British Columbia, 2424 Main Mall, Vancouver BC V6T 1Z4, Canada
3
Canadian Food Inspection Agency, 93 Mount Edward Road, Charlottetown, PEI C1A 5T1, Canada
4
Department of Environmental Science, Policy and Management, University of California, 54 Mulford Hall, Berkeley, CA 94720, USA
*
Author to whom correspondence should be addressed.
Received: 13 August 2019 / Accepted: 13 September 2019 / Published: 18 September 2019

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.
Keywords:
elongation factor 1 alpha; glyceraldehyde 3-phosphate dehydrogenase; Heterobasidion species; molecular identification; phytosanitary; wood products trade

1. 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 the most important disease of conifers worldwide. Globalization has resulted in human-induced long-distance movement of this pathogen [2], and climate change has increased disease risk in sites that historically have not been challenged by this pathogen [3].
The genus Heterobasidion has been recently split into three genetically distinct clusters (H. annosum, H. insulare and H. araucariae) containing a total of 12 species [4] and a newly described hybrid taxon [5]. The H. annosum cluster includes two North American partially intersterile species, namely H. irregulare and H. occidentale [6], and a newly described hybrid taxon genetically distinct from the other two [5] In Europe, three species are currently recognized within the H. annosum species complex, namely H. parviporum, H. annosum sensu stricto (s.s.) and H. abietinum [7]. The Asian H. insulare cluster includes six species, all believed to be mostly saprophytic [4], while the species H. araucariae appears to represent a third monophyletic cluster with a geographic range spanning over Australia, New Zealand and adjacent regions [8].
Heterobasidion irregulare is widespread across Eastern North America forests from Quebec south to Florida and Central Mexico, in US Midwestern forests, [9], and in Pacific forests from Washington State (USA) to Baja California [10]. Its primary hosts are pine (Pinus) species, although scented cedar (Calocedrus) and juniper (Juniperus) are also common hosts. It can also be locally abundant on Pacific madrones (Arbutus menziesii) and Manzanitas (Arcostaphylos spp.). Rare infections of Douglas-fir (Pseudotsuga), a known host of H. occidentale, have also been reported [6]. Heterobasidion irregulare was accidentally introduced into Italy, presumably on infected wood products, during WW2 [11]. It is currently infesting Mediterranean pine forests around Rome, where it has proven to be significantly more widespread than the native H. annosum, due to its increased sporulation rates, and higher wood decay saprobic ability [12,13].
Heterobasidion occidentale is located exclusively on the west coast of North America, from Alaska to southern Mexico, and as far east as Colorado [10]. It has a broader host range than H. irregulare, and it is a pathogen of primary concern for hemlock (Tsuga), and true-fir species. However, it has also been found on several other species, including western red cedar (Thuja plicata), the giant sequoia (Sequoiadendron), coast sequoia (Sequoia) [14], Douglas-fir, spruce, larch, and even broadleaf species, such as alder (Alnus).
The morphology of the fruiting bodies of the two North American species is primarily driven by site ecology [6], and many traits are overlapping between the two, making their diagnosis based on morphology alone quite challenging. A preliminary differentiation may be done through host, symptoms, and location; however, true differentiation can only be achieved through inter-sterility mating or DNA sequencing, both of which are time-consuming, and may require culturing in vitro of the pathogen. Differentiation based on mating tests can also be confusing because intersterility between the two is only partial [10,15]. To further complicate matters, hybrids between the two North American species have been identified in California and Montana [16,17].
Molecular diagnostic methods, including conventional and real-time PCR approaches, have been a heavily researched topic for the potential identification and differentiation of species in the Heterobasidion species complex [16,18,19,20]. Studies on the North American species have identified indels in parts of the ITS sequence that could distinguish the species using DNA extracts from pure cultures [16]. Likewise, polymorphisms in alleles of the Elongation Factor alpha and presence/absence of DNA insertions in the ML5-ML6 region of the mitochondrion can be used to differentiate among European species and between European and North American species [18,19]. Other assays can differentiate Eurasian species using taxon-specific primers [21]. These primers are simple to use and have made forest management much easier in the Baltic region, as well as having some forensic applications [22,23].
The objective of this investigation was to develop PCR primers in order to detect and differentiate the North American Heterobasidion species collected from various substrates, including infected wood, sporocarps, and cultured isolates.

2. 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 (Figure 2 and Figure 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).
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, Figure 2, Figure 5, Figure 6 and Figure 7) on Abies and Pinus strobus and P. ponderosa. Basidiocarps collected from Abies in 2013 (DAVFP29738; Table 1) only amplified using the occidentale-specific primers (Occ-0 For and Occ-0 Rev), while all three basidiocarps collected from Pinus in 2013 and 1997 (DAVFP29739, DAVFP29740 and DAVFP25395, respectively) only amplified using the irregulare-specific primers (Irr-1 For and Irr-1 Rev) (Figure 6 and Figure 7; data not shown for DAVFP29740). This is the first report of H. irregulare in British Columbia.
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 (Figure 5, Figure 6 and Figure 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).

3. 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.

4. Materials and Methods

4.1. 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.

4.2. 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].

4.3. 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.

4.4. 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). Amplification was performed using differentiation primer sets Irr-1 For and Irr-1 Rev and Occ-0 For and Occ-0 Rev in duplex. An aliquot of 1.0 µL of diluted genomic DNA was included in each 25.00 µL PCR reaction (1X PCR reaction buffer, 1.5 mM MgCl2, 1U of Platinum Taq Polymerase [Invitrogen, Carlsbad, CA], 0.25 µM of each primer (Integrated DNA Technologies), 0.3 mM dNTPs [Invitrogen]). A minimum of one negative water control was included with each PCR run. Amplifications were carried out in a Veriti 96-well thermal cycler (Applied Biosystems, Carlsbad, CA)) under the following conditions: Initial denaturation at 94.0 °C for 5 min; then 35 cycles of denaturation at 94.0 °C for 30 s, annealing for 40 s (annealing temperatures in 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.

Author Contributions

S.F.S. conceived and designed the experiments of the research study. C.H. and G.S. performed and validated the results of the experiments. S.F.S., X.L., M.G. and G.S. analyzed the data, wrote the manuscript with further editing and writing contribution by M.G. Further revisions of the manuscript were conducted by all authors.

Funding

We would like to acknowledge NRCan- CFS Forest Pest Risk Management—Forest Invasive Alien and Phytosanitary research program, CFIA- CFS/PFC grant # CHA-P-1011 for funding and supporting this project.

Acknowledgments

We thank Irina Kassatenko for their technical support, and all the colleagues who shared their culture collections of Heterobasidion spp. with us, as listed in Table 1 and Table 2.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Woodward, S.; Stenlid, J.; Karjalainen, R.; Hüttermann, A. Heterobasidion Annosum: Biology, Ecology, Impact and Control; CABI (Cab International): Wallingford, UK, 1998. [Google Scholar]
  2. Gonthier, P.; Anselmi, N.; Capretti, P.; Bussotti, F.; Feducci, M.; Giordano, L.; Honorati, T.; Lione, G.; Luchi, N.; Michelozzi, M.; et al. An integrated approach to control the introduced forest pathogen Heterobasidion irregulare in Europe. Forestry 2014, 87, 471–481. [Google Scholar] [CrossRef]
  3. La Porta, N.; Capretti, P.; Thomsen, I.M.; Kasanen, R.; Hietala, A.M.; Von Weissenberg, K. Forest pathogens with higher damage potential due to climate change in Europe. Can. J. Plant Pathol. 2008, 30, 177–195. [Google Scholar] [CrossRef]
  4. Chen, J.J.; Cui, B.K.; Zhou, L.W.; Korhonen, K.; Dai, Y.C. Phylogeny, divergence time estimation, and biogeography of the genus Heterobasidion (Basidiomycota, Russulales). Fungal Divers. 2015, 71, 185–200. [Google Scholar] [CrossRef]
  5. Sillo, F.; Gonthier, P.; Lockman, B.; Kasuga, T.; Garbelotto, M. Molecular analyses identify hybridization-mediated nuclear evolution in newly discovered fungal hybrids. Ecol. Evol. 2019. [Google Scholar] [CrossRef]
  6. Otrosina, W.J.; Garbelotto, M. Heterobasidion occidentale sp. nov. and Heterobasidion irregulare nom. nov.: A disposition of North American Heterobasidion biological species. Fungal Biol. 2010, 114, 16–25. [Google Scholar] [CrossRef]
  7. Niemelä, T.; Korhonen, K. Taxonomy of the genus Heterobasidion. In Heterobasidion annosum Biology, Ecology, Impact and Control; Woodward, S., Stenlid, J., Karjalainen, R., Hüttermann, A., Eds.; CAB International, University Press: Cambridge, UK, 1998; pp. 27–33. [Google Scholar]
  8. Buchanan, P. A new species of Heterobasidion (Polyporaceae) from Australia. Mycotaxon 1988, 32, 325–337. [Google Scholar]
  9. Linzer, R.E.; Otrosina, W.J.; Gonthier, P.; Bruhn, J.; Laflamme, G.; Bussières, G.; Garbelotto, M. Inferences on the phylogeography of the fungal pathogen Heterobasidion annosum, including evidence of interspecific horizontal genetic transfer and of human-mediated, long-range dispersal. Mol. Phylogenet. Evol. 2008, 46, 844–862. [Google Scholar] [CrossRef]
  10. Garbelotto, M.; Gonthier, P. Biology, epidemiology, and control of Heterobasidion species worldwide. Annu. Rev. Phytopathol. 2013, 51, 39–59. [Google Scholar] [CrossRef]
  11. Gonthier, P.; Warner, R.; Nicolotti, G.; Mazzaglia, A.; Garbelotto, M.M. Pathogen introduction as a collateral effect of military activity. Mycol. Res. 2004, 108, 468–470. [Google Scholar] [CrossRef]
  12. Giordano, L.; Gonthier, P.; Lione, G.; Capretti, P.; Garbelotto, M. The saprobic and fruiting abilities of the exotic pathogen Heterobasidion irregulare may explain its invasiveness. Biol. Invasions 2013. [Google Scholar] [CrossRef]
  13. Sillo, F.; Garbelotto, M.; Friedman, M.; Gonthier, P. Comparative genomics of sibling fungal pathogenic taxa identifies adaptive evolution without divergence in pathogenicity genes or genomic structure. Genome Biol. Evol. 2015, 7, 3190–3206. [Google Scholar] [CrossRef]
  14. Garbelotto, M.; Friedman, M.; Bedell, W.; Henkel, T. First report of Heterobasidion occidentale on Sequoia sempervirens in Northern California. Plant Dis. 2017, 101, 2152. [Google Scholar] [CrossRef]
  15. Harrington, T.C.; Worrall, J.J.; Rizzo, D.M. Compatibility among host-specialized isolates of Heterobasidion annosum from western North America. Phytopathology 1989, 79, 290–296. [Google Scholar] [CrossRef]
  16. Garbelotto, M.; Ratcliff, A.; Bruns, T.D.; Cobb, F.W.; Otrosina, W.J. Use of taxon-specific competitive-priming PCR to study host specificity, hybridization, and intergroup gene flow in intersterility groups of Heterobasidion annosum. Phytopathology 1996, 66, 543–551. [Google Scholar] [CrossRef]
  17. Lockman, B.; Mascheretti, S.; Schechter, S.; Garbelotto, M. A first generation Heterobasidion hybrid discovered in Larix lyalli in Montana. Plant Dis. 2014, 98, 1003. [Google Scholar] [CrossRef]
  18. Gonthier, P.; Garbelotto, M.; Varese, G.C.; Nicolotti, G. Relative abundance and potential dispersal range of intersterility groups of Heterobasidion annosum in pure and mixed forests. Can. J. Bot. 2001, 79, 1057–1065. [Google Scholar]
  19. Gonthier, P.; Guglielmo, F.; Sillo, F.; Giordano, L.; Garbelotto, M. A molecular diagnostic assay for the detection and identification of wood decay fungi of conifers. For. Pathol. 2015, 45, 89–101. [Google Scholar] [CrossRef]
  20. Lamarche, J.; Potvin, A.; Stewart, D.; Blais, M.; Pelletier, G.; Shamoun, S.F.; Hamelin, R.C.; Tanguay, P. Real-time PCR assays for the detection of Heterobasidion irregulare, H. occidentale, H. annosum sensu stricto and the Heterobasidion annosum complex. For. Pathol. 2016. [Google Scholar] [CrossRef]
  21. Hantula, J.; Vainio, E. Specific primers for the differentiation of Heterobasidion annosum (s. str.) and H. parviporum infected stumps in northern Europe. Silva Fenn. 2003, 37, 181–187. [Google Scholar] [CrossRef]
  22. Ðíipars, V.; Ruòìis, D. Detection of Heterobasidion annosum in Scots pine trees using a polymerase chain reaction based method. Balt. For. 2011, 17, 2–7. [Google Scholar]
  23. Nowakowska, J.A.; Borys, M.; Oszako, T. DNA markers used to identify pathogens causing root rot disease in spruce (Picea abies L. Krast) for forensic purposes. J. Forensic Sci. 2013, 93, 465–474. [Google Scholar]
  24. Gardes, M.; Bruns, T.D. ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts. Mol. Ecol. 1993, 2, 113–118. [Google Scholar] [CrossRef] [PubMed]
  25. White, T.J.; Bruns, T.; Lee, S.J.W.T.; Taylor, J.W. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protoc. A Guide Methods Appl. 1990, 18, 315–322. [Google Scholar]
  26. Johannesson, H.; Stenlid, J. Molecular markers reveal genetic isolation and phylogeography of the S and F intersterility groups of the wood-decay fungus Heterobasidion annosum. Mol. Phylogenet. Evol. 2003, 29, 94–101. [Google Scholar] [CrossRef]
  27. Heinze, B. A database of PCR primers for the chloroplast genomes of higher plants. Plant Methods 2007, 3, 4. [Google Scholar] [CrossRef] [PubMed]
  28. Guglielmo, F.; Gonthier, P.; Garbelotto, M.; Nicolotti, G. Optimization of sampling procedures for DNA-based diagnosis of wood decay fungi in standing trees. Lett. Appl. Microbiol. 2010. [Google Scholar] [CrossRef] [PubMed]
  29. Banik, M.T.; Lindner, D.L.; Juzwik, J.; Glaeser, J.A. Development of a DNA sampling kit to detect pathogenic, saprotrophic, and stain fungi in sapwood of declining red pine (Pinus resinosa) in the upper midwest. In Browning, John; Palacios, Patsy, comps. Proceedings of the 60th Annual Western International Forest Disease Work Conference, Lake Tahoe, CA, USA, 8–12 October 2012; pp. 101–110. [Google Scholar]
  30. Särkinen, T.; Staats, M.; Richardson, J.E.; Cowan, R.S.; Bakker, F.T. How to open the treasure chest? Optimising DNA extraction from herbarium specimens. PLoS ONE 2012, 7, e43808. [Google Scholar] [CrossRef]
Figure 1. Consensus sequence alignment of H. irregulare and H. occidentale with the occidentale-specific (Occ-0 For and Occ-0 Rev) and the irregulare-specific (Irr-1 For and Irr-1 Rev) primers.
Figure 1. Consensus sequence alignment of H. irregulare and H. occidentale with the occidentale-specific (Occ-0 For and Occ-0 Rev) and the irregulare-specific (Irr-1 For and Irr-1 Rev) primers.
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Figure 2. Differentiation of Heterobasidion species from pure cultures and herbarium collection with isolates collected across their range in North America. (Note: Isolate 25395 is a herbarium collection- DAVFP25395).
Figure 2. Differentiation of Heterobasidion species from pure cultures and herbarium collection with isolates collected across their range in North America. (Note: Isolate 25395 is a herbarium collection- DAVFP25395).
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Figure 3. Verification of primers from the infected root and heartwood tissue of diseased trees infected with Heterobasidion irregulare or H. occidentale.
Figure 3. Verification of primers from the infected root and heartwood tissue of diseased trees infected with Heterobasidion irregulare or H. occidentale.
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Figure 4. The specificity of the differentiation primers using closely related species from around the world, including other common North American heartwood and root rot fungi.
Figure 4. The specificity of the differentiation primers using closely related species from around the world, including other common North American heartwood and root rot fungi.
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Figure 5. Validation of specificity of universal plant primers PC-UPC-7 For and PC-UPC-7 Rev. irr = Heterobasidion irregulare isolates; occ = Heterobasidion occidentale isolates; WRC = western red cedar; WWP = western white pine; DF = Douglas fir; NTC = no template control.
Figure 5. Validation of specificity of universal plant primers PC-UPC-7 For and PC-UPC-7 Rev. irr = Heterobasidion irregulare isolates; occ = Heterobasidion occidentale isolates; WRC = western red cedar; WWP = western white pine; DF = Douglas fir; NTC = no template control.
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Figure 6. Duplex-PCR using universal plant primers PUC-UPC7 For and PUC-UPC7 Rev and occidentale-specific primers Occ-0 For and Occ-0 Rev (A) and universal plant primer PUC-UPC7 For and PUC-UPC7 Rev and irregulare-specific primers Irr-1 For and Irr-1 Rev (B). irr = Heterobasidion irregulare isolates; occ = Heterobasidion occidentale isolates; WRC = western red cedar; WWP = western white pine; DF = Douglas fir; NTC = no template control.
Figure 6. Duplex-PCR using universal plant primers PUC-UPC7 For and PUC-UPC7 Rev and occidentale-specific primers Occ-0 For and Occ-0 Rev (A) and universal plant primer PUC-UPC7 For and PUC-UPC7 Rev and irregulare-specific primers Irr-1 For and Irr-1 Rev (B). irr = Heterobasidion irregulare isolates; occ = Heterobasidion occidentale isolates; WRC = western red cedar; WWP = western white pine; DF = Douglas fir; NTC = no template control.
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Figure 7. Triplex PCR using universal plant primers PUC-UPC7 For and PUC-UC7 Rev, occidentale-specific primers Occ-0 For and Occ-0 Rev and irregulare-specific primers Irr-1 For and Irr-1 Rev. irr = Heterobasidion irregulare isolates; occ = Heterobasidion occidentale isolates; WRC = western red cedar; WWP = western white pine; DF = Douglas fir; NTC = no template control.
Figure 7. Triplex PCR using universal plant primers PUC-UPC7 For and PUC-UC7 Rev, occidentale-specific primers Occ-0 For and Occ-0 Rev and irregulare-specific primers Irr-1 For and Irr-1 Rev. irr = Heterobasidion irregulare isolates; occ = Heterobasidion occidentale isolates; WRC = western red cedar; WWP = western white pine; DF = Douglas fir; NTC = no template control.
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Figure 8. Agarose gel electrophoresis. DNA extracted from colonized wood tissue by North American Heterobasidion species. Lane 1—100 bp ladder; lane 2—red alder (band 664 bp); lane 3—H. occidentale (band 365 bp); lane 4—red alder and H. occidentale (bands 664 bp and 365 bp); lane 5—red pine (band 664 bp); lane 6—H. irregulare (band 160 bp); lane 7—red pine and H. irregulare (bands 664 bp and 160 bp); lane 8—H. occidentale and H. irregulare (bands 365 bp and 160 bp); lane 9—red alder, H. occidentale and H. irregulare (bands 664 bp, 365 bp and 160 bp); lane 10—negative control, no DNA (no bands).
Figure 8. Agarose gel electrophoresis. DNA extracted from colonized wood tissue by North American Heterobasidion species. Lane 1—100 bp ladder; lane 2—red alder (band 664 bp); lane 3—H. occidentale (band 365 bp); lane 4—red alder and H. occidentale (bands 664 bp and 365 bp); lane 5—red pine (band 664 bp); lane 6—H. irregulare (band 160 bp); lane 7—red pine and H. irregulare (bands 664 bp and 160 bp); lane 8—H. occidentale and H. irregulare (bands 365 bp and 160 bp); lane 9—red alder, H. occidentale and H. irregulare (bands 664 bp, 365 bp and 160 bp); lane 10—negative control, no DNA (no bands).
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Table 1. North American Heterobasidion culture isolates and herbarium samples used in this study.
Table 1. North American Heterobasidion culture isolates and herbarium samples used in this study.
SpeciesIsolate or Herbarium Collection NumberGeographic OriginHostYear CollectedSource/CollectorITSEFAGPD
irregularePFC5201Limerick, ON, CanadaPinus resinosa2003M. DumasKP863563KP863594KP863623
irregularePFC5205York, ON, CanadaP. resinosa2005M. DumasKP863564KP863596KP863625
irregularePFC5214St. Philippe, QC, CanadaP. resinosa2002G. LaflammeKP863565KP863597KP863626
irregularePFC5217Lac La Blanche, QC, CanadaP. resinosa2002G. LaflammeKP863566KP863598KP863627
irregularePFC5227Iowa Co, WI, USAP. resinosa or P. strobus1994G. StanoszKP863567KP863599KP863629
irregularePFC5229Union Co, IL, USAP. echinata1994G. StanoszKP863568KP863600KP863630
irregularePFC5241Portage Co, WI, USAAbies balsamea2010G. StanoszKP863571KP863602KP863634
irregularePFC5276S. Pines, NC, USAP. taeda1967J.S. BoyleKP863572KP863603KP863635
irregularePFC5279Lassen Natl. Forest, CA, USAP. ponderosa1981J. WorrallKP863573KP863604KP863636
irregularePFC5284San Bernardino Mtns., CA, USAP. jeffreyii1975J. WorrallKP863574KP863605KP863639
irregularePFC5288Durham College Woods, NH, USAJuniperus virginiana1987T. HarringtonKP863575KP863606KP863641
irregularePFC5290Nebraska, USAP. ponderosa J. BlodgettKP863576KP863607KP863642
irregularePFC5319Montrose Co, CO, USAP. ponderosa2006J. WorrallKP863581KP863611KP863647
irregularePFC5401St. Williams, ON, CanadaThuja plicata D.C. ConstableKP863587KP863617KP863653
irregularePFC5416Warren Co, GA, USASpore trap2007M. CramKP863589KP863619KP863655
irregulareDAVFP25395Okanagan Falls, BC, CanadaP. ponderosa1997J. HodgesKP863588KP863618KP863654
irregulareDAVFP29739Summarland, BC, CanadaP. strobus2013J.H. Ginns
irregulareDAVFP29740Summerland, BC, CanadaP. strobus2013J.H. Ginns
occidentalePFC5190Ladysmith, BC, CanadaTsuga heterophylla B. CallanKP863561KP863592KP863621
occidentalePFC5192Jordan River, BC, CanadaT. heterophylla B. CallanKP863562KP863593KP863622
occidentalePFC5282Modoc Natl Forest, CA, USAA. concolor1981J. WorrallKP492941.1KP571672.1KP863638
occidentalePFC5312King Co, WA, USAT. heterophylla2011R. EdmondsKP863578KP863608KP863644
occidentalePFC5315Ouray Co, CO, USAA. concolor2005J. WorrallKP863579KP863609KP863645
occidentalePFC5318Mineral CO, CO, USAA. concolor2005J. WorrallKP863580KP863610KP863646
occidentalePFC5339OR, USAA. concolor2009E. GoheenKP863582KP863612KP863648
occidentalePFC5357Clallam Co, WA, USAT. heterophylla2011R. EdmondsKP863583KP863613KP863649
occidentalePFC5362Lincoln Co, OR, USAT. heterophylla2011M. ElliottKP863584KP863614KP863650
occidentalePFC5388San Bernardino, CA, USAA. concolor2011P. ZambinoKP863585KP863615KP863651
occidentaleDAVFP29738Summerland, BC, CanadaAbies stump2013J.H. Ginns
Isolate preceded by PFC (Pacific Forestry Centre) are pure culture sample, and DAVFP (Department of Agriculture, Victoria, Forest Pathology) are herbarium samples.
Table 2. List of Eurasian isolates used in this study.
Table 2. List of Eurasian isolates used in this study.
SpeciesIsolate or Herbarium Collection NumberGeographic OriginHostYear CollectedSource/CollectorITSEFAGPD
abietinumPFC5247PolandAbies alba H. SolheimKC492895.1KC571636.1KP863657
abietinumPFC5249AustriaPicea abies H. SolheimKC492896.1KC571637.1KP863658
abietinumPFC5373GreeceAbies cephallonica1993P. TsopelasKC492956.1KC571687.1KP863664
annosumPFC5252NorwayPinus sylvestris1937R.H. Roll-HansenKC492906.1KC571646.1KP863659
annosumPFC5257ItalyPinus pinaster2008A. BiraghiKC492909.1KC571649.1KP863660
annosumPFC5260SerbiaPinus nigra2003D. DubakKC492911.1KC571651.1KP863661
araucariaePFC5434New ZealandAgathis australis1958J.W. GilmourKX130098KX130101KX130104
ecrustosumPFC5438JapanPinus thunbergii P.K. BuchananKX130099KX130102KX130105
orientalePFC5439JapanTsuga sp. P.K. BuchananKX130100KX130103KX130106
parviporumPFC5262NorwayPicea abies2004R. SaursaunetKC492957.1KC571688.1KP863662
parviporumPFC5269JapanAbies mayriana1942S. KameiKC492951.1KC571682.1KP863663
parviporumPFC5293RussiaAbies sibirica K. KorhonenKC492913.1KC571653.1KP863665
Table 3. Details about the primers used in this study.
Table 3. Details about the primers used in this study.
PrimerSequenceGeneProduct (bp)Tm (°C)Reference
Irr-1 ForTGGCGGTCGTGGTGTTAACGPD16564This study
Irr-1 RevGAATGAGAGACCACTGGAGGTAAACGPD16564This study
Occ-0 ForCGAGAGAATCCTCGATCAGCCTGEFA36564This study
Occ-0 RevTGTGAAAAACGATACAAGCACGEFA36564This study
ITS1-FCTTGGTCATTTAGAGGAAGTAAITS486/51555[24]
ITS4TCCTCCGCTTATTGATATGCITS486/51555[25]
EFA ForTCAACGTGGTCGGTGAGCAGGTAEFA447-5466[26]
EFA RevAAGTCACGATGTCCAGGAGCATCEFA447-5466[26]
GPD-Seq ForCAGAGCCTCTGCCCACTTGAAGGGPD666/75459This study
GPD-Seq RevGCCGGGTGGCCGACAAAGTCGPD66659This study
PC-UPC7 ForGGATTRCGTATGGGMAATATTGAAACCHLOROPLAST66464[27]
PC-UPC7 RevCCCCTTGGACTRCTACGAAAAACACCCHLOROPLAST66464[27]

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