18 spp.-specific amplicons, respectively. The absence of taxon- specific PCR products in any multiplex PCR, and the simulta- neous presence of the amplified DNA fragment corresponding to the ITS region in M1, may indicate the detection of a nontar- get fungal taxon. In this case, it could be advisable to sequence the PCR product to ascertain, after BLAST search analysis, what fungus is involved. Species-level diagnostic primers were developed for three fun- gal taxa frequently reported for their important role in wood decay processes: L. sulphureus , P. fraxinea, and U. deusta (Lonsdale 1999; Nicolotti et al. 2004). Likewise, Mhyme and the Mgano were developed to identify at a subgeneric rank the most hazard- ous taxa within these genera and to differentiate them from fun- gal species known to be less hazardous. Mhyme-specific primers allowed to identify, at the level of the groups described in the classification of Hymenochaetales re-examined by Wagner and Fischer (2001, 2002), very active decay species that can seriously affect the stability of trees in urban landscape such as I. dryadeus , I. hispidus , P. gilvus, and P. robustus . Through Mgano, European G. lucidum , reported as a slow white rot fungus of many broad- leaved trees rarely associated with their failures (Bernicchia 2005), is well distinguishable from other Ganoderma species such as G. adspersum , G. applanatum, and G. resinaceum known to significantly affect tree stability (Lonsdale 1999; Bernicchia 2005). Moreover, G. lucidum from North America, reported to cause rapid and extensive rot in Quercus spp. and Acer spp. (Hickman and Perry 1997; Dreistadt et al. 2004), is identified, through Mgano, as a G. resinaceum group, as already inferred by a phylogenetic study based on small-subunit ribosomal DNA sequences (Hong and Jung 2004). Validation assay on field samples highlights the specificity and efficiency of the PCR-based method, as suggested by the consis- tent matches between PCR-based results and the expected data. The occurrence of unexpected fungi were attributable either to visual misidentification of the emerging fruiting bodies such as in the case of P. fraxinea incorrectly identified as P. torulo- sus or the association between different fungal taxa in a same tree as reported in this study with Ganoderma spp. and P. frax- inea . In this case, patchy patterns of wood localization of decay fungi may allow the detection of only one of the different fungi involved depending on the wood sample. PCR-based identification proved to be efficient starting from decayed wood samples taken at the point of fruiting body emer- gence as well as from wood core samples extracted with a Swedish increment borer. Such results suggest that this PCR- based method may be used by arborists as a complement to VTA analysis. The method described here can be easily performed in disease diagnostic clinics equipped with basic molecular biology instruments. Further studies, aimed at setting up and standardiz- ing field samples, are in progress. LITERATURE CITED Anselmi, N., and M. Bragaloni. 1992. A method to identify wood decay Basidiomycetes by using enzymatic comparisons. Micologia Italiana 2:15–20. Bahnweg, G., E.M. Möller, S. Anegg, C. Langebartels, O. Wienhaus, and H. Sandermann Jr. 2002. Detection of Heterobasidion annosum s.l. [(Fr.) Bref.] in Norway spruce by polymerase chain reaction. Journal of Phytopathology 150:382–389. Bernicchia, A. 2005. Polyporaceae s.l. Ed. Candusso, Alassio, Italy. 808 pp. ©2009 International Society of Arboriculture Nicolotti et al.: Detection of Wood Decay Fungi Blanchette, R.A. 1991. Delignification by wood-decay fungi. Annual Review of Phytopathology 29:381–398. Clausen, C.A. 2003. Detecting decay fungi with antibody-based tests and immunoassays. Wood Deterioration and Preservation 845:325–336. Corbiere Morot-Bizot, S., R. Talon, and S. Leroy. 2004. Development of a multiplex PCR for the identification of Staphylococcus genus and four staphylococcal species isolated from food. Journal of Applied Microbiology 97:1087–1094. Dreistadt, S.H., J.K. Clark, and M.L. Flint. 2004. Pests of Landscape Trees and Shrubs: An Integrated Pest Management Guide, UC Statewide IPM Program. Univ. Calif. Agric. Nat. Res. Publ. 3359; Oakland, CA. Elnifro, E.M., A.M. Ashshi, R.J. Cooper, and P.E. Klapper. 2000. Multiplex PCR: Optimization and application in diagnostic virology. Clinical Microbiology Reviews 13:559–570. Gardes, M., and T.D. Bruns. 1993. ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts. Molecular Ecology 2:113–118. Gonthier, P., M. Garbelotto, and G. Nicolotti. 2003. Swiss stone pine trees and spruce stumps represent an important habitat for Heterobasidion spp. in subalpine forests. Forest Pathology 33:191–203. Guglielmo, F., S.E. Bergemann, P. Gonthier, G. Nicolotti, and M. Garbelotto. 2007. A multiplex PCR-based method for the detec- tion and early identification of wood rotting fungi in standing trees. Journal of Applied Microbiology 103:1490–1507. Guglielmo, F., P. Gonthier, M. Garbelotto, and G. Nicolotti. 2008. A PCR-based method for the identification of important wood rotting fungal taxa within Ganoderma, Inonotus s.l. and Phellinus s.l. FEMS Letters in Microbiology 282:228–237. Habermehl, A., H.W. Ridder, and P. Seidl. 1999. Computerized tomo- graphic systems as tools for diagnosing urban tree health. Acta Horticulturae 496:261–268. Hayden, K.J., D. Rizzo, J. Tse, and M. Garbelotto. 2004. Detection and quantification of Phytophthora ramorum from California forests using a Real-time polymerase chain reaction assay. Phytopathology 94:1075–1083. Hickman, G.W., and E.J. Perry. 1997. Ten Common Wood Decay Fungi on Landscape Trees—Identification Handbook. Western Chapter, ISA, Sacramento, CA. Hong, S.G., and H.S. Jung. 2004. Phylogenetic analysis of Ganoderma based on nearly complete mitochondrial small-subunit ribosomal DNA sequences. Mycologia 96:742–755. Jasalavich, C.A., A. Ostrofsky, and J. Jellison. 2000. Detection and iden- tification of decay fungi in spruce wood by restriction fragment length polymorphism analysis of amplified genes encoding rRNA. Applied and Environmental Microbiology 66:4725–4734. Jellison, J., and C. Jasalavich. 2000. A review of selected methods for the detection of degradative fungi. International Biodeterioration & Biodegradation 46:241–244. Lonsdale, D. 1999. Principles of Tree Hazard Assessment and Management. Research for Amenity Trees 7. The Stationery Office, London, UK. 388 pp. Mattheck, C., and H. Breloer. 1992. Feldaleitung fur Baumkkontrollen mit VTA. Landschaftarchitektur. 6 pp. Moreth, U., and O. Schmidt. 2001. Identification of indoor rot fungi by taxon-specific priming polymerase chain reaction. Holzforschung 54:1–8. Müller, U., R. Bammer, E. Halmschlager, and R. Wimmer. 2001. Detection of fungal wood decay using magnetic resonance imaging. Holz Roh-Werkstoff 59:190–194. Nicolotti, G., P. Gonthier, and D. Pecollo. 2004. Ecologia e grado di preferenza d’ospite dei funghi agenti di carie/I parte. Acer 1:47–51. Nobles, M.K. 1965. Identification of cultures of wood-inhabiting hymenomycetes. Canadian Journal of Botany 44:1097–1165. O’Donnell, K. 1993. Fusarium and its near relatives, pp. 225–233. In: Reynolds, D.R., and Taylor, J.W. (Eds.). The Fungal Holomorph: Mitotic, Meiotic and Pleomorphic Speciation in Fungal Systematics. CAB International, Wallingford, UK.
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