Arboriculture & Urban Forestry 35(1): January 2009 wood-rotting fungi in standing trees. This article also reports on the validation of such methods on wood samples. MATERIALS AND METHODS Target Decay Fungi, Sampling, and Culturing We selected the target fungi based on their frequency in the north- ern temperate areas and their aggressiveness (Hickman and Perry 1997; Lonsdale 1999; Nicolotti et al. 2004; Bernicchia 2005). Selected taxa included Armillaria spp., Ganoderma spp., Hericium spp., Inonotus spp., Laetiporus sulphureus , Perenniporia frax- inea , Phellinus spp., Pleurotus spp., Schizophyllum spp., Stereum spp., Trametes spp., and Ustulina deusta . The design and the initial validation of taxon-specific diagnostic PCR assays were based on 80 fungal collections, either pure cultures or fruiting bodies specimens, encompassing the most representative species per each taxon ( Table 1 ). Fungal collections were either provided by CABI Bioscience National Center of Wood Rotting Fungi–Garston (U.K.), CAS Institute of Microbiology–Videnska (CR), METLA Finnish Forest Research Institute-Vantaa (FIN), MUAF Faculty of Forestry and Wood Technology–Brn o (CR), USDA Forest Products Laboratory–Madison (WI, U.S.), USDA Agricultural Service–Davis (CA, U.S.) and WSL Swiss Federal Research Institute–Birmensdorf (CH) or obtained from fruiting bodies col- lected in California and Italy and identified through analytical keys (Hickman and Perry 1997; Bernicchia 2005). Pure fungal cultures were isolated from the context of the fruiting bodies by using potato dextrose agar (Merck KGaA, Darmstadt, Germany) medium with 0.2 g/L (1.8 10 −3 15 oz/gal) of streptomycin sulphate. Before DNA extraction, isolates were subcultured in a 2% (w/v) liquid malt extract (AppliChem GmbH, Darmstadt, Germany) medium for approximately 2 weeks at room temperature. Cultures were harvested by filtration and lyophilized. Taxon-Specific Primers Design and Testing DNA was extracted from lyophilized mycelia or dried fruiting bodies specimens using a modified cetyltrimethylammonium bromide extraction method (Hayden et al. 2004). Ribosomal DNA amplifications of the 5´ end portion of the nuclear large subunit and of the portion including the internal transcribed spacers (ITSI and ITSII) were performed using the fungal-specific primers pairs ctb6 and tw13 (White et al. 1990; O’Donnell 1993) and ITS1-F and ITS4 (White et al. 1990; Gardes and Bruns 1993), respectively. For taxa lacking suitable portions for taxon - specific primer design in the previously mentioned regions, a portion of mitochondrial small subunit was amplified using primers MS1 and MS2 (White et al. 1990). PCR products were cleaned by using Qia-quick purification kit (Qiagen, Valencia, CA) and cycle-sequenced with a BigDye Terminator v. 3.1 cycle-sequencing kit (Applied Biosystems, Foster City, CA). Sequencing reactions were loaded on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). Additional sequences available in the European Bioin- formatics Institute nucleotide sequences database (EMBL-EBI; www.ebi.ac.uk ) were used to increase the sample size of target Table 1. Multiplex polymerase chain reaction (PCR) primer combinations, diagnostic purposes, and positively tested species. Multiplex PCR Primers combination z M1 Forward ITS1-F F115 M2 ITS3 25sF M3 ITS3 Reverse ITS4 Gano2R Hyme2R Armi2R LaetR Pleu2R Heri2R PerR Schi2R Ste2R MS1 Mgano ITS1-F Ustu2R TraR GadR GapR GlR GrR Mhyme 25sF FomR FuscR IdryaR InocuR InssR PhssR Taxon-specific amplicon size /taxon identified 600–850 bp/fungi 226–228 bp/ Ganoderma spp. 111 bp/ Inonotus spp.– Phellinus spp. 185 bp/ Armillaria spp. 146 bp/ L . sulphureus 158 bp/ Pleurotus spp. 200 bp/ Hericium spp. 152 bp/ P. fraxinea 190 bp/ Schizophyllum spp. 231–236 bp/ Stereum spp. 260 bp/ U. deusta 220 bp/ Trametes spp. 211 bp/ G. adspersum 200 bp/ G. applanatum 193 bp/European G. lucidum 178 bp/ G. resinaceum 258 bp/ Fomitiporia 225 bp/ Fuscoporia 254 bp/ Pseudoinonotus 265 bp/ Inocutis 214 bp /Inonotus s.s. 173 bp/ Phellinus s.s. G. adspersum , G. applanatum , G. lucidum , G. resinaceum I. andersonii , I. dryophilus , I. dryadeus , I. hispidus , I. radiatus , I. tamaricis , P. gilvus , P. igniarius , P. pini , P. punctatus , P. robustus , P. torulosus , P. tremulae , P. tuberculosus A. gallica , A. mellea , A. nabsnona L. sulphureus P. ostreatus , P. pulmonarius H. coralloides , H. erinaceum P. fraxinea S. commune , S. radiatum S. hirsutum , S. rugosum , S. sanguinolentum U. deusta T. cervina , T. versicolor , T. zonatella G. adspersum G. applanatum G. lucidum (from Europe) G. resinaceum , G. lucidum (from North America) P. punctatus , P. robustus P. gilvus , P. torulosus I. dryadeus I. dryophilus I. andersonii , I. hispidus P. igniarius, P. tremulae, P. tuberculosus z Primers are reported in Guglielmo et al. (2007, 2008). Primer for Ustulina deusta is reported in this study. ©2009 International Society of Arboriculture Positively tested fungal species
January 2009
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