16 taxa and ensure specificity of primers by including as many rep- resentatives as possible of the target taxon and closely related nontarget taxa. Domains conserved within a taxon, but variable among taxa, were chosen as target regions for taxon-specific primer design. To identify these regions, all sequences from a given taxon were aligned using CLUSTALW (Thompson et al. 1994) with one sequence of a closely related species used for out- group comparison. Taxon-specific primers to be used in multiplex PCR were designed on the selected regions using the software PRIMER3 ( www.genome.wi.mit.edu/cgi-bin/primer/primer3 ) to be highly specific for the target fungal taxa. The annealing temperature of each primer pair was optimized using a thermocycling gradient to improve PCR efficiency with the highest stringency. Multiplex Polymerase Chain Reaction Development Multiplex PCR was performed by combining taxon-specific reverse primers with similar annealing temperatures and variable amplicon sizes (Guglielmo et al. 2007). Amplified DNA frag- ments were visualized on agarose gel. Validation of the Polymerase Chain Reaction- Based Method on Field Samples Wood samples from trees with evident decay symptoms were analyzed to test the sensitivity and reliability of the method. Sampled trees belonged to 19 species and 15 genera, including Acer , Aesculus , Cedrus , Celtis , Fagus , Juglans , Malus , Platanus , Populus , Prunus , Quercus , Robinia , Sophora , Tilia , and Ulmus . Wood samples were obtained either through a Swedish increment borer near the point of fruiting body emergence or through a scal- pel where incipient decay was visible. Wood samples, approximately 150 mg (5.25 10 −3 oz fresh weight) each, were lyophilized, homogenized through a FastPrep FP120 Cell Disrupter (Qbiogene, Irvine, CA) and extracted with the QIAamp DNA Stool Mini Kit (Qiagen). Each DNA extract was diluted 100-fold and amplified through the multiplex PCR- based method. The expected fungal taxon per each sample was assessed either through the analysis of macroscopic features of the fruit- ing bodies or through BLAST search analysis of the sequenced ITS region amplified with the primers pair ITS1-F and ITS4. The diagnostic efficiency of the method was determined by evaluating its ability to detect and correctly identify the fun- gal taxon expected per each sample (Hickman and Perry 1997; Bernicchia 2005). RESULTS Taxon-Specific Priming Multiplex Polymerase Chain Reactions Taxon-specific primers (Guglielmo et al. 2007, 2008) were combined in five multiplex PCR reactions ( Table 1 ). Taxon- specific primer for Ustulina deusta was designed for this study (5'-GCTCATCTCTACAGGCGAGAA-3'). The universal fungal primers ITS1-F and ITS4 were used in one multiplex (M1) to evaluate the efficiency of fungal DNA extraction, avoiding, thus, ©2009 International Society of Arboriculture Nicolotti et al.: Detection of Wood Decay Fungi possible false-negatives resulting from either undetectable DNA quantities or PCR inhibitory compounds. M1 also allows identi- fication of fungi belonging to the genera Ganoderma and Inon- otus or Phellinus . M2 allows identifying Armillaria , Hericium , Laetiporus sulphureus, and Pleurotus , M3 was suitable for the detection of Perenniporia fraxinea , Schizophyllum , Stereum , Trametes, and Ustulina deusta . Mhyme and Mgano have been developed to identify at a subgeneric rank taxa belonging to the Inonotus – Phellinus group and Ganoderma , respectively. Using the optimized reaction parameters, each multiplex PCR allowed to efficiently amplify the corresponding target taxon DNA fragment without crossreacting with nontarget DNA or pro- ducing any ambiguous or extra amplicons (data not shown). The amplified fragments in multiplex reactions were easily differentiated and scored according to the size using gel con- taining 1% (w/v) of high-resolution MetaPhor and 1% (w/v) of standard agarose after a 2 hr or 2 hr 30 min electrophoretic migration at 4 V/cm (1.6 V/in) depending on the multiplex PCR ( Figures 1 and 2 ). Validation of the Polymerase Chain Reaction- Based Method on Field Samples By using the PCR-based method developed in this study ( Figure 3 ), in 83% of the samples, at least one of the target decay fungi was detected ( Figure 4 ). In 66% of the samples, we found consistent matches between multiplex PCR-based results and the expected data. In 17% of the samples, an unexpected target decay fungi was found. Among the unexpected target taxa detected through the PCR-based method, the most common were P. fraxinea instead of Ganoderma spp. and P. torulosus and Stereum spp. instead of the Inonotus–Phellinus group. In 9% of the samples, other nontarget taxa corresponding, after BLAST search analysis, to anamorphic ascomycetes were detected. Figure 1. The results of M1, M2, and M3 visualized on an ultra- violet gel documentation system after 2 hr electrophoresis at 4 V/cm on a 1% Metaphor 1% standard agarose gel. M1 = Polymerase chain reaction (PCR) products from DNA extracts of Trametes versicolor (lane 1), Ganoderma adspersum (lane 2), Inonotus hispidus (lane 3), and Phellinus torulosus (lane 4), M2: PCR products from DNA extracts of Hericium erinaceum (lane 1), Armillaria mellea (lane 2), Pleurotus ostreatus (lane 3), and Laetiporus sulphureus (lane 4). M3: PCR products from DNA extracts of Ustulina deusta (lane 1), Stereum spp. (lane 2), Trametes spp. (lane 3), Schizophyllum spp. (lane 4), and Perenniporia fraxinea (lane 5). M = molec- ular weight marker 100 bp DNA ladder. Molecular weights are indicated in base pairs (bp).
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