14 Nicolotti et al.: Detection of Wood Decay Fungi Arboriculture & Urban Forestry 2009. 35(1):14–19. A Biomolecular Method for the Detection of Wood Decay Fungi: A Focus on Tree Stability Assessment Giovanni Nicolotti , Paolo Gonthier , Fabio Guglielmo , and Matteo M. Garbelotto Abstract. The detection and identification of wood-rotting fungi in standing trees is crucial for the prediction of the severity and evolution of decay. In the case of very active root and butt rot fungi, an early identification method is important to establish the more appropriate failure risk classification. This work is aimed at reviewing the biomolecular methods recently developed to iden- tify, directly from wood, some of the most important and widespread decay fungi. The whole method is based on the use of taxon- specific primers combined in five multiplex polymerase chain reactions (PCRs). Three multiplex PCRs allow identifying Armillaria , Ganoderma , Hericium , Inonotus , Laetiporus sulphureus , Perenniporia fraxinea , Phellinus , Pleurotus , Schizophyllum , Stereum , Trametes , and Ustulina deusta . The two remaining multiplex PCRs were developed for subgeneric identification of fungi belonging to Ganoderma , Inonotus, and Phellinus . In validation assays, multiplex PCRs allowed successfully detecting fungi in 83% of wood samples collected from decay-affected trees. Thus, the methods proved to be efficient and specific for the diagnosis and the early detection of decay fungi on standing trees. Key Words. Identification; molecular diagnosis; tree stability; visual tree assessment (VTA). Wood decay fungi are peculiar because of their ability to decom- pose lignified cell walls using enzymatic and nonenzymatic sys- tems (Blanchette 1991). Although in forest ecosystems wood decay fungi play a positive role as primary biotic decomposers of wood logs and debris, in urban landscapes they pose serious risks by affecting the structural integrity of roots and trunks of standing trees. The loss of wood mechanical strength caused by these organisms is inherently linked to hazardous situations such as tree wind throws or limb failures, often resulting in significant damage of property and injuries. Furthermore, the establishment of decay in living trees is positively affected by urban envi- ronmental stresses that range from a general weak ening of the tree defense system to frequent injuries on branches and roots, allowing the wood-rotting agents to gain entry through wounds (Lonsdale 1999). The detection of potentially hazardous trees is mainly based on visual tree assessment (VTA), an approach based on a visual inspection of signs and symptoms associated with anomalies in the structure of trees (Mattheck and Breloer 1992). However, VTA rarely allows the detection of incipient decay or the iden- tification of the rotting fungi involved. Although modern tech- nologies are improving our ability to detect internal wood decay (Tomikawa et al. 1990; Habermehl et al. 1999; Müller et al. 2001; Sambuelli et al. 2003), the identification of the agents responsible for such decay is not always feasible without the presence of fun- gal fruiting bodies, which are sporadically visible and frequently show overlapping characters that can lead to confusion. Despite it all, because the biology and ecology of these fungi are variable, their identification is important to predict, to some extent, the typical pattern of spread within the tree, the effect on wood strength (Lonsdale 1999), and the risk of spread from one tree to the neighboring ones. This is particularly important for rapidly progressing root and butt rot fungi that can turn a sound tree into a hazard in a short period of time. ©2009 International Society of Arboriculture Several techniques that have been used to detect incipient decay include isolation and characterization of fungal mycelia followed by the comparison of growth rates, enzymatic capabilities, and biochemical and immunologic assays (Nobles 1965; Stalpers 1978; Anselmi and Bragaloni 1992; Jellison and Jasalavich 2000; Clausen 2003). The main drawback of culture diagnosis is the difficult and time-consuming process required to obtain pure culture isolates from incipient decayed wood. Moreover, the identification of closely related taxa based on the examination of mycelial characters is often complicated and impractical in some instances. Biochemical and immunologic techniques were largely developed for the detection and identification of common brown rot fungi from lumber (Jellison and Jasalavich 2000; Clausen 2003). Although such methods do not always require pure cul- ture isolation, they lack sensitivity for detection of fungi in the early stage of infection. As already reported (Guglielmo et al. 2007), the techniques based on fungal DNA detection are a promising alternative for specific, sensitive, and rapid routine diagnoses directly from wood samples. Polymerase chain reaction (PCR) methods based on taxon-specific primers designed on nuclear or mitochon- drial ribosomal DNA (rDNA) loci proved to be effective for the identification of fungi at different taxonomic levels (Moreth and Schmidt 2001; Bahnweg et al. 2002; Gonthier et al. 2003). Furthermore, multicopy arrangement and highly conserved priming sites, typical of both nuclear and mitochondrial rDNA, permit amplification from virtually all fungi, even if the starting sample is lacking in quantity or quality such as DNA extracts directly from wood (Jasalavich et al. 2000). As an added benefit, the simultaneous application of taxon-specific primers in multi- plex PCR reactions has been reported in clinical and food micro- biology to increase the diagnostic capacity of PCR (Elnifro et al. 2000; Corbiere Morot-Bizot et al. 2004). The objective of this study was to develop a multiplex PCR- based method for the identification of some of the most important
January 2009
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