A molecular diagnostic assay for the detection and identification of wood decay fungi of conifers

Abstract

Ten taxon-specific primers were designed to amplify the Internal Transcribed Spacer of the rRNA operon of several important decay fungi of coniferous wood, including Armillaria spp., Echinodontium spp., Fomitopsis pinicola, Fuscoporia torulosa, Heterobasidion annosum sensu lato (s.l.), Onnia spp., Phaeolus schweinitzii, Phellinus weirii s.l., Pholiota spp. and Porodaedalea spp. Primers designed in this study and in a previous one for the identification of Laetiporus sulphureus and Stereum spp. were combined in two multiplex PCRs, which were tested for efficiency and specificity, and detected at least 1 pg of fungal target DNA. Target DNA at concentrations of 10 À1 pg or lower can be detected with this assay using SYBR â Green Real-Time PCR. Validation assays performed on 129 naturally infected wood samples or fruiting bodies confirmed the reliability of the multiplex PCR-based diagnostic method. This method represents a simple and rapid diagnostic tool for the detection of a number of destructive wood decay fungi of conifer wood.

Development of oligonucleotide microarrays for simultaneous multi‐species identification of Phellinus tree‐pathogenic fungi

“…Multiplex PCR-based methods have previously been used to identify Phellinus s.l. at a generic rank (Guglielmo et al, 2007;2008), and ITS sequences have been used in the development of primers for PCR-based Phellinus species identification techniques (Nam et al, 2002;Gonthier et al, 2015). It is known that microarray techniques can facilitate the simultaneous identification of multiple pathogenic species in a high-throughput manner (Martin et al, 2000;L evesque, 2001;Tsui et al, 2011).…”

“…It has been proposed that ITS regions can serve as a universal barcode marker for fungi, as they provide the broadest range of inter-and intra-species differentiation currently known (Schoch et al, 2012). Internal transcribed spacer sequences have previously been used to develop primers for PCR-based identification of Phellinus species (Nam et al, 2002;Gonthier et al, 2015), but to the best of our knowledge, this is the first study to utilize ITS regions in the development of DNA microarrays for detection of the Phellinus genus s.l. We were able to specifically resolve 17 key Phellinus species on our microarray system, including Phellinus apiahynus, P. cesatii, P. gilvus, P. linteus, P. inermis, P. laevigatus, P. melleoporus, P. membranaceus, P. noxius, P. pini, P. quercinus, P. ribis, P. igniarius, P. formosanus, P. pachyphloeus, P. torulosus (now reclassified as Fuscoporia torulosa) and P. weirii.…”

Multiplex real‐time PCR assays for the detection and identification of Heterobasidion species attacking conifers in Europe

“…The presence of Heterobasidion spp. in these samples was confirmed through molecular analysis performed using the tool developed by Gonthier et al (2015). Three out of five samples had tested positive to H. irregulare based on the LAMP assay described by Sillo et al (2018).…”

Application of MALDI‐TOF mass spectrometry for in vitro identification of wood decay polypores

“…Until recently, fungal identification in phytopathological laboratories has mainly relied on conventional phenotypic and modern molecular techniques (Gonthier, Guglielmo, Sillo, Giordano, & Garbelotto, 2015). Moreover, certain traditionally recognized morphological species are known to occur as complexes of cryptic species, and some of them have been identified using molecular markers in recent decades.…”