Detecting and Identifying Wood Decay Fungi Using ... - ACS Publications

Chapter 20

Detecting and Identifying Wood Decay Fungi Using DNA Analysis

Downloaded by FUDAN UNIV on April 27, 2017 | http://pubs.acs.org Publication Date: March 31, 2003 | doi: 10.1021/bk-2003-0845.ch020

Jody Jellison, Claudia Jasalavich, and Andrea Ostrofsky Biological Sciences Department, University of Maine, Orono, ME 04469

Amplification of genomic DNA by the polymerase chain reaction (PCR) is a sensitive and specific tool that can be used to detect degradative fungi in wood. PCR coupled with other molecular methods has been used to detect specific fungal products and to identify and survey fungi in the environment. Researchers have used PCR methods to detect and monitor specific fungal degradative genes, to detect and identify basidiomycetes in culture, and to sequence ribosomal DNA for taxonomic studies of selected wood decay fungi. In our work basidiomycete specific primers derived from fungal ribosomal DNA sequences were shown to detect the presence of fungal DNA in spruce wood, even at very early stages of decay. When used in conjunction with restriction fragment length polymorphism (RFLP) analysis, this procedure was able to provide reliable fungal species identification. The assay is currently being developed for use in wood of other species and in wood composite materials. The ultimate goal of this work is to develop a reliable and sensitiveDNA-based assay to detect incipient decay in wood and identify the fungi involved.

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© 2003 American Chemical Society Goodell et al.; Wood Deterioration and Preservation ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Introduction The primary decomposers of wood in non-aquatic environments are the white and brown rot fimgi. Thesefimgiare capable of causing significant modification of the wood lignocellulose and also cause dramatic reductions in wood strength properties. With brown rotfimgisignificant strength loss can occur during early stages of decay, before visual indications of infection and degradation become apparent. Thus, it is necessary to identify the presence of incipient decay in structures for control and/or remediation or replacement purposes. However, the detection and identification of wood degrading fungi can be both difficult and time consuming. Earlier methods include isolation and culturing of the fungus, sonics, electrical resistance, dyes, ergosterol and chitin assays, immunoassays and other techniques (1). However, many methods are either unreliable or necessitate extensive destructive sampling. More recently our laboratory and others have explored the use of the polymerase chain reaction (PCR) (2) and other molecular techniques to aid in the detection and identification of degradativefimgiin the early stages of fungal attack. PCR amplification of genomic DNA can be both specific and sensitive. Useful strategies include RAPD analysis (3), targeting specific degradative genes, e.g. genes coding for enzymes involved in wood decay or bioremedialion, or targeting the ribosomal DNA (rDNA). Ribosomal DNA is found universally and exists in high copy number as tandem repeats of several hundred copies; thus it is a good target for the detection of dilute or partially degraded DNA. Ribosomal DNA contains both variable and conserved regions; this makes it useful for comparing organisms at different taxonomic levels. Each tandemly repeated unit of nuclear rDNA contains the coding regions for the 18S, 5.8S and 28S rRNAs separated by the internal transcribed spacers (ITS), with ITS I lying between the 18S and 5.8S rDNAs, and ITS II between the 5.8S and 28S rDNAs. These tandemly repeated units are separated by the intergenic spacers (IGS). The l&S, 5.8S and 28S rDNAs are generally highly conserved, while the ITS and IGS show greater variability and, therefore, are potentially useful for interspecific and intraspecific studies, respectively. In particular, a portion of the tandem repeat, the ITS region (ITS I-5.8S rDNA-ITS II) has been extensively used for detection and identification of fimgi. White et al (4) established the utility of rDNA amplification and sequencing. Additional researchers have developed rDNA PCR protocols for use in combination with restriction fragment length polymorphism (RFLP) analysis (5, 6 7, 8\ temperature gradient gel electrophoresis (9), ELISA amplicon detection (10), automated fluorescent capillary electrophoresis (//), and single-strandKX>nformation polymorphism (SSCP) analysis (12). t

Goodell et al.; Wood Deterioration and Preservation ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Detection of Specific Degradative Genes Detection of the genes or messenger RNAs (mRNA) of specific fimgal degradative enzymes in culture and in soil has been reviewed by Reddy and D'Souza (75). Degradative enzymes of interest include lignin peroxidase, manganese peroxidase, laccase and selected cellulases. Lamar et al (14) demonstrated the use of reverse transcription PCR (RT-PCR) to quantify specific fungal mRNAs for lignin peroxidase (lip) genes and cellobiohydrolase (cbh) genes in soil colonized by Phanerochaete chrysosporium. Subsequently, the expression of all ten lip genes in soil (both mRNA transcripts and translated proteins) and concurrent oxidation of anthracene by P. chrysosporium were studied (15). Bogan et al. (16) monitored manganese peroxidase mRNA and enzyme levels in soil during bioremediation studies of polycyclic aromatic hydrocarbons. D'Souza et al (17) used primers based on conserved sequences around two copper binding regions to isolate laccase genesfromwhite rot fimgi. Our laboratory has used degenerate laccase primers to amplify DNAfromwhite rot, brown rot and assorted wood-inhabiting ascomycetes (Jasalavich, unpublished). Catechol 2,3-dioxygenase specific primers have also been used to monitor bioremediation processes in petroleum-amended soil by Pseudomonas sp. using competitive quantitative PCR (18).

Environmental Sampling PCR technology has been used to sample the environment for the presence of specific fungi and fungal products. Johnston and Aust (6) amplified ligninase H8 DNA and the nuclear ITS region to monitor P. chrysosporium in the soil. Lamar et al (14) quantified fungal mRNAs for lignin peroxidases, cellobiohydrolases, and B-tubulin using RT-PCRfromsoil colonized by P. chrysosporium. They also used this method to monitor the differential expression of complex gene families in soil versus liquid culture in organopollutant degradation studies (75). Bacterial and fungal soil communities have been characterized using ribosomal DNA. Ranjard et al (18) exploited length polymorphisms in die nuclear ITS region to profile soil fungal communities and IGS fingerprints to profile soil bacterial communities. Van Tuinen et al. (20) used taxon-specific primers based on two variable regions in the 28S rDNA to monitor patterns of colonization of onion and leek roots by four different species of arbuscular mycorrhizae from soil Smit et al (9) coupled PCR amplification usingftmgalspecific primers based on the 18S rDNA with temperature gradient gel electrophoresis (TGGE) to profile fungal communities in the wheat rhizosphere. Schmalenberger et al (12) studied how


349 primer selection and the targeting of different variable regions of the 16S r D N A (analogous to die 18S r D N A i n fimgi) affect the detected composition o f bacterial profiles i n soil i n P C R based microbial community analysis. Amplification of the nuclear ITS region has also been used to identify ancient fungi trapped in the ice of the Greenland glacier (21).


Detection and Identification of Basidiomycetes in Culture The use of P C R amplification specifically for the detection and identification of basidiomycete fungi was aided by the early work of Gardes and Brims (22) who reported the use of a fungal specific primer ITS1-F i n combination with a basidiomycete specific primer ITS4-B for the detection erf mycorrhizal fimgi. The tree parasites and wood-attacking basidiomycetes Heterobasidium annosum mdArmillaria ostoyae have been identified by taxon specific P C R (8, 23, 24). Zaremski (25) characterized tropical wood-decaying fungi by R F L P analysis of the ITS region amplified with the universal primers ITS 1 and ITS 4 from D N A isolated from pure fungal cultures. Since differences i n the original amplicon lengths were not sufficient to distinguish among the five brown rot and three white rot species examined, amplicons were digested with restriction enzymes and electrophoresed in gels to generate R F L P profiles of the fiingi. The eight species were separated by their R F L P profiles. Subsequent work allowed the design of species-specific primers. Moreth and Schmidt (26, 27) have used species-specific P C R primers as a diagnostic tool for the European dry rot fungus Serpula lacrymcms. They amplified and sequenced the ITS region from several isolates of each of seven species o f indoor rot basidiomycetes. Species-specific primers based on the sequence o f internal transcribed spacer II (ITS II) were designed for differential diagnosis of Serpula lacrymcms, Serpula himantioides, Donkioporia expansa, Coniophora puteanna, Antrodia vailantii, Tyromyces placenta and Gloeophyllum sepiarium*

Taxonomic Relationships Taxonomic relationships among fungi have also been examined using similar P C R techniques. Ribosomal D N A has been used to estimate phylogenetic relationships in plant pathogenic fimgi (28, 29) and wood decay fungi (30, 31, 32). Lieckfeldt et al. (33) used phylogenetic analyses o f D N A sequences of the nuclear ITS region and the 5'-end of the 28S r D N A , along with R F L P analysis of the endochitinase gene and P C R fingerprinting, to separate two morphological types of the wood-inhabiting ascomycete Trichoderma viride



350 into two species. Harrington and Wingfield (34) developed a PGR~based method to identify isolates of the tree pathogen Armillaria to species. They separated 74 isolates into 11 Armillaria species by RFLP analysis of the amplified intergenic spacer (IGS). Chillali et al (35) detailed the delineation of European Armillaria species based on ITSI and ITSII polymorphisms. Phylogenetic analysis of nuclear 28S rDNA sequence data supports division of the genus Paxittus into three genera, Paxillus, Tapinella and a new genus Austropaxillus (30). The genus Leucogyrophana is polyphyletic based on analysis of the nuclear 28S rDNA (31). Hibbett and Donoghue (32) have generated a phylogenetic framework based on sequences of the mitochondrial small subunit rDNA and the nuclear 18S rDNA for wood decay fimgi in several families of Homobasidiomycetes. Significant work has also been done on the taxonomic characterization of the European dry rot fungus Serpula. Theodore et al. (36) reported on RAPD polymorphisms among isolates of S. lacrymans. Schmidt and Moreth (37) found RAPD homogeneity within S. lacrymans and polymorphism within SL himantioides. More recently White et al (38) reported on the molecular analysis of intraspecific variation between building and wild isolates of S. lacrymans and their relationship to S. himantioides. Random amplified polymorphic DNA (RAPD) analysis did not distinguish between wild Himalayan and building isolates of S. lacrymans, as there was very little intraspecific variability in this species. However, the two species S. lacrymans and S. himantioides were clearly resolved by both RAPD and ITS rDNA sequence analysis. ITS sequence data suggest that an American isolate of S. lacrymansfromMount Shasta in California is intermediate between European S. lacrymans and S. himantioides (39).

Detection of Degradative Fungi in Wood Detection and Identification in Spruce Wood

Much of the work detailed above focuses on the detection of gene transcripts of specific fungal products, or the identification and analysis of fungal species and/or isolates in culture. The use of universal primers makes detection and identification in the field problematic due to potential amplification of DNA from microbial contaminants (40) or constitutive members of the microbial community in the wood or soil environment being sampled Our recent work has focused on the use of basidiomycete specific primers to detect degradativefimgiin the wood substrate. Basidiomycete specific primers were used because of their greater utility in diagnosing decay organisms in general versus targeting a specific species with the risk of missing infection by numerous other degradative organisms. The ITS region of the



351 rDNA was selected because its high copy number makes it a more sensitive and appropriate target for detection in environments such as wood where total fungal DNA levels could be low during incipient decay as well as late term decay when DNA degradation can occur. Protocols for DNA extraction, PCR amplification and restriction digestion to generate RFLPs have been previously outlined (5, 4l\ Initial work indicated that primer pairs ITS1-F (specific for higher fimgi) and ITS4 (universal primer) and ITS1-F and ITS4-B (specific for basidiomycetes) could be used effectively to detect and differentiate decay fungi and non-degradative wood-inhabiting ascomycetes both in pure culture and in spruce wood. Fifty seven isolates representing 14 species of decay fungi and 25 species of woodinhabiting ascomycetes were used to demonstrate assay specificity with DNA isolated from pure culture. Seven brown rot fungi, 13 white rot fungi and 10 wood-inhabiting ascomycetes were used to test the utility of this approach for the detection and identification of the fungi in spruce wood Primer pair ITS1-F and ITS-4 successfully amplified DNAfromall samples, as expected. Primer pair ITS1-F and ITS4-B amplified DNAfromonly the white and brown rot fimgi and not from the wood-inhabiting ascomycetes (Figure 1). Although amplicon length, as estimated by gel electrophoresis, ranged from 850 to 1,460 bp, this characteristic was not diagnostic for individual fungal species, since several fungal species had amplicons of the same or similar length. The identities of the white and brown rotfimgiwere confirmed by comparing RFLP profiles (Figure 2). Time studies demonstrated that the fungi could be detected very early in the decay process, before significant weight and strength loss to the wood had occurred (5). Detection in Oak Wood and in Wood Composites Subsequent work has suggested that caution may be warranted when attempting to amplify DNA directly out of some wood species, as the DNA extraction method developed for red spruce (5) does. not always yield amplifiable DNAfromother wood species. For example, attempts to detect both brown rot and white rotfimgiin red oak using our original DNA extraction method were poor and inconsistent. Recovery of amplifiable DNAfromred oak can be improved by modification of this DNA extraction protocol to reduce phenol polymerization (42\ However, additional work is needed to make detection of fungi in red oak reliable. Detection of decay in oriented strand board (OSB) was also examined because of the increased utilization of composite wood products, and concerns that glues and other additives might inhibit the detection of fungal DNA. The OSB contained a phenolic resin. Poplar wood comprised 80-90% of the wood component of the OSB with the remaining 10-20% a mix of northern conifer



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Figure 1. PCR amplification of nuclear rDNAfromtotal DNA isolatedfromred spruce wood blocks colonized by wood decay fungi or endophytes. Electrophoresis in 2% (w/v) agarose in 1X TBE. The two outer lanes contain molecular weight markers. Inner even-numbered lanes contain samples amplified by the primer pair ITS1-F and ITS4-B, and the odd-numbered lanes contain samples amplified by primers ITS1-F andTTS4. Lanes 2 to 7, brown rot basidiomycetes; lanes 8 to 13, white rot basidiomycetes; lanes 14 to 17, endophytic ascomycetes. Lanes 1 and 20, PCR markers (Promega); lanes 2 and 3, Postia placenta Mad-698-R; lanes 4 and 5, Gloeophyllum trabeum Mad-617-R; lanes 6 and 7, Leucogyrophana pinastrr, lanes 8 and 9, Lentinula edodes 117=lt(d); lanes 10 and 11, Trametes versicolor, lanes 12 and 13, Scytinostroma galactinum ATCC 64896; lanes 14 and 15, Hormonema dematiodesr, lanes 16 and 17, Pestalotiopsis sp.; lanes 18 and 19, no template DNA (i.e., negative controls). (Reproduced with permissionfromreference 5. Copyright 2000 Society for Microbiol ogy, Journals Department.)



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Figure 2. Taql restriction digests of the PCR product amplified by the primer pair ITS1-F and ITS4-B from DNA isolated from pure cultures of basidiomycetes. Electrophoresis in 2% (w/v) Sepharide Gel Matrix (GibcoBRL) in 1 X TAE. The two outer lanes contain molecular weight markers. Each inner lane contains a different fungal species; lanes 2 to 8 contain brown rot fungi, and lanes 9 to 15 contain white rot fungi Lanes 1 and 18, PCR markers (Promega); lane 2, Coniphora puteana Fp-90099-Sp; lane 3, Fomitopsis pinicola K8Sp; lane 4, Gloeophyllum sepiarum 10-BS2-2; lane 5, Gloeophyllum trabeum Mad-617-R; lane 6, Leucogyrophana pinastri; lane 7, Postia placenta Mad-698-R; lane 8, Serpula lacrymans Harm-888-R; lane9, Irpex lacteus KTS003; lane 10, Lentinula edodes 117=lt(d); lane 11, Phanerochaete chrysosporium ATCC 24725; lane 12, Resinicium bicolor ATCC 64897; lane 13, Scytinostroma galactinum ATCC 648%; tone 14, Trametes versicolor Fp-101664-Sp; lane 15, Trichaptum abietinum 1247 MIL; lane 16, Pisolithus tinctorium ATCC 38054, an ectomycorrhiza; lane 17, Rhizoctonia solani 1AP, a pathogen of herbaceous plants.

(Reproduced with permission from reference 5. Copyright 2000 Society for Micro ogy, Journals Department.)


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wood Initially, we used the DNA extraction method developed for red spruce


(5). The brown rot fungi G . sepiarium, Gloeophyllum trabeum and S. lacrymans, and the white rot fungi Irpex lacteus, P. chrysosporium and

Trametes versicolor were detectedfromcolonized OSB blocks with both primer pairs. We were unable to detect Postia placenta and Leucogyrophana pinastri using either primer pair. These problematic samples were heavily degraded and darkly colored. Modification of die original DNA extraction procedure to reduce phenol polymerization (42) was required to achieve reliable detection of all wood decayfiingitested in OSB (Table I). A time course study of OSB blocks infected with the white rot fungus T. versicolor or the brown rot fungus G. trabeum showed reliable detection at all time points for the representative white rot and reliable detection of the brown rot at weeks 1, 2, 3 and 4, but variable detection at week 8 with difficulty amplifying fungal DNAfromthe OSB samples which had heavy degradation. Diagnostically, this is not a practical problem as wood and manufactured wood products at these later stages of fungal degradation show extensive structural modification, weight loss and dark coloration which are easily detected.

a

Table L Detection of wood decayfiingiin oriented strandboard (OSB) Species

% Weight Loss PCR Amplification Mean±SD JTS1-F/JTS4 ITS1-F/ITS4-B h

Brown rot basidiomycetes Gloeophyllum sepiarum Gloeophyllum trabeum Leucogyrophana pinastri Postia placenta Serpula lacrymans

32.4 ± L8 36.1 ± 4.0 44.3 ± 6.9 48.8 ± 7.5 19.9 ± 4.5

• i i I•

••Jil

MM

(Ml MM

++++ -H-H-

1111

++++ ++++

White rot basidiomycetes Irpex lacteus Phanerochaete chrysosporium Trametes versicolor

27.9 ± 8.1 20.3 ± 14.3 25.6 ± 4.5

MM

++++

+-H-+ -H-H-

-H-HM M

a

OSB blocks were harvested after 12 weeks of colonization, and die DNA was isolated using an extraction procedure modified to reduce phenol polymerization (55). ^ e a n of four replicate blocks. °Each plus or minus sign represents the PCR amplification results for one OSB block. Negatives confirmed by reamplification.


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Sequence Data and Specific Primers We have sequenced the nuclear ITS region for a number of wood decay basidiomycetes for use in specific primer design and taxonomic analysis. Amplicon lengths obtained using primers ITS1-F and ITS4-B rangedfrom800 to 911 base pairs. All insertions and deletions were observed in the internal transcribed spacers ITS I and ITS II, with a few base substitutions occurring in the 5.8S rDNA. Sequences from our isolates of S. lacrymans are similar to those of isolates sequenced by Palfreyman's group (43) and Schmidt's group (27). Both of these laboratories have recendy submitted DNA sequences of die ITS region for a number of wood decay basidiomycetes to international nucleotide databases (26, 27, 38, 43, 44). We have also sequenced a portion of the mitochondrial small subunit rDNA for several wood decay basidiomycetes.


:

Applications and Limitations Primers of varying specificity can be designed to suit differing applications. Our work using basidiomycete specific primers clearly indicates the specificity and sensitivity of this approach. Particularly encouraging was our ability to detect degradative organisms in wood at the incipient decay stage, suggesting that the sensitivity of molecular assays could be utilized to allow decay detection in in-service wood products before significant strength loss or other damage had occurred Where necessary, individual degradative species could be identified based upon amplification and subsequent restriction digest patterns. Sequence information or amplification by species-specific primers was done by direcdy sampling the wood products without the need for culturing and traditional identification procedures. Work by us and many others has demonstrated the utility of molecular approaches in the detection and identification of wood decay fungi and fungal metabolites in their natural environments of soil (14) and wood (5). DNA-based molecular techniques provide a powerful tool to examine wood degradative processes (6, 13) and taxonomic relationships (38) among the wood degrading fimgi.

Acknowledgements The authors would like to acknowledge the support of the Maine Agricultural and Forestry Experiment Station and USDA-WUR grants #: 95-34158-1347 and #: 99-34158-8489. This is publication # 2527 of the Maine Agricultural and Forestry Experiment Statioa


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