Detection and Identification of Decay Fungi in Spruce Wood by Restriction Fragment Length Polymorphism Analysis of Amplified Genes Encoding

rRNA
Claudia A. Jasalavich, Andrea Ostrofsky and Jody Jellison Appl. Environ. Microbiol. 2000, 66(11):4725. DOI: 10.1128/AEM.66.11.4725-4734.2000.

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2000, p. 47254734 0099-2240/00/$04.00 0 Copyright 2000, American Society for Microbiology. All Rights Reserved.

Vol. 66, No. 11

Detection and Identication of Decay Fungi in Spruce Wood by Restriction Fragment Length Polymorphism Analysis of Amplied Genes Encoding rRNA
CLAUDIA A. JASALAVICH, ANDREA OSTROFSKY,
AND

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JODY JELLISON*

Department of Biological Sciences, University of Maine, Orono, Maine 04469-5735

Received 6 March 2000/Accepted 12 July 2000

We have developed a DNA-based assay to reliably detect brown rot and white rot fungi in wood at different stages of decay. DNA, isolated by a series of CTAB (cetyltrimethylammonium bromide) and organic extractions, was amplied by the PCR using published universal primers and basidiomycete-specic primers derived from ribosomal DNA sequences. We surveyed 14 species of wood-decaying basidiomycetes (brown-rot and white-rot fungi), as well as 25 species of wood-inhabiting ascomycetes (pathogens, endophytes, and saprophytes). DNA was isolated from pure cultures of these fungi and also from spruce wood blocks colonized by individual isolates of wood decay basidiomycetes or wood-inhabiting ascomycetes. The primer pair ITS1-F (specic for higher fungi) and ITS4 (universal primer) amplied the internal transcribed spacer region from both ascomycetes and basidiomycetes from both pure culture and wood, as expected. The primer pair ITS1-F (specic for higher fungi) and ITS4-B (specic for basidiomycetes) was shown to reliably detect the presence of wood decay basidiomycetes in both pure culture and wood; ascomycetes were not detected by this primer pair. We detected the presence of decay fungi in wood by PCR before measurable weight loss had occurred to the wood. Basidiomycetes were identied to the species level by restriction fragment length polymorphisms of the internal transcribed spacer region. Wood is an important renewable and biodegradable natural resource with a multitude of uses. Wood is used extensively as a structural material for buildings, wharves, telephone poles, and furniture due to its high strength per unit weight, its versatility, and its variety. Wood also serves as the industrial raw material for the manufacture of paper and paper products, wood composites, and other products made from cellulose, such as textiles and cellophane. In many parts of the world wood is used as a fuel for heating and cooking. The primary biotic decomposers of wood are basidiomycete decay fungi, which can attack and degrade both wood in the forest and wood in service. In the forest ecosystem wood decay fungi play an important role in carbon and nitrogen cycling and help to convert organic debris into the humus layer of the soil. Some fungi attack living trees; others invade downed timber and slash on the forest oor, lumber, and wood in service. Wood decay basidiomycetes colonize and degrade wood using enzymatic and nonenzymatic processes. Brown-rot fungi preferentially attack and rapidly depolymerize the structural carbohydrates (cellulose and hemicellulose) in the cell wall leaving the modied lignin behind. White-rot fungi can progressively utilize all major cell wall components, including both the carbohydrates and the lignin. As decay progresses the wood becomes discolored and loses strength, weight, and density. Decay and discoloration caused by fungi are major sources of loss in both timber production and wood use, with losses of 15 to 25% marketable wood volume in standing timber and of 10 to 15% in wood products during storage and conversion. Each year ca. 10% of the timber cut in the United States is used to replace wood in service that has decayed, resulting in the
* Corresponding author. Mailing address: Department of Biological Sciences, University of Maine, Orono, ME 04469-5735. Phone: (207) 581-2995. Fax: (207) 581-2969. E-mail: jellison@maine.maine.edu. This work is contribution no. 2407 from the Maine Agricultural and Forest Experiment Station, Orono, Maine. 4725

expenditure of hundreds of millions of dollars for raw materials, labor, and liability (22). Brown rots rapidly and drastically reduce wood strength early in the decay process, while white rots cause a slower progressive decrease in wood strength. Brown-rot fungi can reduce wood strength by as much as 75% at less than 5% weight loss of the wood (22). For this reason it is important to develop methods which can detect wood decay very early, at the incipient stage prior to the occurrence of signicant strength loss. Techniques which have been used to detect incipient decay include isolation and culturing of fungi, chemical staining, nuclear magnetic resonance, and electrical resistance, as well as serological methods, such as immunoblotting and enzyme-linked immunosorbent assay (ELISA) (3). ELISA has been found to be a sensitive method for detecting incipient decay (4, 11), but the assay sensitivity can be inhibited by wood extractives (12). Optimal methods for early detection of decay for wood in service have not been developed. The development of the DNA-based PCR (14) and taxonspecic primers (2, 6, 7, 16, 17) is making it increasingly feasible to detect and study fungi in their natural substrates. A DNA-based method to detect the presence of wood decay fungi would potentially use only small amounts of wood, thus allowing for nondestructive sampling. The extreme sensitivity and potential specicity of the assay would theoretically allow for the detection of fungal decay agents at an incipient stage enabling remedial biocidal treatments to be applied before signicant strength loss had occurred. Detection of specic decay agents is also a necessary prerequisite to allow evaluation of fungal colonization and proliferation in preservativetreated woods undergoing remediation. Specic and sensitive assay procedures would aid in the monitoring and development of successful fungus-based bioremediation technologies. For our DNA-based detection method, we selected the internal transcribed spacer (ITS) region (ITSI, the 5.8S ribosomal DNA [rDNA], and ITSII) as the target sequence for

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at all steps of processing. Mycelia on the surface of the block were removed by gently scraping them with a razor blade, a new blade being used for each block. The fresh weight was recorded. The block was cut in half vertically, i.e., perpendicular to the face of the block that had contacted the colonized feeder strip. One-half was placed in a small plastic sample bag, labeled, and stored at 70C for DNA isolation. The fresh weight of the other half block was recorded, after which it was oven dried at 100C for 48 h, and its dry weight recorded to allow calculation of the nal dry weight of the whole block at harvest based on the following ratio: total fresh weight/calculated total dry weight partial fresh weight/partial dry weight. Wood decay was estimated as the percent dry weight loss as follows: percent weight loss [1 (nal dry weight/initial dry wt)] 100. DNA isolation. DNA was isolated from fresh mycelia taken from the surface of plate cultures, from lyophilized mycelia, or from infected wood by extraction with cetyltrimethylammonium bromide (CTAB) in the presence of -mercaptoethanol, followed by organic extractions and isopropanol precipitation of the DNA. Our method is based on those of Taylor et al. (19) and Wilson (21). For fresh mycelia a 2 CTAB extraction buffer (2% [wt/vol] CTAB; 100 mM Tris HCl, pH 8.0; 1.4 M NaCl; 20 mM EDTA; 0.2% [vol/vol] -mercaptoethanol) was used, with the -mercaptoethanol being added just prior to use. For lyophilized mycelia or dry tissues such as wood samples, a 1 CTAB extraction buffer (diluted 2 buffer) was used. It is especially important to use the 1 CTAB extraction buffer for wood samples; otherwise, the aqueous and organic phases invert due to rehydration of the wood when the 2 CTAB extraction buffer is used. Wood blocks were sampled by drilling through noninoculated wood surfaces. Precautions were observed during drilling of the wood blocks to prevent crosscontamination of samples. Both the work table and gloves were swabbed with 70% ethanol to surface sterilize them and to collect any bits of sawdust before and after drilling each sample. A rechargeable cordless drill was used because it has less surface area, fewer crevices, and no cord to collect dirt and sawdust, and a molded housing which can be easily wiped clean with 70% ethanol. Drill bits were carefully cleaned with laboratory detergent, rinsed, soaked in 95% ethanol, and ame sterilized. A drill bit was inserted through a cone of lter paper (new for each sample), positioned so as to cover the chuck and prevent sawdust from entering it. We drilled through each wood block, on a line perpendicular to the face of the block that had contacted the colonized feeder strip, with a 1/8-in. diameter bit to generate a ne sawdust from which DNA could be isolated directly; no further grinding of the sawdust was needed to achieve good DNA extraction. Once a prepared drill bit was used to drill a wood sample, it was not reused until it had been recleaned and resterilized by the procedure described above. Fresh or lyophilized mycelia was simply ground to a ne powder with liquid nitrogen in a mortar and pestle for use in DNA extraction. Ground or drilled material (100 to 200 l) was transferred to a sterile microfuge tube. Then, 600 l of the appropriate CTAB extraction buffer was added, and the sample was mixed to resuspend the powdered tissue in the buffer and incubated at 65C for 2 h. The sample was extracted with 1 volume of chloroform-isoamyl alcohol (24:1, vol/vol) and centrifuged at 10,000 g for 10 min at room temperature. The aqueous phase was transferred to a new tube, and a 1/10 volume of 10% (wt/vol) CTAB in 0.7 M NaCl was added. After mixing, the sample was incubated at 65C for 1 h. Once again the sample was extracted with chloroform-isoamyl alcohol (24:1, vol/vol) and centrifuged as described above. The aqueous phase was transferred to a new tube and extracted with 1 volume of phenol-chloroform-isoamyl alcohol (25:24:1, vol/vol/vol), followed by centrifugation at 10,000 g for 10 min at room temperature. The aqueous phase was transferred to a new tube, and the DNA was precipitated by the addition of 0.6 volume of ice-cold isopropanol. After incubation at 20C for 30 min, the DNA precipitate was collected by centrifugation at 12,000 g for 15 min. The pellet was washed twice with ice-cold 70% (vol/vol) ethanol and dried. The pellet was resuspended in DNA storage buffer (6 mM Tris HCl, 0.1 mM EDTA; pH 7.5); 100 l was used for DNA isolated from mycelia, and 50 l was used for DNA isolated from wood samples. Incubation at 65C speeded up resuspension of the DNA. PCR amplication. The ITS region was amplied by PCR from DNA isolated from pure cultures of each of the fungi listed in Table 1 and from wood blocks colonized by individual isolates of wood decay basidiomycetes or wood-inhabiting ascomycetes. Primers ITS1-F (CTT GGT CAT TTA GAG GAA GTA A), which is specic for the higher fungi (7), and ITS4 (TCC TCC GCT TAT TGA TAT GC), the universal primer (20), were used together as a positive control for amplication, since they would be expected to amplify the ITS region from both ascomycetes and basidiomycetes. The primer pair ITS1-F and ITS4-B (CAG GAG ACT TGT ACA CGG TCC AG), which is specic for basidiomycetes (7), were used to specically amplify the ITS region from only basidiomycetes. Amplications were performed in 50- l reactions of PCR buffer (10 mM Tris HCl, pH 8.3; 50 mM KCl; 0.001% [wt/vol] gelatin [Perkin-Elmer]), 200 M concentrations of each deoxyribonucleotide triphosphate, and 200 nM concentrations of each of the appropriate primers, with nonacetylated bovine serum albumin (BSA; Sigma A-7906) at 250 ng/ l, total DNA isolated from a pure fungal culture or from a wood decay sample, 0.056 M TaqStart antibody (Clontech), and 0.002 M AmpliTaq DNA polymerase (Perkin-Elmer), i.e., 2 U per 50- l reaction. The TaqStart antibody and AmpliTaq DNA polymerase were mixed together and preincubated prior to being added to the rest of the reaction components as per the manufacturers instructions (Clontech). Samples were

amplication for three reasons. The ITS region is present at a very high copy number in the genome of fungi, as part of the tandemly repeated nuclear rDNA; this, coupled with PCR amplication, should produce a highly sensitive assay. The DNA sequences of the ITSI and ITSII are highly variable; this feature can be exploited to generate restriction fragment length polymorphism (RFLP) patterns to identify wood decay fungi or to design taxon-specic primers. The European Armillaria species Armillaria cepistipes, A. gallica, A. borealis, A. ostoyae, and A. mellea are clearly delimited by RFLPs of rDNA (18). RFLPs generated by restriction digestion of the PCRamplied ITS region have been used successfully to study intraspecic variation in A. ostoyae (17), to identify ectomycorrhizal fungi to the genera and/or species level (5, 7, 8, 9, 10), and to identify intersterility groups of Heterobasidion annosum (6). In designing an assay to detect fungi by PCR amplication using total DNA isolated from infected plant material as the template, it is important to be able to discriminate between DNAs of fungal and plant origin. Primers which specically amplify the ITS region from only fungal DNA (7) and not plant DNA are available. These fungus-specic primers were originally designed to identify fungal symbionts directly from ectomycorrhizae and to identify rusts, which are obligate parasites, in the host tissue (7). More recently, these primers have been used to study the community structure of ectomycorrhizal fungi in a pine forest (8) and the genetic structure of a natural population of Suillus pungens (2). The objectives of our study were (i) to rigorously test the specicity of the basidiomycete-specic primer (7) by surveying a large number of wood decay basidiomycetes, as well as woodinhabiting ascomycetes (pathogens, endophytes, and saprophytes); (ii) to optimize the PCR assay conditions for specic detection of brown-rot and white-rot fungi in wood; (iii) to identify the PCR-detected fungi to species via RFLPs of the amplied internal transcribed spacer region; and (iv) to develop a DNA-based method to detect incipient stages of wood decay, thus allowing remedial treatments to be applied to wooden structural members before substantial strength loss has occurred.
MATERIALS AND METHODS Fungal culture. The fungi used in this study and their sources are given in Table 1. Cultures were grown on plates of malt agar at room temperature for use in DNA isolation or as inocula for soil block jars. Soil block culture. Modied ASTM soil block jars (1) were set up as follows. A soil mix (1:1:1 by volume) was prepared by mixing equal volumes of potting soil, sphagnum moss, and vermiculite and then moistened with deionized distilled water. About 1 cup of the mix was placed in each pint-sized Mason jar, and water was allowed to absorb overnight. The next day, 20 ml of water was added per jar so that the soil mix was moist and a little free water was present. Two pieces of birch tongue depressor were placed on the soil surface to serve as feeder strips. Lids were inverted to prevent sealing and screwed onto the jars with rings. Jars were autoclaved for 30 min. Two days later the jars were again autoclaved for 30 min. The feeder strips in each jar were inoculated with culture blocks (ca. 0.5 cm3) of the appropriate fungal isolate, and one culture block was placed at each end of each feeder strip. In uninoculated control jars, blocks of sterile malt agar were used. Jars were incubated at room temperature to allow fungal colonization of feeder strips. Radial or longitudinal sections of spruce sapwood (1 by 1 by 0.25 in. [1 in. 25.4 mm]) cut from the same tree were oven dried at 100C for 48 h, weighed, and then autoclaved for 30 min enclosed in glass petri dishes. After cooling, the wood blocks were added aseptically to the jars at one block per jar. Each experiment used wood blocks cut from only radial sections or from only longitudinal sections. Wood blocks cut from radial sections were placed so that a transverse face contacted the top of the colonized feeder strips. Wood blocks cut from longitudinal sections were placed so that a longitudinal face contacted the top of the colonized feeder strip. Jars were incubated at room temperature to allow colonization of the spruce blocks. After the appropriate colonization time, the wood blocks were harvested aseptically, observing precautions to prevent cross-contamination of the samples

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VOL. 66, 2000

PCR DETECTION OF WOOD DECAY FUNGI TABLE 1. Amplication of ITS region from DNA isolated from pure fungal cultures

4727

Species

Isolate

Sourcea

Ecologyb

PCR amplicationc with primer pair: ITS1-FITS4 ITS1-FITS4-B

Basidiomycetes Coniophora puteana Fomitopsis pinicola Gloeophyllum sepiarum Gloeophyllum trabeum Gloeophyllum trabeum Gloeophyllum trabeum Leucogyrophana pinastri Postia placenta Postia placenta Serpula lacrimans Serpula lacrimans Irpex lacteus Irpex lacteus Lentinula edodes Phanerochaete chrysosporium Phanerochaete chrysosporium Resinicium bicolor Resinicium bicolor Resinicium bicolor Scytinostroma galactinum Scytinostroma galactinum Scytinostroma galactinum Trametes versicolor Trametes versicolor Trichaptum abietinum Pisolithus tinctorius Rhizoctonia solani Ascomycetes Aureobasidium pullulans Ceratocystis pilifera Cytospora eutypelloides Diatrypella favacea Diatrypella prominens Hormonema dematiodes Hormonema dematiodes Leucostoma cincta Leucostoma cincta Leucostoma kunzei Leucostoma persoonii Ophiostoma ulmi Pestalotiopsis sp. Phaeocryptopus gaeumannii Phialocephala fusca Phialophora mutabilis Rhizosphaera kalkhofi Scleroderris lagerbergii Scleroderris lagerbergii Sirococcus conigenus Sphaeropsis sapinea Sphaeropsis sapinea Sphaeropsis sapinea Sphaeropsis sapinea Trichoderma reesei Trichoderma viride Valsa ceratophora Valsa germanica Valsa nivea Valsa sordida Xenomeris abietis Chaetomium globosum Sordaria sp.

Fp-90099-Sp K8sp 10-BS2-2 Mad-617-R ATCC 11539 Mad-698-R Harm-888-R ATCC 36335 KTS 003 ATCC 60993 117 1t(d) ATCC 24725 HHB-8850-sp ATCC 44175 ATCC 64897 MB-1880-sp ATCC 44178 ATCC 64896 Fp-101664-Sp 1247 MJL ATCC 38054 1AP ATCC 34621 ATCC 60758 CMI 140798

b This h This b a i This b b a g a b This a b a a b a a This b b a c a a c c c c c c c c c a c c a a c c c c c c c c a a c c c c c d f

lab lab

lab

lab

lab

Brown rot, C Brown rot, C Brown rot, C Brown rot, HCS Brown rot, HCS Brown rot, HCS Brown rot, C Brown rot, C Brown rot, C Brown rot, CS Brown rot, CS White rot, CH White rot, CH White rot, CH White rot, H White rot, H White rot, C White rot, C White rot, C White rot, CH White rot, CH White rot, CH White rot, H White rot, H White rot, C Ectomycorrhiza Pathogen of herbaceous plants Saprophyte, CH Wood stain, CH Canker Dead wood, H Canker, H Endophyte, C Endophyte, C Canker, R Canker, R Canker, C Canker, RH Dutch elm disease Endophyte, C Needle blight, C Softrot, C Softrot, H Needle blight, C Canker, C Canker, C Canker, C Canker, C Canker, C Canker, C Canker, C Saprophyte, CH Saprophyte, CH Canker, H Canker, H Canker, H Canker, H Canker, C Cosmopolitan Dung

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Scots pine MI Ontario/Biggs Lp66 LCN ATCC 32439 ATCC 24725 ATCC 62326 ATCC 42792 Spruce MI 1877 28379 A411 A472 B416 B468 ATCC 26921 ATCC 32630

a a, American Type Culture Collection; b, Forest Products Laboratory, Madison, Wis.; c, Gerard Adams, Michigan State University; d, Barry Goodell, University of Maine, Orono; e, Dilip Lakshman, University of Maine, Orono; f, David Lambert, University of Maine, Orono; g, Kevin Smith, University of New Hampshire; h, Swedish Agricultural University, Uppsala, Sweden; i, Paul I. Morris, Forintek Canada Corp., Vancouver, British Columbia, Canada. b C, conifers; H, hardwoods; R, Rosaceae; S, wood in service. c Primer ITS1-F is specic for higher fungi, primer ITS4 is a universal primer, and primer ITS4-B is specic for basidiomycetes.

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RESULTS DNA isolation from decayed wood. CTAB extraction in the presence of -mercaptoethanol followed by organic extractions and isopropanol precipitation of the DNA yielded DNA clean enough to amplify by PCR regardless of whether the starting material was fungal mycelia or decayed wood. The more decayed the wood, the more pigmented was the DNA-containing aqueous phase. Subsequent extractions with chloroformisoamyl alcohol and phenol-chloroform-isoamyl alcohol removed some of the pigmented by-products of wood decay, and more remained behind in the aqueous isopropanol phase upon precipitation of the DNA. However, substances inhibitory to PCR could carry through the purication procedure. For example, in preliminary experiments when DNA was isolated from replicate sets of drilled samples from highly decayed wood blocks (60% plus weight loss), the aqueous phase of samples in which the initial CTAB extraction had lasted overnight were much more strongly pigmented than those initially extracted for only 2 h, as in the standard protocol (see Materials and Methods); we would expect more by-products of wood decay to be extracted in an overnight versus a 2-h incubation. All of the 2-h CTAB-extracted DNA preparations were amplied by PCR; however, several of the overnight CTABextracted DNA preparations did not amplify, probably due to a higher concentration of compounds inhibitory to PCR remaining after purication. Optimization of PCR assay conditions for detection of basidiomycetes. The primers ITS1-F (higher fungus specic) and ITS4 (universal primer) amplied only one band (500 to 1,300 bp, depending on the fungal species) from DNA isolated from pure cultures of both ascomycetes and basidiomycetes via an ordinary PCR protocol, i.e., no hot start was needed. However, when we amplied the ITS region with the primers ITS1-F and ITS4-B (basidiomycete specic) from total DNA isolated from pure cultures, we obtained a number of minor amplication bands in both basidiomycetes and ascomycetes with the published amplication protocol that used an annealing temperature of 55C (7). Although the main product (850 to 1,460 bp, depending on the fungal species) was not amplied from as-

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FIG. 1. PCR amplication of nuclear rDNA from total DNA isolated from pure cultures of basidiomycetes (A) and ascomycetes (B). Electrophoresis in 2% (wt/vol) agarose in 1 TBE. The two outer lanes contain molecular weight markers. Inner even-numbered lanes contain samples amplied by the primer pair ITS1-F and ITS4-B, and the odd-numbered lanes contain samples amplied by primers ITS1-F and ITS4. (A) Lanes 2 to 9 contain brown-rot fungi, and lanes 10 to 17 contain white-rot fungi. Lanes 1 and 20, PCR markers (Promega); lanes 2 and 3, Coniophora puteana Fp-90099-Sp; lanes 4 and 5, Gloeophyllum trabeum Mad-617-R; lanes 6 and 7, Postia placenta Mad-698-R; lanes 8 and 9, Serpula lacrimans Harm-888-R; lanes 10 and 11, Lentinula edodes 117 1t(d); lanes 12 and 13, Resinicium bicolor ATCC 64897; lanes 14 and 15, Scytinostroma galactinum ATCC 64896; lanes 16 and 17, Trametes versicolor Fp-101664-Sp; lanes 18 and 19, no template DNA (i.e., negative controls). (B) Lanes 1 and 20, PCR markers (Promega); lanes 2 and 3, Aureobasidium pullulans ATCC 34621; lanes 4 and 5, Hormonema dematiodes; lanes 6 and 7, Pestalotiopsis sp.; lanes 8 and 9, Leucostoma kunzei; lanes 10 and 11, Scleroderris lagerbergii 1877; lanes 12 and 13, Sirococcus conigenus; lanes 14 and 15, Sphaeropsis sapinea B468; lanes 16 and 17, Xenomeris abietis; lanes 18 and 19, no template DNA (i.e., negative controls).

overlaid with mineral oil and amplied in a MJ Research Thermocycler Model PTC-100. PCR reactions consisted of an initial denaturation at 94C for 1 min 25 s, 35 cycles of amplication, and a nal extension at 72C for 10 min; each cycle of amplication consisted of denaturation at 95C for 35 s, annealing for 55 s (at 55C for reactions with ITS1-F and ITS4 and at 60C for reactions with ITS1-F and ITS4-B), and extension at 72C for 1 min. Weakly positive or negative amplications were reconrmed as positive or negative by taking an aliquot of the PCR reaction and reamplifying it with the primer pair used in the original reaction. Aliquots of the PCR reaction using template DNA isolated from wood and the primers ITS1-F and ITS4-B were also reamplied to ensure ample amplicon DNA for multiple restriction digestions; this allowed us to identify the fungus present in the wood, even when there were few to no physical signs of decay. PCR products were separated by electrophoresis in 2% (wt/vol) agarose gels in 1 TBE (89 mM Tris-borate, 89 mM boric acid, 2 mM EDTA) with ethidium bromide (EtBr) at 100 ng/ml in the gel and running buffer; DNA bands were visualized by the uorescence of the intercalated EtBr under UV light and photographed. Restriction digestion of PCR products. PCR reaction products were digested directly without further purication with restriction endonucleases to obtain RFLPs; each sample was digested with AluI, HaeIII, TaqI, or RsaI in singleenzyme digests, as well as in a double digest with TaqI and HaeIII. Per each 20- l restriction digest, 10 l of unpuried, amplied PCR reaction was mixed with the appropriate restriction reaction buffer and 10 U of the appropriate enzyme and then incubated for 6 h at 37C for the AluI, HaeIII, or RsaI digests or at 65C for the TaqI digests. Restriction fragments were separated by electrophoresis in 2% (wt/vol) and 2.5% (wt/vol) Sepharide Gel Matrix (Gibco-BRL) in 1 TAE (40 mM Tris acetate, 1 mM sodium EDTA) with EtBr at 100 ng/ml in the gel and running buffer. DNA bands were visualized by uorescence under UV light and photographed.

FIG. 2. PCR amplication of nuclear rDNA from total DNA isolated from wood blocks colonized by wood decay fungi or endophytes. Electrophoresis in 2% (wt/vol) agarose in 1 TBE. The two outer lanes contain molecular weight markers. Inner even-numbered lanes contain samples amplied by the primer pair ITS1-F and ITS4-B, and the odd-numbered lanes contain samples amplied by primers ITS1-F and ITS4. 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 pinastri; lanes 8 and 9, Lentinula edodes 117 1t(d); lanes 10 and 11, Trametes versicolor; lanes 12 and 13, Scytinostroma galactinum ATCC 64896; lanes 14 and 15, Hormonema dematiodes; lanes 16 and 17, Pestalotiopsis sp.; lanes 18 and 19, no template DNA (i.e., negative controls).

VOL. 66, 2000

PCR DETECTION OF WOOD DECAY FUNGI TABLE 2. Wood decay and detection of fungal species in wood blocks after 8 months of colonization

4729

Species

Isolate

Mean % wt loss of wood SDa

PCR amplicationb with primer pair: ITS1-FITS4 ITS1-FITS4-B

Uninoculated control wood Brown-rot basidiomycetes Coniophora puteana Postia placenta Postia placenta Gloeophyllum trabeum Gloeophyllum trabeum Gloeophyllum sepiarum Leucogyrophana pinastri Serpula lacrimans White-rot basidiomycetes Lentinula edodes Trametes versicolor Trametes versicolor Irpex lacteus Resinicium bicolor Resinicium bicolor Resinicium bicolor Scytinostroma galactinum Scytinostroma galactinum Scytinostroma galactinum Phanerochaete chrysosporium Phanerochaete chrysosporium Wood-inhabiting ascomycetes Phialophora mutabilis Trichoderma reesei Trichoderma viride Hormonema dematiodes Pestolotiopsis sp. Xenomeris abietis Aureobasidium pullulans Phialocephala fusca Ceratocystis pilifera Ophiostoma ulmi
a b

0.1 Fp-90099-Sp Mad-698-R Mad-617-R 10-BS2-2 Harm-888-R 117 1t(d) Fp-101664-Sp KTS 003 HHB-8850-sp ATCC 44175 ATCC 64897 MB-1880-sp ATCC 64896 ATCC 44178 ATCC 24725 ATCC 42792 ATCC 26921 ATCC 32630 0.1 65.5 65.8 67.3 69.6 68.1 68.1 67.5 3.0 35.1 0.0 40.1 10.4 3.7 12.1 0.8 1.1 0.2 9.9 14.2 0.4 0.2 0.1 0.4 0.6 0.5 0.2 0.2 2.7 0.1

0.3

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0.3 1.2 1.4 3.3 1.8 0.9 4.8 1.9 1.7 13.6 0.1 12.1 0.9 3.6 5.7 0.5 0.3 0.7 11.8 5.7 0.2 0.4 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0

ATCC ATCC ATCC ATCC

34621 62326 60758 32439

Mean of two replicate spruce wood blocks. Primer ITS1-F is specic for higher fungi, primer ITS4 is a universal primer, and primer ITS4-B is specic for basidiomycetes. Each plus or minus sign represents the amplication results for an individual wood block.

comycetes, a small band amplied very strongly in certain species of ascomycetes, e.g., a 210-bp band from Phialocephala fusca and a 330-bp band from Ophiostoma ulmi, in addition to the other minor bands. We tested a series of incrementally higher annealing temperatures and found that 60C gave the cleanest results. The other minor bands were no longer produced in amplications from basidiomycetes and ascomycetes, but the small strong band was still amplied from P. fusca and O. ulmi; the addition of a hot start to the PCR protocol eliminated this band. The primers ITS1-F and ITS4-B amplify a product from only basidiomycetes when a hot start and an annealing temperature of 60C are used. Substitution of the TaqStart antibody system for the traditional hot start method produced the same amplication results. The TaqStart antibody system mimics a traditional hot start and yet is much simpler to use for processing large numbers of samples at one time and with less risk of introducing contaminating DNA. In order to achieve specic amplication of DNA isolated from decayed wood, further adjustments to the PCR protocol were needed. Addition of nonacetylated BSA to PCR reactions, which is known to relieve inhibition of amplication by humic acids, fulvic acids, and organic components of soils and

manure (13), allowed some amplication to occur from samples containing inhibitory wood decay by-products; but this amplication was often nonspecic. Additionally, a hot-start protocol, either the traditional method or the TaqStart antibody system, was required to obtain specic amplication of DNA isolated from wood decay samples. We adopted as our standard PCR amplication conditions the inclusion of nonacetylated BSA at 250 ng/ l and the use of a hot start, the TaqStart antibody system for all reactions regardless of the tissue source of template DNA or primers used. These conditions are described in detail in Materials and Methods. Fungal species survey. We surveyed a total of 43 species (60 isolates) of fungi. These included 16 species of basidiomycetes, 14 of which are wood decay fungi, both brown rot and white rot, and 27 species of ascomycetes, 25 of which are wood inhabiters (pathogens, endophytes, and saprophytes). For the initial survey (Table 1), PCR amplications were performed using total DNA isolated from pure cultures of the fungi as the DNA template. Primers ITS1-F and ITS4 amplied the ITS region from all of these fungi, both ascomycetes and basidio-

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FIG. 3. TaqI restriction digests of the PCR product amplied by the primer pair ITS1-F and ITS4-B from DNA isolated from pure cultures of basidiomycetes. Electrophoresis in 2% (wt/vol) Sepharide Gel Matrix (Gibco-BRL) in 1 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, Coniophora 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 lacrimans Harm-888-R; lane 9, Irpex lacteus KTS 003; lane 10, Lentinula edodes 117 1t(d); lane 11, Phanerochaete chrysosporium ATCC 24725; lane 12, Resinicium bicolor ATCC 64897; lane 13, Scytinostroma galactinum ATCC 64896; lane 14, Trametes versicolor Fp-101664-Sp; lane 15, Trichaptum abietinum 1247 MJL; lane 16, Pisolithus tinctorium ATCC 38054, an ectomycorrhiza; lane 17, Rhizoctonia solani 1AP, a pathogen of herbaceous plants.

mycetes, as expected. Primers ITS1-F and ITS4-B amplied the ITS region from only the basidiomycetes (Fig. 1, Table 1). Detection of decay fungi in wood. The next step was to see if we could detect fungi in spruce wood by PCR amplication (Fig. 2, Table 2) using total DNA isolated from colonized wood blocks as the DNA template. We surveyed 30 species of fungi colonizing spruce wood. Two replicate jars were set up and inoculated, as described in Materials and Methods, for each of the wood-decaying basidiomycete species listed in Table 1 (excluding Fomitopsis pinicola and Trichaptum abietinum) and for each of the following wood-inhabiting ascomycetes: Ceratocystis pilifera ATCC 60758, Ophiostoma ulmi ATCC 32439, Phialocephala fusca ATCC 62326, Phialophora mutabilis ATCC 42792, Trichoderma reesei ATCC 26921, Trichoderma viride ATCC 32630, Aureobasidium pullulans, Hormonema dematiodes, Pestalotiopsis sp., and Xenomeris abietis. Wood blocks cut from radial sections of spruce were added to the colonized feeder strips and harvested after 8 months of colonization. Wood blocks with ascomycetes had a negligible weight loss, the majority by less than 0.5%, and seemed unchanged in appearance. The weight losses of wood blocks with white-rot fungi were very variable and ranged from negligible to approximately 40%; there was little to no change in the color of the wood, but some of the more decayed ones, e.g., replicate blocks colonized by one of the isolates of Trametes versicolor had become stringy in texture. Wood blocks with brown-rot fungi had decayed the most and were very brown in color; all but one isolate had caused a weight loss of 65 to 70%, the exception being Coniophora puteana isolate Fp-90099-Sp. In all of the wood block treatments, we had weight losses ranging between 0 and 70%. Primers ITS1-F and ITS4 amplied DNA from all of the samples, including the uninoculated control blocks. Primers ITS-1F and ITS4-B amplied DNA from wood blocks that had been inoculated with only basidiomycetes, i.e., the brown-rot isolates and white-rot isolates; ITS1-F and ITS4-B did not amplify DNA from uninoculated control blocks nor from any of the wood blocks inoculated with wood-inhabiting ascomycetes. The unknown contaminant fungus detected

in the uninoculated control blocks is probably an ascomycete, since no amplication occurs when the basidiomycete-specic primer ITS4-B is present in the PCR reaction; we suspect it may be a mold known to survive in wood upon repeated autoclaving. We could reliably detect the presence of wood decay fungi by PCR with the primers ITS1-F and ITS4-B in spruce blocks exhibiting a range of degradation states. Fungal identication. In order to identify the basidiomycetes detected by PCR, we generated RFLPs of the ITS region, the product amplied by primers ITS1-F and ITS4-B, by restriction digestion with RsaI, AluI, HaeIII, TaqI, or TaqIHaeIII. RsaI was not very useful because it did not cut the amplicon from 10 out of the 14 species of wood-decaying basidiomycetes tested nor that from the ectomycorrhizal Pisolithus tinctorius and the soil-borne Rhizoctonia solani, which do not decay wood. The other restriction endonucleases generated more fragments per digest, so that each basidiomycete could be identied to the species level from the combination of its RFLP proles (Fig. 3, Table 3). The majority of RFLP proles generated for any given enzyme were unique for each fungal species. The two Gloeophyllum species, however, had identical AluI RFLP proles and identical HaeIII RFLP proles; G. trabeum and G. sepiarum could be separated by their TaqI RFLP proles and their TaqI-HaeIII RFLP proles. Different isolates of a given fungal species usually had identical RFLP proles for a particular restriction endonuclease; this was true for each enzyme for isolates of G. trabeum, Irpex lacteus, Postia placenta, Resinicium bicolor, and Serpula lacrimans. For other fungi, some enzymes would generate RFLP proles which separated isolates at the species level and other enzymes would generate RFLP proles which separated isolates at the subspecies level. For example, isolates of Scytinostroma galactinum had identical AluI RFLP proles and identical HaeIII RFLP proles, but the isolates could be distinguished from each other by their respective TaqI RFLP proles and TaqI HaeIII RFLP proles. The identities of basidiomycetes detected by PCR from colonized spruce wood blocks were conrmed by comparing the RFLPs of the product amplied by primers ITS1-F and ITS4-B from DNA isolated from wood blocks to that from the respective pure culture of the fungus. Figure 4 demonstrates that the TaqI RFLP prole for any one wood block matches that of the TaqI digest of the amplicon obtained from DNA from a pure culture of that particular fungal isolate; analogous results were also obtained with AluI, HaeIII, and TaqI-HaeIII digestions. Time course studies. In order to determine how early we could detect wood decay fungi in wood, we ran two time course studies with the brown-rot fungi Postia placenta isolate Mad698-R and Gloeophyllum trabeum isolate Mad-617-R. Soil block jars were set up and inoculated as described in Materials and Methods. For each time course experiment, three replicate jars were inoculated for each combination of time and fungal isolate, as well as for a time-zero uninoculated control and an 8-month-incubated uninoculated control. The rst time course used wood blocks cut from radial sections of spruce sapwood, and the second time course used wood blocks cut from longitudinal sections. Wood blocks were harvested after 1, 2, 4, and 8 weeks and after 4 and 8 months of colonization. Wood decay progressed more rapidly in wood blocks cut from radial versus longitudinal sections of spruce sapwood, as evidenced by the change in percent weight loss of the wood over time (Table 4). A few samples from the rst time course and several from the second time course amplied weakly or not at all with primers ITS1-F and ITS4-B; the positive or negative nature of each was conrmed by reamplication of an aliquot of the original PCR reaction. Gloeophyllum trabeum

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4731

Species and isolate(s)a

RFLPs (bp) obtained with: AluI HaeIII TaqI TaqIHaeIII Uncut ampliconb

Species and isolate(s)a

RFLPs (bp) obtained with: AluI HaeIII TaqI TaqIHaeIII Uncut ampliconb

Brown-rot basidiomycetes Coniophora puteana Fp-90099-Sp

500 187 170 91

470 242 114 59 23 908 545 127 98 62 832 670 107 72 849 670 107 72 849 320 245 170 140 63 55 993 610 110d 72 902 800 108

530 185 71

Total Fomitopsis pinicola K8Sp Total Gloeophyllum trabeum This lab Mad-617-R ATCC 11539 Total Gloeophyllum sepiarum 10-BS2-2 Total Leucogyrophana pinastri This lab

948 380 200 90 52 722 435 155 105 86 781 435 155 105 86 781 580 300 100

786 310 203 165 130 870 500 318 72 890 500 170 140 72 882 400 180 127 95 78 63 943 530 335 72 937 485 215 81 68 50 899

247 197 182 69 59 41 23 813 248 165 107c 62 796 420 215 107 72 814 420 140 107 72 739 245 127 95 72 63 37 639 445 115d 72 747 485 215 77 68 39 884

Resinicium bicolor HHB-8850-sp ATCC 44175 ATCC 64897 Total Scytinostroma galactinum MB-1880-sp

380 150 98 69 697 595 204 94

800 50 850 540 268 60

270 245 215 60 790 315 200 164 75 60 814 220 200 116 91 75 60 762 200 137 116 91 75 60 679 320 172 74 566 320 172 74

270 245 215 60 790 315 137 116 75 60 50 753 220 137 116 91 75 60 50 749 137 116 91 75 60 50 529 260 135 108 74 50 627 255 205 135 113 74 50 832 335 145 62 542 208 127 100 69 59 44 23 630

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850

970

Total 930 Scytinostroma galactinum ATCC 44178

893 595 204 94

868 540 268 60

930

890

Total Scytinostroma galactinum ATCC 64896

893 595 204 94

868 540 268 60

930

890 Total Trametes versicolor This lab 1,050 Total 930 Trametes versicolor Fp-101664-Sp

893 360 146 93 74 58 731 360 146 93 74 58 731 405 365 215 155 50 1,190 435 182 165 87

868 300d 105 70 775 300d 210 70

930

Total Postia placenta This lab Mad-698-R Total Serpula lacrimans Harm-888-R ATCC 36335 Total White-rot basidiomycetes Irpex lacteus KTS 003 ATCC 60993

980 448 240 100 788 810 100

910

Total 930 Trichaptum abietinum 1247 MJL

880 900 310 145 53 1,408 605 170 59

566 470 335 53 858 208 150 127 76 59 23 643

910

910

908

500 105 93

425 345 105 61 936 860 102 50 705 98 50 853 705 98 50 853

Total Lentinula edodes 117 lt(d)

698 515 235 105 515 165 94 774 515 165 94 774

270 245 150 108 77 61 911 540 355 60 385 315 98 65 863 330 300 98 59 32 819

245 140 108 77 61 631 540 255 102 60 385 220 98 65 768 330 208 98 59 32 727 960

Total Ectomycorrhiza Pisolithus tinctorius ATCC 38054

1,460

Total Soil-borne pathogen of herbaceous plants Rhizoctonia solani 1AP Total 850

869

834

890

Phanerochaete chrysosporium This lab Total Phanerochaete chrysosporium ATCC 24725

850

415 245 98 83 841

620 260 880

500 335 59 894

335 260 242 59 896

970

Total
a

All isolates of a species which have identical RFLP proles per restriction endonuclease for all enzymes are listed together. If different isolates of a species have different RFLP proles for at least one of the restriction endonucleases, then those isolates are listed separately. b Amplicon of the ITS region amplied by primers ITS1-F (specic for higher fungi) and ITS4-B (specic for basidiomycetes). c This fragment is a triplet, as reected in the fragment total. d This fragment is a doublet, as reected in the fragment total.

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FIG. 4. TaqI restriction digests of the PCR product amplied by the primer pair ITS1-F and ITS4-B from DNA isolated from wood decay basidiomycetes. Electrophoresis in 2% (wt/vol) Sepharide Gel Matrix (Gibco-BRL) in 1 TAE. The two outer lanes contain molecular weight markers. Each group of three inner lanes represents the TaqI digests for one fungal isolate amplied from DNA isolated from each of two different wood blocks and a pure culture from left to right, respectively. (A) Lanes 1 and 20, PCR markers (Promega); lanes 2 to 4, Gloeophyllum trabeum; lanes 5 to 7, Gloeophyllum trabeum, Mad-617-R; lanes 8 to 10, Postia placenta; lanes 11 to 13, Postia placenta Mad-698-R; lanes 14 to 16, Trametes versicolor; lanes 17 to 19, Trametes versicolor Fp-101664-Sp. (B) Lanes 1 and 20, PCR markers (Promega); lanes 2 to 4, Resinicium bicolor; lanes 5 to 7, Resinicium bicolor ATCC 44175; lanes 8 to 10, Resinicium bicolor ATCC 64897; lanes 11 to 13, Scytinostroma galactinum; lanes 14 to 16, Scytinostroma galactinum ATCC 64896; lanes 17 to 19, Scytinostroma galactinum ATCC 44178.

and P. placenta, both brown-rot basidiomycetes, could be detected in wood by PCR amplication using primers ITS1-F (higher fungus specic) and ITS4-B (basidiomycete specic) after 1 week of colonization, the shortest colonization period used in the study. G. trabeum was detected in all replicates of all samples from all colonization times in both cuts of wood and could be detected at a 0.3% mean weight loss of the wood. P. placenta was detected in all of the samples from all of the wood blocks cut from radial sections but not in all of those cut from longitudinal sections. After 1 week of colonization (0.5% mean weight loss), P. placenta could be detected in only one of three of the wood blocks cut from longitudinal sections, but by 2 week (3.0% mean weight loss), it could be detected in three of three blocks; detection was also variable at later time points. DISCUSSION Although our procedure for DNA isolation and purication may be longer than desired to routinely screen large numbers of wood samples, we thought it best to begin the process of assay development with a method highly likely to yield DNA ampliable by PCR, since many by-products of wood decay, if present at too high a concentration in the reaction, would inhibit amplication of the DNA template. When setting up

PCR reactions with wood samples that have very low DNA concentrations, diluting out the inhibitors could also mean diluting out the DNA past the threshhold of detection. So it is better to start with a DNA preparation from which one has removed as much of the inhibitory materials as possible. With the minipreparation procedure described in the Materials and Methods, one person can drill and isolate DNA from 24 wood samples in one work day, observing all the necessary precautions both during drilling of the wood and DNA isolation to prevent any cross-contamination of samples. Avoiding cross-contamination of samples is critical. Early on, we found that preparation of the wood for DNA isolation is the step at which cross-contamination can most easily occur due to the inherent properties of sawdust. For example, a Wiley mill is not a good choice for grinding samples for PCR work. It is very difcult to clean out all of the crevices in which sawdust can be caught and, even after disassembly, careful brushing out of remaining debris, reassembly, and running through several volumes of clean fungus-free wood, and recleaning all the surfaces and crevices with a cotton swab, there is still carryover from one wood decay sample to the next; furthermore, this whole process takes an unacceptably long time. A drill is a much better choice. A rechargeable cordless drill has fewer crevices and surfaces to collect dirt and debris and can be more easily cleaned than a Wiley mill. Drill bits are easy to clean and ame sterilize and are relatively inexpensive, so one can have many of them ready to use. One can prepare wood samples for DNA isolation very rapidly with a drill and at far less risk of sample cross-contamination via sawdust. It is also important to wear gloves and to keep the work area clean, i.e., it is advisable to swab both your gloves and work surface with 70% ethanol to collect any bits of sawdust between drilling each sample. By observing these precautions, as described in detail in Materials and Methods, we have not detected any cross-contamination in samples prepared by drilling and so have adopted this procedure for routine use. We have developed a DNA-based method to reliably detect brown-rot and white-rot fungi in spruce wood using the published (7) primers ITS1-F (higher fungus specic) and ITS4-B (basidiomycete specic) to amplify the ITS region. We have optimized the reaction conditions for PCR with these primers for template DNA isolated from both pure culture and spruce wood and can detect brown-rot and white-rot fungi from incipient through advanced stages of wood decay. Some latestage brown-rot samples appeared to have weaker amplication signals than less-decayed samples (data not shown). This could be due to carryover of by-products of wood decay inhibitory to PCR, degradation of DNA in the late stages of wood decay, or a combination of the two. Currently, our assay is only qualitative; more work needs to be done to make it quantitative. The ability to detect decay fungi in other species of wood, preservative-treated wood, and wood composites should also be examined. The differing chemical compositions of both the undecayed and decayed forms of these substrates could introduce new kinds of PCR-inhibitory compounds that may or may not be eliminated or neutralized by our current methodology. While the primer pair ITS1-F and ITS4-B will detect only basidiomycetes, it will detect any basidiomycete present. For example, if the wood sample were taken from a root, there might be mycorrhizae present that would also be detected. Identity of the basidiomycete present can be achieved by restriction digestion of the PCR product. We could distinguish wood decay basidiomycetes at the species level by comparing the RFLP proles obtained by TaqI digestion of the ITS region amplied by ITS1-F and ITS4-B or by comparing the combination of different RFLP proles generated from this amplicon

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PCR DETECTION OF WOOD DECAY FUNGI TABLE 4. Time courses of wood decay and detection of brown-rot basidiomycetes
Expt and species Time Mean % wt loss of wood SDa

4733

PCR amplicationb with primer pair: ITS1-FITS4 ITS1-FITS4-B

Expt 1 (wood blocks cut from radial sections of spruce) Uninoculated control Uninoculated control Gloeophyllum trabeum Mad-617-R

Postia placenta Mad-698-R

0 8 1 2 4 8 4 8 1 2 4 8 4 8 0 8 1 2 4 8 4 8 1 2 4 8 4 8

mo wk wk wk wk mo mo wk wk wk wk mo mo

0.3 0.4 0.7 15.5 34.1 64.7 70.1 69.3 2.6 11.7 44.2 61.9 66.3 67.6 0.3 0.03 0.3 1.9 13.5 34.5 63.7 70.7 0.5 3.0 14.8 38.7 59.8 60.7

0.4 0.3 1.6 4.2 2.0 1.9 1.0 2.3 0.4 2.8 9.7 0.8 0.5 0.6 0.2 0.1 0.4 1.2 2.1 2.7 8.0 6.5 0.4 0.7 3.1 8.1 3.7 6.1

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Expt 2 (wood blocks cut from longitudinal sections of spruce) Uninoculated control Uninoculated control Gloeophyllum trabeum Mad-617-R

Postia placenta Mad-698-R

mo wk wk wk wk mo mo wk wk wk wk mo mo

Mean of three replicate spruce wood blocks. Primer ITS1-F is specic for higher fungi, primer ITS4 is a universal primer, and primer ITS4-B is specic for basidiomycetes. Each plus or minus sign represents the amplication results for an individual wood block.
b

by a number of different restriction endonucleases. Gardes et al. (8) identied 20 taxa of ectomycorrhizal fungi to the species or species group level from the RFLP proles of the ITS region amplied by these primers using DNA from mycorrhizae and basidiocarps. Using this method to identify all of their samples, these researchers were able to create a snapshot of the community structure of these ectomycorrhizal fungi both above and below ground in natural stands of Pinus muricata. Although PCR amplication followed by digestion with restriction endonucleases worked ne for samples containing only one fungus, eld samples could pose a greater challenge and contain more than one species of wood decay basidiomycete. As the number of different wood decay basidiomycetes contained in a wood sample increases, it would become correspondingly more difcult to identify them all to the species level based on RFLPs. For a more specic and one-step assay for a particular basidiomycete species, it would be better to develop a species-specic PCR primer based on a suitably informative area of the DNA sequence of the ITS region of that species. This concept could be extended to develop an assay in which a number of different species could be identied concurrently in one PCR reaction. Recently, Schmidt and Moreth (16) developed species-specic primers based on the DNA sequence of ITSII for the indoor rot fungi Serpula lacrimans and Serpula himantioides. We are currently designing species-specic primers for other brown-rot fungi. We are also looking at DNA sequences of enzymes thought to be involved in wood decay to see if it is possible to design

primers that would specically detect only wood decay basidiomycetes and not other basidiomycetes. It would be useful to be able to detect several wood decay species concurrently in samples where other non-wood-decaying species are likely to occur, e.g., tree roots and forest soils. However, for the purposes of detecting wood decay fungi in branches, tree trunks, harvested timber, or wood in service, where the probability of nondecay basidiomycetes colonizing the internal wood is very low, the assay we developed using the published primers ITS1-F and ITS4-B is potentially very useful. The very lack of specicity which limits the direct identication of the fungus to species can be an advantage in developing a broad-based assay. Previous workers have used PCR amplication in conjunction with RFLP analysis to identify wood decay fungi (15, 23), but their work has focused on identication of the fungi in culture or fungal material present on the wood versus the direct identication of early stages of decay within the wood. By focusing our efforts on the development of an assay that can sample directly from wood, we hope to eventually eliminate the need to culture the decay fungi as a rst step, so that detection would not be limited by the ability to culture them from a specic wood sample. Although our current PCR method is broad based for basidiomycetes, if used in combination with species-specic primers, one could detect a particular wood decay species of interest and also be alerted to the presence of other basidiomycetes, i.e., other potential decay fungi, in the wood sample.

4734

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APPL. ENVIRON. MICROBIOL.

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This work was supported by grant number 95-34158-1347 from the USDA and by the Maine Agricultural and Forest Experiment Station.
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