10.1128/AEM.66.11.4725-4734.2000. 2000, 66(11):4725. DOI: Appl. Environ. Microbiol. Claudia A. Jasalavich, Andrea Ostrofsky and Jody Jellison Amplified Genes Encoding rRNA Length Polymorphism Analysis of in Spruce Wood by Restriction Fragment Detection and Identification of Decay Fungi http://aem.asm.org/content/66/11/4725 Updated information and services can be found at: These include: REFERENCES http://aem.asm.org/content/66/11/4725#ref-list-1 This article cites 15 articles, 1 of which can be accessed free at: CONTENT ALERTS more» articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new http://aem.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to: on May 4, 2012 by Universitätsbib TECHN.UNIVERSITAET BRAUN http://aem.asm.org/ Downloaded from
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10.1128/AEM.66.11.4725-4734.2000.
2000, 66(11):4725. DOI:Appl. Environ. Microbiol. Claudia A. Jasalavich, Andrea Ostrofsky and Jody Jellison Amplified Genes Encoding rRNALength Polymorphism Analysis ofin Spruce Wood by Restriction Fragment Detection and Identification of Decay Fungi
http://aem.asm.org/content/66/11/4725Updated information and services can be found at:
These include:
REFERENCEShttp://aem.asm.org/content/66/11/4725#ref-list-1This article cites 15 articles, 1 of which can be accessed free at:
CONTENT ALERTS more»articles cite this article),
Receive: RSS Feeds, eTOCs, free email alerts (when new
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Detection and Identification of Decay Fungi in Spruce Wood byRestriction Fragment Length Polymorphism Analysis of
Amplified Genes Encoding rRNA†CLAUDIA A. JASALAVICH, ANDREA OSTROFSKY, AND 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 differentstages of decay. DNA, isolated by a series of CTAB (cetyltrimethylammonium bromide) and organic extrac-tions, was amplified by the PCR using published universal primers and basidiomycete-specific primers derivedfrom ribosomal DNA sequences. We surveyed 14 species of wood-decaying basidiomycetes (brown-rot andwhite-rot fungi), as well as 25 species of wood-inhabiting ascomycetes (pathogens, endophytes, and sapro-phytes). DNA was isolated from pure cultures of these fungi and also from spruce wood blocks colonized byindividual isolates of wood decay basidiomycetes or wood-inhabiting ascomycetes. The primer pair ITS1-F(specific for higher fungi) and ITS4 (universal primer) amplified the internal transcribed spacer region fromboth ascomycetes and basidiomycetes from both pure culture and wood, as expected. The primer pair ITS1-F(specific for higher fungi) and ITS4-B (specific for basidiomycetes) was shown to reliably detect the presenceof wood decay basidiomycetes in both pure culture and wood; ascomycetes were not detected by this primerpair. We detected the presence of decay fungi in wood by PCR before measurable weight loss had occurred tothe wood. Basidiomycetes were identified to the species level by restriction fragment length polymorphisms ofthe internal transcribed spacer region.
Wood is an important renewable and biodegradable naturalresource with a multitude of uses. Wood is used extensively asa structural material for buildings, wharves, telephone poles,and furniture due to its high strength per unit weight, itsversatility, and its variety. Wood also serves as the industrialraw 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 worldwood is used as a fuel for heating and cooking.
The primary biotic decomposers of wood are basidiomycetedecay fungi, which can attack and degrade both wood in theforest and wood in service. In the forest ecosystem wood decayfungi play an important role in carbon and nitrogen cycling andhelp to convert organic debris into the humus layer of the soil.Some fungi attack living trees; others invade downed timberand slash on the forest floor, lumber, and wood in service.Wood decay basidiomycetes colonize and degrade wood usingenzymatic and nonenzymatic processes. Brown-rot fungi pref-erentially attack and rapidly depolymerize the structural car-bohydrates (cellulose and hemicellulose) in the cell wall leav-ing the modified lignin behind. White-rot fungi can progressivelyutilize all major cell wall components, including both the car-bohydrates and the lignin. As decay progresses the wood be-comes discolored and loses strength, weight, and density. De-cay and discoloration caused by fungi are major sources of lossin both timber production and wood use, with losses of 15 to25% marketable wood volume in standing timber and of 10 to15% in wood products during storage and conversion. Eachyear ca. 10% of the timber cut in the United States is used toreplace wood in service that has decayed, resulting in the
expenditure of hundreds of millions of dollars for raw materi-als, labor, and liability (22).
Brown rots rapidly and drastically reduce wood strengthearly in the decay process, while white rots cause a slowerprogressive decrease in wood strength. Brown-rot fungi canreduce wood strength by as much as 75% at less than 5%weight loss of the wood (22). For this reason it is important todevelop methods which can detect wood decay very early, atthe incipient stage prior to the occurrence of significantstrength loss. Techniques which have been used to detect in-cipient decay include isolation and culturing of fungi, chemicalstaining, nuclear magnetic resonance, and electrical resistance,as well as serological methods, such as immunoblotting andenzyme-linked immunosorbent assay (ELISA) (3). ELISA hasbeen found to be a sensitive method for detecting incipientdecay (4, 11), but the assay sensitivity can be inhibited by woodextractives (12). Optimal methods for early detection of decayfor wood in service have not been developed.
The development of the DNA-based PCR (14) and taxon-specific primers (2, 6, 7, 16, 17) is making it increasingly fea-sible to detect and study fungi in their natural substrates. ADNA-based method to detect the presence of wood decayfungi would potentially use only small amounts of wood, thusallowing for nondestructive sampling. The extreme sensitivityand potential specificity of the assay would theoretically allowfor the detection of fungal decay agents at an incipient stageenabling remedial biocidal treatments to be applied beforesignificant strength loss had occurred. Detection of specificdecay agents is also a necessary prerequisite to allow evalua-tion of fungal colonization and proliferation in preservative-treated woods undergoing remediation. Specific and sensitiveassay procedures would aid in the monitoring and develop-ment of successful fungus-based bioremediation technologies.
For our DNA-based detection method, we selected the in-ternal transcribed spacer (ITS) region (ITSI, the 5.8S ribo-somal DNA [rDNA], and ITSII) as the target sequence for
* Corresponding author. Mailing address: Department of BiologicalSciences, University of Maine, Orono, ME 04469-5735. Phone: (207)581-2995. Fax: (207) 581-2969. E-mail: [email protected].
† This work is contribution no. 2407 from the Maine Agriculturaland Forest Experiment Station, Orono, Maine.
amplification for three reasons. The ITS region is present at avery high copy number in the genome of fungi, as part of thetandemly repeated nuclear rDNA; this, coupled with PCRamplification, should produce a highly sensitive assay. TheDNA sequences of the ITSI and ITSII are highly variable; thisfeature can be exploited to generate restriction fragmentlength polymorphism (RFLP) patterns to identify wood decayfungi or to design taxon-specific primers. The European Ar-millaria 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 PCR-amplified ITS region have been used successfully to study in-traspecific variation in A. ostoyae (17), to identify ectomycor-rhizal 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 amplificationusing total DNA isolated from infected plant material as thetemplate, it is important to be able to discriminate betweenDNAs of fungal and plant origin. Primers which specificallyamplify the ITS region from only fungal DNA (7) and not plantDNA are available. These fungus-specific primers were origi-nally designed to identify fungal symbionts directly from ecto-mycorrhizae and to identify rusts, which are obligate parasites,in the host tissue (7). More recently, these primers have beenused to study the community structure of ectomycorrhizalfungi in a pine forest (8) and the genetic structure of a naturalpopulation of Suillus pungens (2).
The objectives of our study were (i) to rigorously test thespecificity of the basidiomycete-specific primer (7) by surveyinga large number of wood decay basidiomycetes, as well as wood-inhabiting ascomycetes (pathogens, endophytes, and sapro-phytes); (ii) to optimize the PCR assay conditions for specificdetection of brown-rot and white-rot fungi in wood; (iii) toidentify the PCR-detected fungi to species via RFLPs of theamplified internal transcribed spacer region; and (iv) to de-velop a DNA-based method to detect incipient stages of wooddecay, thus allowing remedial treatments to be applied towooden structural members before substantial strength losshas occurred.
MATERIALS AND METHODS
Fungal culture. The fungi used in this study and their sources are given inTable 1. Cultures were grown on plates of malt agar at room temperature for usein DNA isolation or as inocula for soil block jars.
Soil block culture. Modified 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 pottingsoil, sphagnum moss, and vermiculite and then moistened with deionized dis-tilled water. About 1 cup of the mix was placed in each pint-sized Mason jar, andwater was allowed to absorb overnight. The next day, 20 ml of water was addedper jar so that the soil mix was moist and a little free water was present. Twopieces of birch tongue depressor were placed on the soil surface to serve asfeeder strips. Lids were inverted to prevent sealing and screwed onto the jars withrings. Jars were autoclaved for 30 min. Two days later the jars were againautoclaved 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 endof each feeder strip. In uninoculated control jars, blocks of sterile malt agar wereused. Jars were incubated at room temperature to allow fungal colonization offeeder strips.
Radial or longitudinal sections of spruce sapwood (1 by 1 by 0.25 in. [1 in. 525.4 mm]) cut from the same tree were oven dried at 100°C for 48 h, weighed,and then autoclaved for 30 min enclosed in glass petri dishes. After cooling, thewood blocks were added aseptically to the jars at one block per jar. Eachexperiment used wood blocks cut from only radial sections or from only longi-tudinal sections. Wood blocks cut from radial sections were placed so that atransverse face contacted the top of the colonized feeder strips. Wood blocks cutfrom longitudinal sections were placed so that a longitudinal face contacted thetop of the colonized feeder strip. Jars were incubated at room temperature toallow colonization of the spruce blocks.
After the appropriate colonization time, the wood blocks were harvestedaseptically, observing precautions to prevent cross-contamination of the samples
at all steps of processing. Mycelia on the surface of the block were removed bygently 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., perpen-dicular 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 270°Cfor DNA isolation. The fresh weight of the other half block was recorded, afterwhich it was oven dried at 100°C for 48 h, and its dry weight recorded to allowcalculation of the final dry weight of the whole block at harvest based on thefollowing ratio: total fresh weight/calculated total dry weight 5 partial freshweight/partial dry weight. Wood decay was estimated as the percent dry weightloss as follows: percent weight loss 5 [1 2 (final dry weight/initial dry wt)] 3 100.
DNA isolation. DNA was isolated from fresh mycelia taken from the surfaceof plate cultures, from lyophilized mycelia, or from infected wood by extractionwith cetyltrimethylammonium bromide (CTAB) in the presence of b-mercapto-ethanol, followed by organic extractions and isopropanol precipitation of theDNA. Our method is based on those of Taylor et al. (19) and Wilson (21). Forfresh mycelia a 23 CTAB extraction buffer (2% [wt/vol] CTAB; 100 mM TrisHCl, pH 8.0; 1.4 M NaCl; 20 mM EDTA; 0.2% [vol/vol] b-mercaptoethanol) wasused, with the b-mercaptoethanol being added just prior to use. For lyophilizedmycelia or dry tissues such as wood samples, a 13 CTAB extraction buffer(diluted 23 buffer) was used. It is especially important to use the 13 CTABextraction buffer for wood samples; otherwise, the aqueous and organic phasesinvert due to rehydration of the wood when the 23 CTAB extraction buffer isused.
Wood blocks were sampled by drilling through noninoculated wood surfaces.Precautions were observed during drilling of the wood blocks to prevent cross-contamination of samples. Both the work table and gloves were swabbed with70% ethanol to surface sterilize them and to collect any bits of sawdust beforeand after drilling each sample. A rechargeable cordless drill was used because ithas less surface area, fewer crevices, and no cord to collect dirt and sawdust, anda molded housing which can be easily wiped clean with 70% ethanol. Drill bitswere carefully cleaned with laboratory detergent, rinsed, soaked in 95% ethanol,and flame sterilized. A drill bit was inserted through a cone of filter paper (newfor each sample), positioned so as to cover the chuck and prevent sawdust fromentering it. We drilled through each wood block, on a line perpendicular to theface of the block that had contacted the colonized feeder strip, with a 1/8-in.diameter bit to generate a fine sawdust from which DNA could be isolateddirectly; no further grinding of the sawdust was needed to achieve good DNAextraction. Once a prepared drill bit was used to drill a wood sample, it was notreused until it had been recleaned and resterilized by the procedure describedabove. Fresh or lyophilized mycelia was simply ground to a fine powder withliquid nitrogen in a mortar and pestle for use in DNA extraction.
Ground or drilled material (100 to 200 ml) was transferred to a sterile mi-crofuge tube. Then, 600 ml of the appropriate CTAB extraction buffer was added,and the sample was mixed to resuspend the powdered tissue in the buffer andincubated at 65°C for 2 h. The sample was extracted with 1 volume of chloro-form-isoamyl alcohol (24:1, vol/vol) and centrifuged at 10,000 3 g for 10 min atroom temperature. The aqueous phase was transferred to a new tube, and a 1/10volume of 10% (wt/vol) CTAB in 0.7 M NaCl was added. After mixing, thesample was incubated at 65°C for 1 h. Once again the sample was extracted withchloroform-isoamyl alcohol (24:1, vol/vol) and centrifuged as described above.The aqueous phase was transferred to a new tube and extracted with 1 volumeof phenol-chloroform-isoamyl alcohol (25:24:1, vol/vol/vol), followed by centrif-ugation at 10,000 3 g for 10 min at room temperature. The aqueous phase wastransferred to a new tube, and the DNA was precipitated by the addition of 0.6volume of ice-cold isopropanol. After incubation at 220°C for 30 min, the DNAprecipitate was collected by centrifugation at 12,000 3 g for 15 min. The pelletwas washed twice with ice-cold 70% (vol/vol) ethanol and dried. The pellet wasresuspended in DNA storage buffer (6 mM Tris HCl, 0.1 mM EDTA; pH 7.5);100 ml was used for DNA isolated from mycelia, and 50 ml was used for DNAisolated from wood samples. Incubation at 65°C speeded up resuspension of theDNA.
PCR amplification. The ITS region was amplified by PCR from DNA isolatedfrom pure cultures of each of the fungi listed in Table 1 and from wood blockscolonized by individual isolates of wood decay basidiomycetes or wood-inhabit-ing ascomycetes. Primers ITS1-F (CTT GGT CAT TTA GAG GAA GTA A),which is specific for the higher fungi (7), and ITS4 (TCC TCC GCT TAT TGATAT GC), the universal primer (20), were used together as a positive control foramplification, since they would be expected to amplify the ITS region from bothascomycetes and basidiomycetes. The primer pair ITS1-F and ITS4-B (CAGGAG ACT TGT ACA CGG TCC AG), which is specific for basidiomycetes (7),were used to specifically amplify the ITS region from only basidiomycetes.
Amplifications were performed in 50-ml reactions of PCR buffer (10 mM TrisHCl, pH 8.3; 50 mM KCl; 0.001% [wt/vol] gelatin [Perkin-Elmer]), 200 mMconcentrations of each deoxyribonucleotide triphosphate, and 200 nM concen-trations of each of the appropriate primers, with nonacetylated bovine serumalbumin (BSA; Sigma A-7906) at 250 ng/ml, total DNA isolated from a purefungal culture or from a wood decay sample, 0.056 mM TaqStart antibody(Clontech), and 0.002 mM AmpliTaq DNA polymerase (Perkin-Elmer), i.e., 2 Uper 50-ml reaction. The TaqStart antibody and AmpliTaq DNA polymerase weremixed together and preincubated prior to being added to the rest of the reactioncomponents as per the manufacturer’s instructions (Clontech). Samples were
TABLE 1. Amplification of ITS region from DNA isolated from pure fungal cultures
Species Isolate Sourcea EcologybPCR amplificationc with primer pair:
ITS1-F–ITS4 ITS1-F–ITS4-B
BasidiomycetesConiophora puteana Fp-90099-Sp b Brown rot, C 1 1Fomitopsis pinicola K8sp This lab Brown rot, C 1 1Gloeophyllum sepiarum 10-BS2-2 h Brown rot, C 1 1Gloeophyllum trabeum This lab Brown rot, HCS 1 1Gloeophyllum trabeum Mad-617-R b Brown rot, HCS 1 1Gloeophyllum trabeum ATCC 11539 a Brown rot, HCS 1 1Leucogyrophana pinastri i Brown rot, C 1 1Postia placenta This lab Brown rot, C 1 1Postia placenta Mad-698-R b Brown rot, C 1 1Serpula lacrimans Harm-888-R b Brown rot, CS 1 1Serpula lacrimans ATCC 36335 a Brown rot, CS 1 1Irpex lacteus KTS 003 g White rot, CH 1 1Irpex lacteus ATCC 60993 a White rot, CH 1 1Lentinula edodes 11751t(d) b White rot, CH 1 1Phanerochaete chrysosporium This lab White rot, H 1 1Phanerochaete chrysosporium ATCC 24725 a White rot, H 1 1Resinicium bicolor HHB-8850-sp b White rot, C 1 1Resinicium bicolor ATCC 44175 a White rot, C 1 1Resinicium bicolor ATCC 64897 a White rot, C 1 1Scytinostroma galactinum MB-1880-sp b White rot, CH 1 1Scytinostroma galactinum ATCC 44178 a White rot, CH 1 1Scytinostroma galactinum ATCC 64896 a White rot, CH 1 1Trametes versicolor This lab White rot, H 1 1Trametes versicolor Fp-101664-Sp b White rot, H 1 1Trichaptum abietinum 1247 MJL b White rot, C 1 1Pisolithus tinctorius ATCC 38054 a Ectomycorrhiza 1 1Rhizoctonia solani 1AP c Pathogen of herbaceous plants 1 1
AscomycetesAureobasidium pullulans ATCC 34621 a Saprophyte, CH 1 2Ceratocystis pilifera ATCC 60758 a Wood stain, CH 1 2Cytospora eutypelloides CMI 140798 c Canker 1 2Diatrypella favacea c Dead wood, H 1 2Diatrypella prominens c Canker, H 1 2Hormonema dematiodes c Endophyte, C 1 2Hormonema dematiodes Scots pine MI c Endophyte, C 1 2Leucostoma cincta Ontario/Biggs c Canker, R 1 2Leucostoma cincta Lp66 c Canker, R 1 2Leucostoma kunzei c Canker, C 1 2Leucostoma persoonii LCN c Canker, RH 1 2Ophiostoma ulmi ATCC 32439 a Dutch elm disease 1 2Pestalotiopsis sp. c Endophyte, C 1 2Phaeocryptopus gaeumannii ATCC 24725 c Needle blight, C 1 2Phialocephala fusca ATCC 62326 a Softrot, C 1 2Phialophora mutabilis ATCC 42792 a Softrot, H 1 2Rhizosphaera kalkhoffii Spruce MI c Needle blight, C 1 2Scleroderris lagerbergii 1877 c Canker, C 1 2Scleroderris lagerbergii 28379 c Canker, C 1 2Sirococcus conigenus c Canker, C 1 2Sphaeropsis sapinea A411 c Canker, C 1 2Sphaeropsis sapinea A472 c Canker, C 1 2Sphaeropsis sapinea B416 c Canker, C 1 2Sphaeropsis sapinea B468 c Canker, C 1 2Trichoderma reesei ATCC 26921 a Saprophyte, CH 1 2Trichoderma viride ATCC 32630 a Saprophyte, CH 1 2Valsa ceratophora c Canker, H 1 2Valsa germanica c Canker, H 1 2Valsa nivea c Canker, H 1 2Valsa sordida c Canker, H 1 2Xenomeris abietis c Canker, C 1 2Chaetomium globosum d Cosmopolitan 1 2Sordaria sp. f Dung 1 2
a a, American Type Culture Collection; b, Forest Products Laboratory, Madison, Wis.; c, Gerard Adams, Michigan State University; d, Barry Goodell, University ofMaine, 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 specific for higher fungi, primer ITS4 is a universal primer, and primer ITS4-B is specific for basidiomycetes.
VOL. 66, 2000 PCR DETECTION OF WOOD DECAY FUNGI 4727
overlaid with mineral oil and amplified in a MJ Research Thermocycler ModelPTC-100. PCR reactions consisted of an initial denaturation at 94°C for 1 min25 s, 35 cycles of amplification, and a final extension at 72°C for 10 min; eachcycle of amplification consisted of denaturation at 95°C for 35 s, annealing for55 s (at 55°C for reactions with ITS1-F and ITS4 and at 60°C for reactions withITS1-F and ITS4-B), and extension at 72°C for 1 min.
Weakly positive or negative amplifications were reconfirmed as positive ornegative by taking an aliquot of the PCR reaction and reamplifying it with theprimer pair used in the original reaction. Aliquots of the PCR reaction usingtemplate DNA isolated from wood and the primers ITS1-F and ITS4-B were alsoreamplified to ensure ample amplicon DNA for multiple restriction digestions;this allowed us to identify the fungus present in the wood, even when there werefew to no physical signs of decay.
PCR products were separated by electrophoresis in 2% (wt/vol) agarose gelsin 13 TBE (89 mM Tris-borate, 89 mM boric acid, 2 mM EDTA) with ethidiumbromide (EtBr) at 100 ng/ml in the gel and running buffer; DNA bands werevisualized by the fluorescence of the intercalated EtBr under UV light andphotographed.
Restriction digestion of PCR products. PCR reaction products were digesteddirectly without further purification with restriction endonucleases to obtainRFLPs; each sample was digested with AluI, HaeIII, TaqI, or RsaI in single-enzyme digests, as well as in a double digest with TaqI and HaeIII. Per each 20-mlrestriction digest, 10 ml of unpurified, amplified PCR reaction was mixed with theappropriate restriction reaction buffer and 10 U of the appropriate enzyme andthen incubated for 6 h at 37°C for the AluI, HaeIII, or RsaI digests or at 65°C forthe TaqI digests.
Restriction fragments were separated by electrophoresis in 2% (wt/vol) and2.5% (wt/vol) Sepharide Gel Matrix (Gibco-BRL) in 13 TAE (40 mM Trisacetate, 1 mM sodium EDTA) with EtBr at 100 ng/ml in the gel and runningbuffer. DNA bands were visualized by fluorescence under UV light and photo-graphed.
RESULTS
DNA isolation from decayed wood. CTAB extraction in thepresence of b-mercaptoethanol followed by organic extractionsand isopropanol precipitation of the DNA yielded DNA cleanenough to amplify by PCR regardless of whether the startingmaterial was fungal mycelia or decayed wood. The more de-cayed the wood, the more pigmented was the DNA-containingaqueous phase. Subsequent extractions with chloroform-isoamyl alcohol and phenol-chloroform-isoamyl alcohol re-moved some of the pigmented by-products of wood decay, andmore remained behind in the aqueous isopropanol phase uponprecipitation of the DNA. However, substances inhibitory toPCR could carry through the purification procedure. For ex-ample, in preliminary experiments when DNA was isolatedfrom replicate sets of drilled samples from highly decayedwood blocks (60% plus weight loss), the aqueous phase ofsamples in which the initial CTAB extraction had lasted over-night were much more strongly pigmented than those initiallyextracted for only 2 h, as in the standard protocol (see Mate-rials and Methods); we would expect more by-products ofwood decay to be extracted in an overnight versus a 2-h incu-bation. All of the 2-h CTAB-extracted DNA preparations wereamplified by PCR; however, several of the overnight CTAB-extracted DNA preparations did not amplify, probably due toa higher concentration of compounds inhibitory to PCR re-maining after purification.
Optimization of PCR assay conditions for detection of ba-sidiomycetes. The primers ITS1-F (higher fungus specific) andITS4 (universal primer) amplified only one band (500 to 1,300bp, depending on the fungal species) from DNA isolated frompure cultures of both ascomycetes and basidiomycetes via anordinary PCR protocol, i.e., no hot start was needed. However,when we amplified the ITS region with the primers ITS1-F andITS4-B (basidiomycete specific) from total DNA isolated frompure cultures, we obtained a number of minor amplificationbands in both basidiomycetes and ascomycetes with the pub-lished amplification protocol that used an annealing tempera-ture of 55°C (7). Although the main product (850 to 1,460 bp,depending on the fungal species) was not amplified from as-
FIG. 1. PCR amplification of nuclear rDNA from total DNA isolated frompure cultures of basidiomycetes (A) and ascomycetes (B). Electrophoresis in 2%(wt/vol) agarose in 13 TBE. The two outer lanes contain molecular weightmarkers. Inner even-numbered lanes contain samples amplified by the primerpair ITS1-F and ITS4-B, and the odd-numbered lanes contain samples amplifiedby primers ITS1-F and ITS4. (A) Lanes 2 to 9 contain brown-rot fungi, and lanes10 to 17 contain white-rot fungi. Lanes 1 and 20, PCR markers (Promega); lanes2 and 3, Coniophora puteana Fp-90099-Sp; lanes 4 and 5, Gloeophyllum trabeumMad-617-R; lanes 6 and 7, Postia placenta Mad-698-R; lanes 8 and 9, Serpulalacrimans Harm-888-R; lanes 10 and 11, Lentinula edodes 11751t(d); lanes 12and 13, Resinicium bicolor ATCC 64897; lanes 14 and 15, Scytinostroma galacti-num ATCC 64896; lanes 16 and 17, Trametes versicolor Fp-101664-Sp; lanes 18and 19, no template DNA (i.e., negative controls). (B) Lanes 1 and 20, PCRmarkers (Promega); lanes 2 and 3, Aureobasidium pullulans ATCC 34621; lanes4 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).
FIG. 2. PCR amplification of nuclear rDNA from total DNA isolated fromwood blocks colonized by wood decay fungi or endophytes. Electrophoresis in2% (wt/vol) agarose in 13 TBE. The two outer lanes contain molecular weightmarkers. Inner even-numbered lanes contain samples amplified by the primerpair ITS1-F and ITS4-B, and the odd-numbered lanes contain samples amplifiedby primers ITS1-F and ITS4. Lanes 2 to 7, brown-rot basidiomycetes; lanes 8 to13, white-rot basidiomycetes; lanes 14 to 17, endophytic ascomycetes. Lanes 1and 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, Leucogyrophanapinastri; lanes 8 and 9, Lentinula edodes 11751t(d); lanes 10 and 11, Trametesversicolor; lanes 12 and 13, Scytinostroma galactinum ATCC 64896; lanes 14 and15, Hormonema dematiodes; lanes 16 and 17, Pestalotiopsis sp.; lanes 18 and 19,no template DNA (i.e., negative controls).
comycetes, a small band amplified very strongly in certainspecies of ascomycetes, e.g., a 210-bp band from Phialocephalafusca and a 330-bp band from Ophiostoma ulmi, in addition tothe other minor bands. We tested a series of incrementallyhigher annealing temperatures and found that 60°C gave thecleanest results. The other minor bands were no longer pro-duced in amplifications from basidiomycetes and ascomycetes,but the small strong band was still amplified from P. fusca andO. ulmi; the addition of a hot start to the PCR protocol elim-inated this band. The primers ITS1-F and ITS4-B amplify aproduct from only basidiomycetes when a hot start and anannealing temperature of 60°C are used. Substitution of theTaqStart antibody system for the traditional hot start methodproduced the same amplification results. The TaqStart anti-body system mimics a traditional hot start and yet is muchsimpler to use for processing large numbers of samples at onetime and with less risk of introducing contaminating DNA.
In order to achieve specific amplification of DNA isolatedfrom decayed wood, further adjustments to the PCR protocolwere needed. Addition of nonacetylated BSA to PCR reac-tions, which is known to relieve inhibition of amplification byhumic acids, fulvic acids, and organic components of soils and
manure (13), allowed some amplification to occur from sam-ples containing inhibitory wood decay by-products; but thisamplification was often nonspecific. Additionally, a hot-startprotocol, either the traditional method or the TaqStart anti-body system, was required to obtain specific amplification ofDNA isolated from wood decay samples.
We adopted as our standard PCR amplification conditionsthe inclusion of nonacetylated BSA at 250 ng/ml and the use ofa hot start, the TaqStart antibody system for all reactionsregardless of the tissue source of template DNA or primersused. These conditions are described in detail in Materials andMethods.
Fungal species survey. We surveyed a total of 43 species (60isolates) of fungi. These included 16 species of basidiomycetes,14 of which are wood decay fungi, both brown rot and whiterot, and 27 species of ascomycetes, 25 of which are woodinhabiters (pathogens, endophytes, and saprophytes). For theinitial survey (Table 1), PCR amplifications were performedusing total DNA isolated from pure cultures of the fungi as theDNA template. Primers ITS1-F and ITS4 amplified the ITSregion from all of these fungi, both ascomycetes and basidio-
TABLE 2. Wood decay and detection of fungal species in wood blocks after 8 months of colonization
a Mean of two replicate spruce wood blocks.b Primer ITS1-F is specific for higher fungi, primer ITS4 is a universal primer, and primer ITS4-B is specific for basidiomycetes. Each plus or minus sign represents
the amplification results for an individual wood block.
VOL. 66, 2000 PCR DETECTION OF WOOD DECAY FUNGI 4729
mycetes, as expected. Primers ITS1-F and ITS4-B amplifiedthe ITS region from only the basidiomycetes (Fig. 1, Table 1).
Detection of decay fungi in wood. The next step was to see ifwe could detect fungi in spruce wood by PCR amplification(Fig. 2, Table 2) using total DNA isolated from colonized woodblocks as the DNA template. We surveyed 30 species of fungicolonizing spruce wood. Two replicate jars were set up andinoculated, as described in Materials and Methods, for each ofthe wood-decaying basidiomycete species listed in Table 1 (ex-cluding Fomitopsis pinicola and Trichaptum abietinum) and foreach of the following wood-inhabiting ascomycetes: Ceratocys-tis pilifera ATCC 60758, Ophiostoma ulmi ATCC 32439, Phia-locephala fusca ATCC 62326, Phialophora mutabilis ATCC42792, Trichoderma reesei ATCC 26921, Trichoderma virideATCC 32630, Aureobasidium pullulans, Hormonema dema-tiodes, Pestalotiopsis sp., and Xenomeris abietis. Wood blockscut from radial sections of spruce were added to the colonizedfeeder 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 inappearance. The weight losses of wood blocks with white-rotfungi were very variable and ranged from negligible to approx-imately 40%; there was little to no change in the color of thewood, but some of the more decayed ones, e.g., replicateblocks colonized by one of the isolates of Trametes versicolorhad become stringy in texture. Wood blocks with brown-rotfungi had decayed the most and were very brown in color; allbut one isolate had caused a weight loss of 65 to 70%, theexception being Coniophora puteana isolate Fp-90099-Sp. Inall of the wood block treatments, we had weight losses rangingbetween 0 and 70%. Primers ITS1-F and ITS4 amplified DNAfrom all of the samples, including the uninoculated controlblocks. Primers ITS-1F and ITS4-B amplified DNA from woodblocks that had been inoculated with only basidiomycetes, i.e.,the brown-rot isolates and white-rot isolates; ITS1-F andITS4-B did not amplify DNA from uninoculated control blocksnor from any of the wood blocks inoculated with wood-inhab-iting ascomycetes. The unknown contaminant fungus detected
in the uninoculated control blocks is probably an ascomycete,since no amplification occurs when the basidiomycete-specificprimer ITS4-B is present in the PCR reaction; we suspect itmay be a mold known to survive in wood upon repeated au-toclaving. We could reliably detect the presence of wood decayfungi by PCR with the primers ITS1-F and ITS4-B in spruceblocks exhibiting a range of degradation states.
Fungal identification. In order to identify the basidiomyce-tes detected by PCR, we generated RFLPs of the ITS region,the product amplified by primers ITS1-F and ITS4-B, by re-striction digestion with RsaI, AluI, HaeIII, TaqI, or TaqI-HaeIII. RsaI was not very useful because it did not cut theamplicon from 10 out of the 14 species of wood-decayingbasidiomycetes tested nor that from the ectomycorrhizal Piso-lithus tinctorius and the soil-borne Rhizoctonia solani, which donot decay wood. The other restriction endonucleases gener-ated more fragments per digest, so that each basidiomycetecould be identified to the species level from the combination ofits RFLP profiles (Fig. 3, Table 3). The majority of RFLPprofiles generated for any given enzyme were unique for eachfungal species. The two Gloeophyllum species, however, hadidentical AluI RFLP profiles and identical HaeIII RFLP pro-files; G. trabeum and G. sepiarum could be separated by theirTaqI RFLP profiles and their TaqI-HaeIII RFLP profiles. Dif-ferent isolates of a given fungal species usually had identicalRFLP profiles for a particular restriction endonuclease; thiswas true for each enzyme for isolates of G. trabeum, Irpexlacteus, Postia placenta, Resinicium bicolor, and Serpula lacri-mans. For other fungi, some enzymes would generate RFLPprofiles which separated isolates at the species level and otherenzymes would generate RFLP profiles which separated iso-lates at the subspecies level. For example, isolates of Scytinos-troma galactinum had identical AluI RFLP profiles and iden-tical HaeIII RFLP profiles, but the isolates could bedistinguished from each other by their respective TaqI RFLPprofiles and TaqI HaeIII RFLP profiles.
The identities of basidiomycetes detected by PCR from col-onized spruce wood blocks were confirmed by comparing theRFLPs of the product amplified by primers ITS1-F and ITS4-Bfrom DNA isolated from wood blocks to that from the respec-tive pure culture of the fungus. Figure 4 demonstrates that theTaqI RFLP profile for any one wood block matches that of theTaqI digest of the amplicon obtained from DNA from a pureculture of that particular fungal isolate; analogous results werealso obtained with AluI, HaeIII, and TaqI-HaeIII digestions.
Time course studies. In order to determine how early wecould detect wood decay fungi in wood, we ran two time coursestudies with the brown-rot fungi Postia placenta isolate Mad-698-R and Gloeophyllum trabeum isolate Mad-617-R. Soilblock jars were set up and inoculated as described in Materialsand Methods. For each time course experiment, three replicatejars were inoculated for each combination of time and fungalisolate, as well as for a time-zero uninoculated control and an8-month-incubated uninoculated control. The first time courseused wood blocks cut from radial sections of spruce sapwood,and the second time course used wood blocks cut from longi-tudinal sections. Wood blocks were harvested after 1, 2, 4, and8 weeks and after 4 and 8 months of colonization.
Wood decay progressed more rapidly in wood blocks cutfrom radial versus longitudinal sections of spruce sapwood, asevidenced by the change in percent weight loss of the woodover time (Table 4). A few samples from the first time courseand several from the second time course amplified weakly ornot at all with primers ITS1-F and ITS4-B; the positive ornegative nature of each was confirmed by reamplification of analiquot of the original PCR reaction. Gloeophyllum trabeum
FIG. 3. TaqI restriction digests of the PCR product amplified by the primerpair ITS1-F and ITS4-B from DNA isolated from pure cultures of basidiomyce-tes. Electrophoresis in 2% (wt/vol) Sepharide Gel Matrix (Gibco-BRL) in 13TAE. The two outer lanes contain molecular weight markers. Each inner lanecontains a different fungal species; lanes 2 to 8 contain brown-rot fungi, and lanes9 to 15 contain white-rot fungi. Lanes 1 and 18, PCR markers (Promega); lane2, 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, Lentinulaedodes 11751t(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 1247MJL; lane 16, Pisolithus tinctorium ATCC 38054, an ectomycorrhiza; lane 17,Rhizoctonia solani 1AP, a pathogen of herbaceous plants.
a All isolates of a species which have identical RFLP profiles per restriction endonuclease for all enzymes are listed together. If different isolates of a species havedifferent RFLP profiles for at least one of the restriction endonucleases, then those isolates are listed separately.
b Amplicon of the ITS region amplified by primers ITS1-F (specific for higher fungi) and ITS4-B (specific for basidiomycetes).c This fragment is a triplet, as reflected in the fragment total.d This fragment is a doublet, as reflected in the fragment total.
and P. placenta, both brown-rot basidiomycetes, could be de-tected in wood by PCR amplification using primers ITS1-F(higher fungus specific) and ITS4-B (basidiomycete specific)after 1 week of colonization, the shortest colonization periodused in the study. G. trabeum was detected in all replicates ofall samples from all colonization times in both cuts of woodand 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 thewood blocks cut from radial sections but not in all of those cutfrom longitudinal sections. After 1 week of colonization (0.5%mean weight loss), P. placenta could be detected in only one ofthree of the wood blocks cut from longitudinal sections, but by2 week (3.0% mean weight loss), it could be detected in threeof three blocks; detection was also variable at later time points.
DISCUSSION
Although our procedure for DNA isolation and purificationmay be longer than desired to routinely screen large numbersof wood samples, we thought it best to begin the process ofassay development with a method highly likely to yield DNAamplifiable by PCR, since many by-products of wood decay, ifpresent at too high a concentration in the reaction, wouldinhibit amplification of the DNA template. When setting up
PCR reactions with wood samples that have very low DNAconcentrations, diluting out the inhibitors could also meandiluting out the DNA past the threshhold of detection. So it isbetter to start with a DNA preparation from which one hasremoved as much of the inhibitory materials as possible. Withthe minipreparation procedure described in the Materials andMethods, one person can drill and isolate DNA from 24 woodsamples in one work day, observing all the necessary precau-tions both during drilling of the wood and DNA isolation toprevent any cross-contamination of samples.
Avoiding cross-contamination of samples is critical. Earlyon, we found that preparation of the wood for DNA isolationis the step at which cross-contamination can most easily occurdue to the inherent properties of sawdust. For example, aWiley mill is not a good choice for grinding samples for PCRwork. It is very difficult to clean out all of the crevices in whichsawdust can be caught and, even after disassembly, carefulbrushing out of remaining debris, reassembly, and runningthrough several volumes of clean fungus-free wood, and re-cleaning all the surfaces and crevices with a cotton swab, thereis still carryover from one wood decay sample to the next;furthermore, this whole process takes an unacceptably longtime. A drill is a much better choice. A rechargeable cordlessdrill has fewer crevices and surfaces to collect dirt and debrisand can be more easily cleaned than a Wiley mill. Drill bits areeasy to clean and flame sterilize and are relatively inexpensive,so one can have many of them ready to use. One can preparewood samples for DNA isolation very rapidly with a drill andat far less risk of sample cross-contamination via sawdust. It isalso important to wear gloves and to keep the work area clean,i.e., it is advisable to swab both your gloves and work surfacewith 70% ethanol to collect any bits of sawdust between dril-ling each sample. By observing these precautions, as describedin detail in Materials and Methods, we have not detected anycross-contamination in samples prepared by drilling and sohave adopted this procedure for routine use.
We have developed a DNA-based method to reliably detectbrown-rot and white-rot fungi in spruce wood using the pub-lished (7) primers ITS1-F (higher fungus specific) and ITS4-B(basidiomycete specific) to amplify the ITS region. We haveoptimized the reaction conditions for PCR with these primersfor template DNA isolated from both pure culture and sprucewood and can detect brown-rot and white-rot fungi from in-cipient through advanced stages of wood decay. Some late-stage brown-rot samples appeared to have weaker amplifica-tion signals than less-decayed samples (data not shown). Thiscould be due to carryover of by-products of wood decay inhib-itory to PCR, degradation of DNA in the late stages of wooddecay, or a combination of the two. Currently, our assay is onlyqualitative; more work needs to be done to make it quantita-tive. The ability to detect decay fungi in other species of wood,preservative-treated wood, and wood composites should alsobe examined. The differing chemical compositions of both theundecayed and decayed forms of these substrates could intro-duce new kinds of PCR-inhibitory compounds that may or maynot be eliminated or neutralized by our current methodology.
While the primer pair ITS1-F and ITS4-B will detect onlybasidiomycetes, it will detect any basidiomycete present. Forexample, if the wood sample were taken from a root, theremight be mycorrhizae present that would also be detected.Identity of the basidiomycete present can be achieved by re-striction digestion of the PCR product. We could distinguishwood decay basidiomycetes at the species level by comparingthe RFLP profiles obtained by TaqI digestion of the ITS regionamplified by ITS1-F and ITS4-B or by comparing the combi-nation of different RFLP profiles generated from this amplicon
FIG. 4. TaqI restriction digests of the PCR product amplified by the primerpair ITS1-F and ITS4-B from DNA isolated from wood decay basidiomycetes.Electrophoresis in 2% (wt/vol) Sepharide Gel Matrix (Gibco-BRL) in 13 TAE.The two outer lanes contain molecular weight markers. Each group of threeinner lanes represents the TaqI digests for one fungal isolate amplified fromDNA isolated from each of two different wood blocks and a pure culture fromleft to right, respectively. (A) Lanes 1 and 20, PCR markers (Promega); lanes 2to 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 14to 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; lanes5 to 7, Resinicium bicolor ATCC 44175; lanes 8 to 10, Resinicium bicolor ATCC64897; lanes 11 to 13, Scytinostroma galactinum; lanes 14 to 16, Scytinostromagalactinum ATCC 64896; lanes 17 to 19, Scytinostroma galactinum ATCC 44178.
by a number of different restriction endonucleases. Gardes etal. (8) identified 20 taxa of ectomycorrhizal fungi to the speciesor species group level from the RFLP profiles of the ITS regionamplified by these primers using DNA from mycorrhizae andbasidiocarps. Using this method to identify all of their samples,these researchers were able to create a snapshot of the com-munity structure of these ectomycorrhizal fungi both aboveand below ground in natural stands of Pinus muricata.
Although PCR amplification followed by digestion with re-striction endonucleases worked fine for samples containingonly one fungus, field samples could pose a greater challengeand contain more than one species of wood decay basidiomy-cete. As the number of different wood decay basidiomycetescontained in a wood sample increases, it would become corre-spondingly more difficult to identify them all to the specieslevel based on RFLPs. For a more specific and one-step assayfor a particular basidiomycete species, it would be better todevelop a species-specific PCR primer based on a suitablyinformative area of the DNA sequence of the ITS region ofthat species. This concept could be extended to develop anassay in which a number of different species could be identifiedconcurrently in one PCR reaction. Recently, Schmidt andMoreth (16) developed species-specific primers based on theDNA sequence of ITSII for the indoor rot fungi Serpula lac-rimans and Serpula himantioides. We are currently designingspecies-specific primers for other brown-rot fungi.
We are also looking at DNA sequences of enzymes thoughtto be involved in wood decay to see if it is possible to design
primers that would specifically detect only wood decay basid-iomycetes and not other basidiomycetes. It would be useful tobe able to detect several wood decay species concurrently insamples where other non-wood-decaying species are likely tooccur, e.g., tree roots and forest soils. However, for the pur-poses of detecting wood decay fungi in branches, tree trunks,harvested timber, or wood in service, where the probability ofnondecay basidiomycetes colonizing the internal wood is verylow, the assay we developed using the published primersITS1-F and ITS4-B is potentially very useful. The very lack ofspecificity which limits the direct identification of the fungus tospecies can be an advantage in developing a broad-based assay.Previous workers have used PCR amplification in conjunctionwith RFLP analysis to identify wood decay fungi (15, 23), buttheir work has focused on identification of the fungi in cultureor fungal material present on the wood versus the direct iden-tification of early stages of decay within the wood. By focusingour efforts on the development of an assay that can sampledirectly from wood, we hope to eventually eliminate the needto culture the decay fungi as a first step, so that detectionwould not be limited by the ability to culture them from aspecific wood sample. Although our current PCR method isbroad based for basidiomycetes, if used in combination withspecies-specific primers, one could detect a particular wooddecay species of interest and also be alerted to the presence ofother basidiomycetes, i.e., other potential decay fungi, in thewood sample.
TABLE 4. Time courses of wood decay and detection of brown-rot basidiomycetes
Expt and species Time Mean % wt loss ofwood 6 SDa
PCR amplificationb with primer pair:
ITS1-F–ITS4 ITS1-F–ITS4-B
Expt 1 (wood blocks cut from radial sections of spruce)Uninoculated control 0 0.3 6 0.4 122 222Uninoculated control 8 mo 0.4 6 0.3 111 222Gloeophyllum trabeum Mad-617-R 1 wk 0.7 6 1.6 111 111
a Mean of three replicate spruce wood blocks.b Primer ITS1-F is specific for higher fungi, primer ITS4 is a universal primer, and primer ITS4-B is specific for basidiomycetes. Each plus or minus sign represents
the amplification results for an individual wood block.
VOL. 66, 2000 PCR DETECTION OF WOOD DECAY FUNGI 4733
This work was supported by grant number 95-34158-1347 from theUSDA and by the Maine Agricultural and Forest Experiment Station.
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