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Applied and Environmental Microbiology, November 2000, p. 4725-4734, Vol. 66, No. 11
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 amplified by the PCR using published universal primers
and basidiomycete-specific 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
(specific for higher fungi) and ITS4 (universal primer) amplified the
internal transcribed spacer region from both 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 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 identified 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 floor, 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 modified 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 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
significant 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 taxon-specific
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 specificity 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 significant strength loss had occurred. Detection of specific
decay agents is also a necessary prerequisite to allow evaluation of
fungal colonization and proliferation in preservative-treated woods
undergoing remediation. Specific 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 amplification 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 amplification, 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-specific 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 PCR-amplified ITS region have been used
successfully to study intraspecific 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
amplification 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 specifically amplify the ITS
region from only fungal DNA (7) and not plant DNA are
available. These fungus-specific 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 specificity
of the basidiomycete-specific primer (7) by surveying a
large number of wood decay basidiomycetes, as well as wood-inhabiting ascomycetes (pathogens, endophytes, and saprophytes); (ii) to optimize
the PCR assay conditions for specific detection of brown-rot and
white-rot fungi in wood; (iii) to identify the PCR-detected fungi to
species via RFLPs of the amplified 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.
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.
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Detection and Identification of Decay Fungi in
Spruce Wood by Restriction Fragment Length Polymorphism Analysis of
Amplified Genes Encoding rRNA
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Amplification of ITS region from DNA isolated from pure
fungal cultures
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 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 100°C 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 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
70°C
for DNA isolation. The fresh weight of the other half block was
recorded, after which it was oven dried at 100°C for 48 h, and
its dry weight recorded to allow calculation of the final 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 (final 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.
20°C 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 µl was used for DNA isolated from mycelia, and 50 µl was used for DNA isolated from wood samples. Incubation at 65°C
speeded up resuspension of the DNA.
PCR amplification. The ITS region was amplified 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 specific 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 amplification, 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 specific for basidiomycetes (7), were used to specifically amplify the ITS region from only basidiomycetes.
Amplifications were performed in 50-µl reactions of PCR buffer (10 mM Tris HCl, pH 8.3; 50 mM KCl; 0.001% [wt/vol] gelatin [Perkin-Elmer]), 200 µM 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/µl, total DNA isolated from a pure fungal culture or from a wood decay sample, 0.056 µM TaqStart antibody (Clontech), and 0.002 µM AmpliTaq DNA polymerase (Perkin-Elmer), i.e., 2 U per 50-µl 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 manufacturer's instructions (Clontech). Samples were overlaid with mineral oil and amplified in a MJ Research Thermocycler Model PTC-100. PCR reactions consisted of an initial denaturation at 94°C for 1 min 25 s, 35 cycles of amplification, and a final extension at 72°C for 10 min; each cycle of amplification consisted of denaturation at 95°C for 35 s, annealing for 55 s (at 55°C for reactions with ITS1-F and ITS4 and at 60°C for reactions with ITS1-F and ITS4-B), and extension at 72°C for 1 min. Weakly positive or negative amplifications were reconfirmed 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 reamplified 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 fluorescence of the intercalated EtBr under UV light and photographed.Restriction digestion of PCR products. PCR reaction products were digested directly without further purification with restriction endonucleases to obtain RFLPs; 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-µl restriction digest, 10 µl of unpurified, amplified PCR reaction was mixed with the appropriate restriction reaction buffer and 10 U of the appropriate enzyme and then incubated for 6 h at 37°C for the AluI, HaeIII, or RsaI digests or at 65°C 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 fluorescence under UV light and photographed.| |
RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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
chloroform-isoamyl 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 purification 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 amplified by PCR; however, several of the overnight
CTAB-extracted DNA preparations did not amplify, probably due to a
higher concentration of compounds inhibitory to PCR remaining after purification.
Optimization of PCR assay conditions for detection of basidiomycetes. The primers ITS1-F (higher fungus specific) and ITS4 (universal primer) amplified 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 amplified the ITS region with the primers ITS1-F and ITS4-B (basidiomycete specific) from total DNA isolated from pure cultures, we obtained a number of minor amplification bands in both basidiomycetes and ascomycetes with the published amplification protocol that used an annealing temperature of 55°C (7). Although the main product (850 to 1,460 bp, depending on the fungal species) was not amplified from ascomycetes, a small band amplified 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 60°C gave the cleanest results. The other minor bands were no longer produced in amplifications from basidiomycetes and ascomycetes, but the small strong band was still amplified 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 60°C are used. Substitution of the TaqStart antibody system for the traditional hot start method produced the same amplification 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 specific amplification 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 amplification by humic acids, fulvic acids, and organic components of soils and manure (13), allowed some amplification to occur from samples containing inhibitory wood decay by-products; but this amplification was often nonspecific. Additionally, a hot-start protocol, either the traditional method or the TaqStart antibody system, was required to obtain specific amplification of DNA isolated from wood decay samples. We adopted as our standard PCR amplification conditions the inclusion of nonacetylated BSA at 250 ng/µl 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
amplifications were performed using total DNA isolated from pure
cultures of the fungi as the DNA template. Primers ITS1-F and ITS4
amplified the ITS region from all of these fungi, both ascomycetes and
basidiomycetes, as expected. Primers ITS1-F and ITS4-B amplified the
ITS region from only the basidiomycetes (Fig.
1, Table 1).
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Detection of decay fungi in wood.
The next step was to see if
we could detect fungi in spruce wood by PCR amplification (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.
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Fungal identification.
In order to identify the basidiomycetes
detected by PCR, we generated RFLPs of the ITS region, the product
amplified by primers ITS1-F and ITS4-B, by restriction digestion with
RsaI, AluI, HaeIII, TaqI,
or TaqI-HaeIII. 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 identified to the species level from the
combination of its RFLP profiles (Fig. 3,
Table 3). The majority of RFLP profiles
generated for any given enzyme were unique for each fungal species. The
two Gloeophyllum species, however, had identical
AluI RFLP profiles and identical HaeIII RFLP
profiles; G. trabeum and G. sepiarum could be
separated by their TaqI RFLP profiles and their
TaqI-HaeIII RFLP profiles. Different isolates of
a given fungal species usually had identical RFLP profiles 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 profiles which separated isolates at the species level
and other enzymes would generate RFLP profiles which separated isolates
at the subspecies level. For example, isolates of Scytinostroma
galactinum had identical AluI RFLP profiles and
identical HaeIII RFLP profiles, but the isolates could be
distinguished from each other by their respective TaqI RFLP
profiles and TaqI HaeIII RFLP profiles.
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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 Mad-698-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 first 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 first time course and several from the second time course amplified weakly or not at all with primers ITS1-F and ITS4-B; the positive or negative nature of each was confirmed by reamplification of an aliquot of the original PCR reaction. Gloeophyllum trabeum and P. placenta, both brown-rot basidiomycetes, could be detected 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 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.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Although our procedure for DNA isolation and purification 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 amplifiable by PCR, since many by-products of wood decay, if present at too high a concentration in the reaction, would inhibit amplification 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 difficult 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 flame 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 specific) and ITS4-B (basidiomycete specific) 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 late-stage brown-rot samples appeared to have weaker amplification 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 profiles obtained by TaqI digestion of the ITS region amplified by ITS1-F and ITS4-B or by comparing the combination of different RFLP profiles generated from this amplicon by a number of different restriction endonucleases. Gardes et al. (8) identified 20 taxa of ectomycorrhizal fungi to the species or species group level from the RFLP profiles of the ITS region amplified 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 amplification followed by digestion with restriction endonucleases worked fine for samples containing only one fungus, field 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 difficult to identify them all to the species level based on RFLPs. For a more specific and one-step assay for a particular basidiomycete species, it would be better to develop a species-specific 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 identified concurrently in one PCR reaction. Recently, Schmidt and Moreth (16) developed species-specific primers based on the DNA sequence of ITSII for the indoor rot fungi Serpula lacrimans and Serpula himantioides. We are currently designing species-specific 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 specifically 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 specificity which limits the direct identification of the fungus to species can be an advantage in developing a broad-based assay. Previous workers have used PCR amplification in conjunction with RFLP analysis to identify wood decay fungi (15, 23), but their work has focused on identification of the fungi in culture or fungal material present on the wood versus the direct identification 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 first step, so that detection would not be limited by the ability to culture them from a specific wood sample. Although our current PCR method is broad based for basidiomycetes, if used in combination with species-specific 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.
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ACKNOWLEDGMENTS |
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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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FOOTNOTES |
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* 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{at}maine.maine.edu.
This work is contribution no. 2407 from the Maine Agricultural and
Forest Experiment Station, Orono, Maine.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, November 2000, p. 4725-4734, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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