| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Biology, University of Oslo, Box 1066 Blindern, N-0316 Oslo, Norway
Marit Lie
Norwegian University of Life Sciences, Department of Ecology and Natural Resource Management, Box 5003, 1432 Ås, Norway
Øyvind Stensrud
Department of Biology, University of Oslo, Box 1066 Blindern, N-0316 Oslo, Norway
Mikael Ohlson
Norwegian University of Life Sciences, Department of Ecology and Natural Resource Management, Box 5003, 1432 Ås, Norway
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
In this study we present a new approach to characterize fungal diversity with DNA sequencing of mycelium grown from trapped airborne spores. Fungal spores were extracted systematically from air in three boreal forest sites (clear-cut, young and old-growth forests) using an air sampling device. Internal transcribed spacer (ITS) sequences from the nuclear ribosomal DNA (nrDNA) were generated, and the sequences most likely taxon affinities were established through DNA homology searches. Phylogenetic analyses were used to classify similar sequences into operational taxonomic units (OTUs). The analyses indicated that a total of 84 different OTUs had been sampled, 24 basidiomycetes and 60 ascomycetes. OTUs belonging to the ascomycete orders Helotiales and Pleosporales were most frequent (31 and 18 respectively). A total of 54, 29 and 33 OTUs were sampled, respectively, in the old-growth, young and clear-cut forest sites. Although heavy generalization should be avoided due to few replicates, the results could indicate that old-growth boreal forests have significantly higher airborne fungal species richness than recently managed forests. The study shows that the spore-trapping approach has a great potential for targeting and studying anonymous fungi.
Key words: diversity, environmental sampling, forest management, Helotiales, ITS
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
), but this number represents apparently only a fraction of the de facto diversity.
Today the use of molecular markers is revolutionizing the field of microbial ecology, and has made it possible to detect the previously unknown fungal diversity (Pennisi 2004, Horton and Bruns 2001). A wide array of DNA based techniques is used for environmental sampling of unknown organisms, including DNA sequencing (often coupled to cloning), T-RFLP, ARISA, DGGE and SSCP. Unknown fungal diversity can be targeted by DNA sequencing and DNA homology searches against public access sequence databases (e.g. GenBank/EMBL) to get a clue which taxon anonymous fungal material belongs to. The internal transcribed spacer (ITS) region of nrDNA is the most used target sequence in molecular detection of fungi and is also the most employed marker to infer lower-levels taxonomy in fungi (Bruns 2001). However, the level of intraspecific ITS divergence varies extensively within species and it can be difficult to assign an anonymous fungal ITS sequence to a certain taxon based on ITS sequence similarity. The term operational taxonomic unit (OTU) might be convenient to use on organisms having a higher or lower level of uncertain taxon affinity.
Recent studies using molecular detection have demonstrated that endophytic and mycorrhizal fungi constitute a huge but largely unknown diversity component in ecosystems. For example, high fungal species richness (49 SSU rRNA phylotypes) was uncovered in a single plant root system (Vandenkoornhuyse et al 2002) and a high diversity of Helotiales taxa occurred as root symbionts belowground (Vrålstad et al 2002). A large and unknown endophytic fungal community was detected in western white pine trees (Ganley et al 2004). The widespread dark septate fungi of Phialocephala in a recent study were shown to occupy a range of different habitats, including dead wood and living plants materials including roots (Menkis et al 2004). However, technically, it can be highly problematic to target anonymous fungal material belowground or within plant substrates (Ranjard et al 2003). In addition to the fungi of interest, a mixture of other types of organisms exist both belowground and within plant materials.
Because the great majority of fungi are dispersed by airborne meio- or mitospores, we have used an alternative and new approach to capture and characterize the cryptic fungal diversity by trapping airborne fungal spores and perform ITS nrDNA characterization of outgrowing mycelium from the trapped spores. Thus, our method is restricted to fungi able to grow in vitro. Until now there has been very few field studies dealing with fungal spore diversity in natural environments (but see Vasiliauskas et al 2005), but some studies have been carried out in indoor environments (e.g. Herbarth et al 2003) or have been directed toward specific plant pathogenic fungi (Schweigkofler et al 2004). A taxon-specific method, where basidiospores are captured with a monokaryotic bait mycelium, has been used successfully to study spore spread in basidiomycetes (Adams et al 1984, James and Vilgalys 2001, Edman et al 2004).
To evaluate the new approach, we have sampled fungal spores in neighboring boreal forests of contrasting human disturbance (i.e. an old forest that has not been affected by logging in the past century, a young forest that was planted approximately 30 y ago and a recently clear-cut forest). We have chosen to study the fungal diversity in forest ecosystems because fragmentation and loss of natural forest habitats pose a major biodiversity threat (Noble and Dirzo 1997). It is well documented that some fungal groups suffer due to habitat loss in boreal forests (e.g. Sippola and Renvall 1999, Penttilä et al 2004), but it is not yet known to what extent the largely unexplored fungal diversity is affected by human land-use, such as forestry. The main aim with our study is twofold; first to present a novel approach to target the cryptic fungal diversity of airborne spores, and second to document anonymous fungal diversity in boreal forests of contrasting human disturbance. Because habitat qualities associated with old forests are known to be important determinants of the species richness of easily detectable life-forms, such as vascular plants, mosses, lichens and macroscopic fruit bodies of wood-decaying fungi (Ohlson et al 1997), we hypothesize that the fungal diversity explored by spore capturing should be highest in the old forest and lowest in the most disturbed clear-cut forest.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Molecular methods.— –
DNA extraction was performed with a 2% CTAB miniprep method (Murray and Thompson 1980) with minor modifications: DNA was resuspended in 100 µL dsH2O at the final step of extraction, and DNA templates were diluted 50-fold before PCR amplification. PCR amplification was accomplished with primers ITS4 and ITS5 (White et al 1990) for the nuclear ITS1-5.8S-ITS2 rDNA region. PCR was performed in 30 µL reactions containing 17.5 µL 50 x diluted template DNA and 12.5 µL reaction mix (final concentrations: 4 x 250 mM dNTPs, 0.625 mM of each primer, 2 mM MgCl2 and 1 unit DyNAzymeTM II DNA polymerase [Finnzymes Oy, Espoo, Finland]) on a Bio-metra PCR machine. The amplification program was initiated by a 4 min denaturation step at 94 C, followed by 37 cycles of 30 s at 94 C, 35 s at 54 C, and 40 s at 72 C. The program was terminated with a 10 min elongation step at 72 C before storage at 4 C. Automated sequencing was performed on a MegaBACETM 500 DNA Analysis System (Amersham Biosciences, Ohio) with the DYEnamicTM ET Dye Terminator Cycle Sequencing Kit (Amersham Biosciences, Buckinghamshire, England) according to the manufacturer’s recommendations. PCR products and cycle sequencing products were purified respectively with the ExoSAP-IT and AutoSeq96TM Dye Terminator Clean-up kits according to the manufacturer’s recommendations (Amersham Biosciences, Ohio). For unknown reasons, 24 of the isolates; 13 from the old-growth, three from the young and four from the clear-cut site, did not yield reliable sequences, possibly due to contamination of the cultures of yeast fungi; these isolates/sequences were discharged from further analyses. The 261 ITS sequences included in this study are deposited in the EMBL/GenBank sequence databases under accession numbers AM084419
[GenBank]
-AM084549 and AM084748
[GenBank]
-AM084977 (cf. supplementary material 1).
Statistical analyses.— –
Sequence chromatograms were inspected visually with the program BioEdit Sequence Alignment Editor version 5.0.9 (Hall 1999). All generated ITS sequences were submitted to Blast searches (at www.ncbi.nlm.nih.gov). The current NCBI taxonomy was used for classification of the OTUs into orders. In six selected taxonomic groups (see below) sequence alignments were established using a combination of Clustal W (www.ebi.ac.uk/clustalw) and manual alignment. Selected ITS GenBank sequences, having best matches with the obtained sequences, where included in the alignments. Phylogenetic analyses of the six ITS datasets were performed with PAUP* version 4.0b10 (Swofford 2003), with all transformations coded as unordered, all characters treated as equally weighted and gaps treated as missing values. The heuristic search option, with the tree bisection-reconnection (TBR) branch swapping algorithm and the random addition sequence option with 100 replicates to find multiple islands, was employed for all searches for most parsimonious tree(s). All other settings were default. Bootstrap support for branching topologies was examined with the same parameter settings, except that simple addition of sequences was used, and with 1000 search replicates. For two of the datasets ("Mollisia" and "Phaeosphaeria"), Neighbor joining bootstrap analyses (1000 replicates) with default settings were employed due to limited data power. EstimateS (Colwell 1997) was used to calculate saturation curves of richness of OTUs in the three different forest sites. One thousand randomized runs were performed on each dataset. A binary OTU/replicate data matrix was constructed for a principal co-ordinate (PCO) analysis, where the OTUs abundance was converted to presence/absence data in the 15 replicates. The PCO analysis was performed in NTSYS-pc (Rholf 1997), using Jaccard’s coefficient for similarity.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
). Forty-six percent of the sequences (119) obtained 99 or 100% sequence similarity with accessioned GenBank sequences (all more or less full-length matches); taxon names are shown (TABLE I). Provisional names were assigned to each sequence according to the best Blast matches. In this way most of the sequences could be assigned to an "operational taxonomic unit", hereafter referred to as an OTU. All the isolates included in the "Cladosporium" morphotype were pooled into one OTU because different isolates of this morphotype obtained best hit with various Cladosporium spp. accessioned in GenBank. In other cases where we obtained multiple closely related sequences, phylogenetic analyses were used to group sequences with high similarity into different OTUs. Six different ITS alignments including sequences matching GenBank accessions of Mollisia spp., Phaeospaeria spp., Lachnum spp., Peniophora spp., Penicillum spp. and Phialocephala spp. were generated, and the resulting phylogenies are shown (FIG. 2).
|
|
|
). Counting just the number of spores, Mycosphaerellales was most frequent due to the high frequency of Cladosporium spp. in the clear-cut site and in the young forest site. A stepwise decline in number of Cladosporium spp. spores was observed from the clear-cut forest site (198 spores) to the young (90) and the old-growth forest site (21). Next to Mycosphaerellales, Helotiales was most frequent concerning number of spores and included most OTUs. Judging from the similarity to GenBank sequences Mollisia, Phialocephala and Lachnum were the most abundant genera within Helotiales, being especially frequent in the old-growth forest site. OTUs belonging to Aphyllophorales were sampled far more frequently in the old-growth forest (nine times) compared to the young (three) and clear-cut (one) forest sites and all OTUs in Agaricales were sampled only in the old growth forest, except one (cf. TABLE II). OTUs belonging to Pleosporales were sampled more frequently in the clear-cut forest site (14 times), compared to the young (nine) and old-growth (four) forest sites.
|
). As shown with saturation curves, a larger fraction of the total diversity of OTU was captured in the clear-cut site, compared to the young and especially the old-growth forest site (FIG. 3B). Furthermore, a principal coordinate (PCO) analysis (FIG. 4) showed that the variation in composition of OTUs among replicates was far greater in the old-growth forest, compared to the young and clear-cut forests. A forest age gradient corresponded perfectly with the first PCO axis and thus accounted for most of the variation in composition of OTUs (FIG. 4). Thirty-five OTUs were sampled in the old-growth forest only, while 15 and nine were sampled in the clear-cut and young forests only (FIG. 5). Seven OTUs occurred in all three forest sites, while four occurred in both the young and clear-cut sites, six in both the clear-cut and old-growth sites and eight in both the young and old-growth sites. Thus, the young and old-growth forest sites had most OTUs in common.
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
, Vrålstad et al 2002) and the diversity of fungi within living or dead plant materials (Ganley et al 2004, Menkis et al 2004), but to our knowledge this is the first attempt to characterize the diversity of fungi in natural ecosystems by targeting airborne fungal spores. The majority of fungal spores fall close to the fruit bodies (Ingold 1965) and it is thus reasonable to conclude that the captured spores largely reflect a site-specific and local species diversity of fungi. With the exception of Glomales, Chytridiomycota and many gasteromycetes, most fungi disperse by airborne spores (Alexopoulos et al 1996) and the approach presented here, hypothetically, is able to reveal a large proportion of the diversity of fungi in natural ecosystems. Some fungi also are spread by insects or are rain splash dispersed; they will not be targeted by our approach. Furthermore, the approach restricts to fungi that are able to grow in vitro on nutrient media. Thus, measuring the diversity of established mycelia is only an indirect way of measuring the diversity of airborne spores. However, a direct molecular characterization of trapped spores also could be conducted using for example a combination of nested-PCR and cloning (Williams et al 2001). In this way, spores from obligate biotrophic fungi not able to grow in culture also could be obtained and a larger fraction of the entire diversity described. However, one advantage of the employed methodology is that by establishing single-spore cultures we avoid mixed DNA samples and the usage of cloning techniques to obtain sequences. Multiple spores also could be characterized simultaneously using techniques such as T-RFLP (Bruce 1997).
This study represents a first examination of the spore-trapping approach, and it includes some methodological limitations that decrease the generality of our results. First, spore-sampling replicates (five) were obtained only within the forest stands and there was no replication of forest stand type. This is a weakness (cf. Hurlbert 1984), but our idea in this first study was to assess the beta diversity among spore sample replicates taken within the same forest stand. For example, the amount of fruiting bodies in the nearby vicinity probably strongly affects the presence of spores in the air. According to the PCO plot, there was a high similarity within the forest stands, especially concerning the clear-cut and young forests. The variation in spore composition was far higher in the old-growth forest stand (cf. FIG. 4
). Another limitation is that only one culture medium (DG18 agar) was employed. No single culture medium will be suitable for all fungi of interest and this will introduce bias into the results. In future studies different types of media should be employed together. Furthermore spore sampling was done during a 2 h window and it certainly is some natural variation in spore composition during such a period. Diurnal (Ingold 1965) and seasonal (e.g. Vasiliauskas et al 2005) variations in the aerial spore composition also are important to consider during spore-sampling experiments. Another methodological weakness is that cultures with similar appearance were pooled and only representative isolates from each morphotype were sequenced. This could be circumvented by DNA sequencing all cultures in future studies or employ PCR-RFLP in conjunction with sequencing to classify the cultures into OTUs. In spite of the mentioned methodological weaknesses we think that some preliminary knowledge can be obtained from this first experiment.
As much as 46% (119) of the obtained ITS sequences had 99 or 100% sequence similarity (obtained by Blast searches) to accessioned GenBank sequences (cf. FIG. 1). Although the intraspecific ITS variation varies a lot, our impression is that a sequence similarity of 99% or higher normally falls within the intraspecific range of ITS variation. This means that a quiet high proportion of the spore diversity in boreal forests, employing the current approach, can be determined at the species level.
Most of the recorded OTUs belonged to the ascomycete orders Helotiales and Pleosporales, followed by the basidiomycete orders Aphyllophorales and Agaricales. Helotiales includes an ecologically diverse group of plant pathogens, wood, debris and soil saprobes, plant endophytes, and mycorrhizal fungi (Alexopoulos et al 1996). Mollisia was the most abundant genus within Helotiales and was especially frequent in the old-growth forest site (cf. FIG. 2A). Mollisia includes species growing on plant debris and decaying wood, but some are obviously also biotrophs (Vrålstad et al 2002). The species delimitation is poorly understood, and in our phylogeny of Mollisia-like sequences (FIG. 1a), GenBank sequences of Mollisia fusca, M. cinerea and M. melaleuca clustered closely together. The Helotiales genus Phialocephala also was captured frequently in the old-growth forest site. Phialocephala spp., often referred to as "dark-septate endophytes" are widespread, have little host or habitat specificities and are conidial or sterile inhabitants of many terrestrial plants ( Jumpponen and Trappe 1998). Phialocephala spp. has apparently a great ability to colonize and persist in living and dead trees under strikingly different ecological conditions (Menkis et al 2004). Four OTUs, apparently belonging to the genus Lachnum, were captured in the old-growth forest, except one, that also was captured in the clear-cut forest site (FIG. 2B). Lachnum spp. grows saprophytically on wood and other plant materials but also has been isolated from living roots (Vrålstad et al 2002). Three of the obtained Lachnum sequences had a high similarity to L. virgineum GenBank sequence AJ430221
[GenBank]
. However, judged from the placement of Lachnum GenBank sequences in the phylogenetic tree (FIG. 2B), species delimitations in the genus are questionable. The Helotiales species Cistella acuum and Sclerotinia sclerotiorum, producing ascocarps on dead plant materials, were captured several times, the former at all sites and the latter only in the young and clear-cut sites. Sclerotinia sclerotiorum is a widespread plant pathogen with a conidial stadium attacking a broad range of host plants. OTUs belonging to the ascomycete order Pleosporales, which includes plant pathogens, saprobes and lichen-forming fungi, were captured most frequently at the clear-cut site.
Most of the basidiomycetes were captured in the old growth forest site (cf. TABLE I, supplementary material 2). Several Peniophora species, being wood decomposers of various tree species, were solely detected in the old-growth forest site (cf. FIG. 2C). The root-rot pathogen Heterobasidon annosum and the wood-decomposer Phellinus ferrogineofuscus (both with 100/99% match in GenBank) likewise were captured frequently at this site.
Traditional inventories of visible macroscopic fruit bodies have demonstrated a negative correlation between forestry impact and fungal diversity in boreal forests (Sippola and Renvall 1999, Penttilä et al 2004). Our preliminary results support this relationship and suggest that the aerial spore sampling approach might have a great potential as a complementary method in monitoring fungal diversity and spread. We found a higher number of OTUs in the old-growth forest site (56), compared to the young (28) and clear cut (32) forest sites, and there was a significant difference in the mean number of OTU per spore sample replicate among the sites (cf. FIG. 3A). It is noteworthy that as much as 41.7% of the OTUs (35) were unique to the old-growth forest site, indicating that a high proportion of the fungal diversity in boreal forest could be associated with the old-growth forest habitat (cf. FIG. 5). However, we want to emphasize that our results are based on three forest sites only, with five replicates in each. Thus, due to the laborious methodology and the fact that we also wanted to investigate the level of beta diversity in each site by replicated measurements, we had no replicates of each forest type. This means that generalizations should be avoided and results dealing with the differences among forest types should be considered as preliminary. In one of the few other studies measuring fungal spore spread in natural forest environments, it was shown that the spore deposition (and indirectly the abundance of fruit bodies) for three selected fungal species was higher in landscapes characterized by a large proportion of old spruce forests than in landscapes with a lower proportion of old forests (Edman et al 2004). This indicates that the spore deposition, and the frequencies of fruit bodies, strongly depends on the landscape composition at both regional and local scales and corresponds well with our preliminary results.
This study represents a first examination of this new approach, and we believe that the spore-trapping approach has a great potential in future studies to analyze the largely unexplored and highly anonymous species richness of fungi. The presented approach makes it possible to study the organisms further in vitro and to establish a link between genotype and cultural phenotypic traits. The spore capturing approach thus provides a good starting point to study life-history characteristics of anonymous fungal groups difficult to target in other ways (e.g. species of Lachnum, Mollisia and Phialocephala). Population genetic analyses may be carried out on the captured populations to explore their population structure. The presented method opens for several new questions to be studied, including change of diversity along a time-axis and various ecological gradients. Finally, the approach also could encourage attempts to relate the aerial spore diversity with corresponding mycelia and sporocarps to approach a better understanding of dispersal dynamics in different groups of fungi.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Corresponding author. E-mail: haavarka{at}bio.uio.no
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Alexopoulos CJ, Mims CW, Blackwell M. 1996. Introductory Mycology. New York: John Wiley & Sons.
Bruce KD. 1997. Analysis of mer gene subclasses within bacterial communities in soils and sediments resolved by fluorescent-PCR-restriction fragment length polymorphism profiling. Appl Environ Microbiol 63:4914–4919.[Abstract]
Bruns T. 2001. ITS reality. Inoculum, 2–3 Supplement to Mycologia 52.
Colwell RK. 1997. EstimateS: Statistical estimation of species richness and shared species from samples. Version 5 http://viceroy.eeb.uconn.edu/estimates
Edman M, Gustafsson M, Stenlid J, Jonsson BG, Ericson L. 2004. Spore deposition of wood-decaying fungi: importance of landscape composition. Ecography 27:103–111.[CrossRef]
Ganley RJ, Brunsfeld SJ, Newcombe G. 2004. A community of unknown, endophytic fungi in western white pine. Proc Natl Acad Sci USA 101:10107–10112.
Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98.
Herbarth O, Schlink U, Mueller A, Richter M. 2003. Spatiotemporal distribution of airborne mould spores in apartments. Mycol Res 107:1361–1371.[CrossRef][Medline]
Horton TR, Bruns T. 2001. The molecular revolution in ectomycorrhizal ecology: peeking into the black-box. Mol Ecol 10:1855–1871.[CrossRef][Medline]
Hurlbert SH. 1984. Pseudoreplication and the design of ecological field experiments. Ecol Monog 54:187–211.[CrossRef]
Ingold CT. 1965. Spore liberation. London, UK: Oxford University Press.
James TY, Vilgalys R. 2001. Abundance and diversity of Schizophyllum commune spore clouds in the Caribbean detected by selective sampling. Mol Ecol 10:471–479.[CrossRef][Medline]
Jumpponen A, Trappe JM. 1998. Dark septate endophytes: a review of facultative biotrophic root-colonising fungi. New Phytol 140:295–310.[CrossRef]
Kirk PM, Cannon PF, David JC, Stalpers JA. 2001. Ainsworth & Bisby’s dictionary of the fungi. 9th ed. Wallingford, UK: CABI Publishing.
Menkis A, Allmer J, Vasiliauskas R, et al. 2004. Ecology and molecular characterization of dark septate fungi from roots, living stems coarse and fine woody debris. Mycol Res 108:965–973.[CrossRef][Medline]
Murray MG, Thompson WF. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acid Res 8:4321–4325.
Noble IR, Dirzo R. 1997. Forests as human-dominated ecosystems. Science 277:522–525.
Ohlson M, Söderström L, Hörnberg G, Zackrisson O, Hermansson J. 1997. Habitat qualities versus long-term continuity as determinants of biodiversity in boreal old-growth forests. Biol Cons 81:221–231.[CrossRef]
Pennisi E. 2004. The secret life of fungi. Science 304:1620–1622.
Penttilä R, Siitonen J, Kuusinen M. 2004. Polypore diversity in managed and old-growth boreal Picea abies forests in southern Finland. Biol Cons 117:271–283.[CrossRef]
Ranjard L, Lejon DPH, Mougel C, et al. 2003. Sampling strategy in molecular microbial ecology: influence of soil sample size on DNA fingerprinting analysis of fungal and bacterial communities. Environ Microbiol 5:1111–1120.[CrossRef][Medline]
Rholf FJ. 1997. NTSYS-pc-numerical taxonomy and multivariate analysis system. New York: Exeter Software.
Schweigkofler W, O’Donnell K, Garbelotto M. 2004. Detection and quantification of airborne conidia of Fusarium circinatum, the causal agent of pine pitch canker, from two California sites by using a real-time PCR approach combined with a simple spore trapping method. Appl Environ Microbiol 70:3512–3520.
Sippola AL, Renvall P. 1999. Wood-decomposing fungi and seed-tree cutting: a 40-year perspective. For Ecol Manage 115:183–201.[CrossRef]
Swofford DL. 2003. PAUP*. Phylogenetic analysis using parsimony (*and other methods). Version 4. Sunderland: Sinauer.
Vandenkoornhuyse P, Baldauf SL, Leyval C, Straczek J, Young JPW. 2002. Extensive fungal diversity in plant roots. Science 295:2051.
Vasiliauskas R, Lygis V, Larsson KH, Stenlid J. 2005. Airborne fungal colonisation of coarse woody debris in North Temperate Picea abies forest: impact of season and local spatial scale. Mycol Res 109:487–96.[CrossRef][Medline]
Vrålstad T, Myhre E, Schumacher T. 2002. Molecular diversity and phylogenetic affinities of symbiotic root-associated ascomycetes of the Helotiales in burnt and metal polluted habitats. New Phytol 155:131–148.[CrossRef]
White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ., eds. PCR protocols: a guide to methods and applications. San Diego, California: Academic Press. p 315–321.
Williams RH, Ward E, McCartney HA. 2001. Methods for integrated air sampling and DNA analysis for detection of airborne fungal spores. Appl Environ Microbiol 67:2453–2459.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
