DNA Barcoding of Fungi
Preamble
It has been estimated that global fungal diversity totals 1.5 million species, but less than ten percent of these have been formally described (Kirk et al. 2001). Fungi are of considerable economic importance, not only because of their well known role in many fermentation processes, but also as causative agents of plant and animal diseases, as allergens, as sources of antibiotic production, and increasingly for their use in bio-control. Fungi also feature prominently in the list of invasive alien species in Canada, with prominent examples including the pathogens causing white pine blister rust, potato wart, soybean rust, and most recently, Sudden Oak Death. The deployment of DNA barcoding for the efficient detection and identification of both pathogenic and beneficial fungi will be crucial for protecting and enhancing the health of our forests and crops.
The creation of stable taxonomic systems has proven difficult for many fungal groups because of the limited number of distinctive morphological characters exhibited by these organisms. Not only have higher-level taxa been shown to be heterogeneous, but even basic species concepts are commonly called into question by new molecular data (see Taylor et al.2000). In the present context, we use the colloquial term “fungi” to include members of what are now recognized as two distinct kingdoms: the Mycota and Straminipila (Money 1998). Past taxonomic efforts have been directed largely towards Basidiomycetes and Ascomycetes that sporadically produce conspicuous fruiting bodies, but spend most of their life as sterile, microscopic hyphae. However, it is obvious that a system is needed that is capable of identifying all fungi, be they macroscopic or microscopic, and regardless of life stage. This is especially true in practical situations such as the study of asymptomatic material (Hamelin et al. 2000).
Already, there is a large ribosomal DNA (rDNA) database for fungi, representing about 12% of described species, based on sequences from the nuclear large subunit RNA (LSU) gene and the internal transcribed spacer (ITS). These rDNA sequences, particularly ITS, have been successfully exploited as species markers in some fungal groups (e.g. Basidiomycetes and Oomycetes), but these sequences are too conserved in some Ascomycetes and related moulds. In these cases, identifications can currently only be done at taxonomic ranks above the species level. The US-NSF Assembling the Tree of Life Program has funded the development of a comprehensive phylogeny of the Fungi based on 7 genes (but not including COI) of 1,000-2,000 species. However, that program does not consider species level identifications and, consequently, is complementary to our objectives to develop species diagnostics for selected groups.
One potential complication for DNA barcoding of fungal species is the fact that, unlike animals, the mitochondrial genes of fungi are frequently interrupted by introns. One solution to this problem would be to focus on the characterization of sequences obtained from cDNA libraries from which introns have already been spliced, an approach that has shown promise in rusts (Joly et al. 2003). Alternatively, it may be useful to employ RT-PCR methods to amplify mRNA sequences rather than genomic DNA. These methodological challenges aside, the presence of introns does provide some advantages, for example by facilitating the resolution of closely related taxa by virtue of their accelerated rates of sequence divergence. This also makes it possible to carry out surveys of the distribution of mobile mitochondrial introns in natural populations.
Research plan (year 2)
During the first year of the project, we will establish a reference list of DNA barcodes for fungal species. In order to do this, we will exploit several exceptional collections of fungi that are located in museums, government laboratories, and universities in Canada, all of which have already been classified using non-molecular taxonomic methods. These collections include the Canadian Collection of Fungal Cultures (CCFC, the largest collection of living fungi in Canada), the Canadian National Fungal Herbarium (DAOM), and the fungal collections of the Royal Ontario Museum (ROM). The latter collection incorporates the former University of Toronto herbarium, with a rich sampling of the fungi of Ontario.
TopResearch plan (years 3 and 4)
During the second and third years of the project, we will exploit our newly established DNA barcodes for the rapid identification of species collected in nature. We will focus particularly on fungi that are associated with plant roots (the rhizosphere) and leaves (the phyloplane). The rhizosphere includes the economically important and beneficial fungi that are associated with the roots of forest trees, while the phyloplane includes many plant pathogens. This will provide a proof-of-concept that DNA barcoding can be used to study these systems in forestry and agricultural field sites in Ontario. Initially, we will focus on four phylogenetically divergent fungal groups, three from the Mycota (rusts, mycorrhiza forming mushrooms, ascomycetous moulds) and one from the Straminipila (oomycetes). For all groups, taxonomic resolution based on DNA barcodes will be compared with existing methods, including those based on rDNA data.
i. Fungi associated with plant roots – the rhizosphere: First, we will compile a set of DNA barcodes for macroscopic basidiomycetes, focusing particularly on the mushrooms (Agaricales) that have mycorrhizal associations with plants, especially forest trees. Members of our fungal group have already published extensively on rDNA phylogenetics in the Agaricales (Moncalvo et al. 2000a, 2002), including the ectomycorrhizal genera Amanita (Drehmel et al. 1999; Moncalvo et al. 2000b) and Cortinarius (Peintner et al. 2001, 2004). These fungi are widespread in Canadian forests and form an essential component of the forest ecosystem. Molecular markers will be developed for at least 1,000 forest species. Species boundaries will be inferred first from our barcoding surveys and these boundaries will then be compared to multigene phylogenies. Such phylogenies have already been constructed for several common and broadly distributed species complexes, e.g., Laccaria laccata, Amanita vaginata, and Cantharellus spp. (Taylor et al. 2000).
Although the development of DNA barcodes is needed for the rapid identification of macroscopic fungi, this is even more crucial for microscopic species. To address this problem, we will develop DNA barcodes for microscopic ascomycetes, focusing on species that are either known root pathogens, or that may play a role in the rhizosphere. This work will extend ongoing research on the common genus Penicillium (Samson et al. 2004), focussing on the subgenus Furcatum, which is assumed to include much unknown species diversity. We will also develop DNA barcodes for species of Fusarium that cause root rots, along with other common root pathogens such as species of Cylindrocarpon, Plectosporium, and other rhizosphere fungi, including species of Aspergillus. Finally, we will develop barcodes for several genera of recently discovered heat-resistant moulds associated with plant roots, which appear to be a component of the fungi often reported as “unidentified” in studies of environmental DNA (Hambleton et al. 2005).
Our initial studies on the oomyctes will target members of the genus Pythium, which includes ubiquitous species such as P. ultimum (a cause of damping off in many plants) and P. insidiosum (which infects animals including humans). We have previously used rDNA sequences to differentiate Pythium species (Lévesque and de Cock 2004). More recently, COI was sequenced and compared for Phytophthora species (Martin and Tooley 2003; Kroon et al. 2004), although most of the sequences are partial and at the 3' end of the gene. We will develop DNA barcodes for all known species in the genus Pythium as well as those in Phytophthora, along with a wide range of other Oomycota (e.g., Saprolegnia, Achlya). We will use these barcodes in combination with our other sequence data to help determine species boundaries in several species complexes that we have identified by recent molecular work, e.g., P. irregulare, P. vexans, P. ultimum (Lévesque and de Cock 2004). CCFC has a multiple strain coverage of these complexes from an earlier study with isozyme analyses (Barr et al. 1996, 1997a, 1997b).
ii. Fungi associated with plant leaves – the phyloplane: We will develop DNA barcodes for two separate taxonomic groups that are associated with leaves and shoots (the phyloplane). The first group includes many of the forest rusts, one of the most important groups of forest pathogens. Many species of rust fungi have complex life histories that involve their growth on two different plant hosts, and DNA barcodes will be especially useful in linking these different life stages to single species. We will use the relationship among species of the genera Peridermium and Cronartium to test the hypothesis that the former taxa are simply haploid derivatives of the latter. If confirmed, this result would have tremendous implications for the control and epidemiology of these important disease agents because species of Peridermium can maintain themselves on a single host (their common name is pine-to-pine rust), while species of Cronartium must switch hosts to complete their life cycle. We will also carry out work on several taxonomic assemblages of rusts to ascertain the value of DNA barcodes in species recognition.
We will likewise develop DNA barcode data sets for other phyloplane fungi, including the common moulds Cladosporium and Alternaria (in collaboration with the Centraalbureau voor Schimmelcultures (CBS) in The Netherlands) and Epicoccum. These taxa, along with Penicillium and Aspergillus, are important contributors to reduced indoor air quality, and an offshoot of this work could be an improved molecular detection assay for workers in this field. A planned AAFC survey on legume rusts of southern Ontario will also provide data that will be exploited in this study.
In a more applied project, we will explore the role of hybridization and introgression in generating novel fungal diseases of plants. For instance, a new Eurasian poplar rust was recently discovered in eastern North America and it is thought to hybridize with several native species. Mitochondrial markers, in general, have a strong track record of providing useful insights in hybrid systems because of their matrilineal inheritance. Moreover, we recently discovered COI sequence differences among species of this genus in isolates collected prior to the invasion. We will combine analyses of these historical collections with work on newly gathered material to investigate the role of hybridization in generating this rust disease.
Development of microarrays for ecological samples (year 4)
Sequence databases of a single gene or gene region encompassing several species can be used to design DNA oligonucleotide (oligo) microarrays for the identification and detection of organisms in multi-species mixtures. Such microarrays can be used to analyze complex environmental DNA samples that can be processed directly to determine the species present. We have demonstrated the feasibility of this approach in fungi (Lévesque et al. 1998) and it has also been used for the identification of nematodes (Uehara et al. 1999) and bacteria (Fessehaie et al. 2003). These microarrays comprise a collection of short oligos (16-28 bases in length) that match a particular gene. The location of these oligos within the gene can be chosen to be specific to different taxonomic levels such as species, genus, or family. The region of the gene corresponding to the oligos on the microarray is PCR amplified and fluorescently labelled from DNA extracted from an “unknown” sample, and then hybridized to the microarray. RNA can also be used to generate labelled cDNA, an approach similar to what is being used in functional genomics.
DNA oligos for microarrays and for PCR amplification (or cDNA labelling) will be designed based on the species-specific DNA barcodes, using software that was developed to identify oligos of various specificity levels from large sequence databases (by LifeIntel Software Inc., Port Moody, BC, under contract by C.A. Lévesque). The databases developed in this project will provide an opportunity to extend the microarray approach for ecological studies. We will choose a subset of organisms from this project to generate a COI microarray. This will then be tested on a micro-community from which a wide range of species has been barcoded and that can be sampled in small volumes (i.e., soil or leaves). DNA from these samples will be amplified with COI primers for targeted taxonomic groups and the PCR products will be hybridized to the "community microarray". Validation of the microarray data will be done on a limited number of samples by sequencing cloned PCR products and by using species-specific PCR primers for some species. Once the feasibility of such an approach has been established, we will begin work on larger ecological samples of fungi (see Section 6.7).
Personnel: Richard Hamelin (Canadian Forest Service) is actively engaged in the use of molecular techniques to gain insights into the taxonomy of rusts and other forest pathogens. Keith Seifert (Agriculture and Agri-Food Canada) is a world leader in mould systematics, and the chair of the International Commission on the Taxonomy of Fungi. André Lévesque (Agriculture and Agri-Food Canada) is the world authority on the molecular taxonomy of Pythium and an acknowledged leader in developing molecular diagnostics for microorganisms. Jean-Marc Moncalvo (University of Toronto and ROM) was involved in developing the first comprehensive phylogenies of agarics and has ongoing field surveys and environmental DNA studies. Other mycologists at AAFC will contribute to this project by deriving data on fungi in areas of their expertise that may be useful for our environmental analyses (S. Redhead, agarics; S. Hambleton, agricultural rusts and smuts; J. Bissett, leaf pathogens, Trichoderma; Y. Dalpé, arbuscular mycorrhizal fungi). For specific fungal groups, we will collaborate with colleagues at the CBS in The Netherlands, the world’s largest fungal culture collection.
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