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Keywords:

  • COX1;
  • DNA barcoding;
  • fungi;
  • mitochondrial cytochrome oxidase 1;
  • mycotoxins;
  • plant pathogens

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest statement
  9. References
  10. Supporting Information

Using data from published mitochondrial or complete genomes, we developed and tested primers for amplification and sequencing of the barcode region of cytochrome oxidase 1 (COX1) of the fungal genus Fusarium, related genera of the order Hypocreales, and degenerate primers for fungi in the subdivision Pezizomycotina. The primers were successful for amplifying and sequencing COX1 barcodes from 13 genera of Hypocreales (Acremonium, Beauveria, Clonostachys, Emericellopsis, Fusarium, Gliocladium, Hypocrea, Lanatonectria, Lecanicillium, Metarhizium, Monocillium, Neonectria and Stilbella), 22 taxa of Fusarium, and two genera in other orders (Arthrosporium, Monilochaetes). Parologous copies of COX1 occurred in several strains of Fusarium. In some, copies of the same length were detected either by heterozygous bases in otherwise clean sequences or in different replicates of amplification and sequencing events; this may indicate multiple transcribed copies. Other strains included one or two introns. Two intron insertion sites had at least two nonhomologous intron sequences among Fusarium species. Irrespective of whether the multiple copy issue could be resolved by sequencing RNA transcripts, developing a precise COX1-based barcoding system for Fusarium may not be feasible. The overall divergence among homologous COX1 sequences obtained so far is rather low, with many species sharing identical sequences.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest statement
  9. References
  10. Supporting Information

DNA barcoding is the use of a short, easily amplified genetic fragment for species level identification; in animals, this marker is typically from cytochrome oxidase 1 (COX1) (Hebert et al. 2003). The barcoding movement facilitates identification of species across all kingdoms of eukaryotic life, recognition of cryptic species, and identification of organism fragments or life cycle stages not amenable to morphological identification. Although mycology has a long history of DNA sequence-based identification using the nuclear ribosomal internal transcribed spacer (ITS), fungal barcoding with COX1 is in its infancy. Until 2007, most published fungal COX1 sequences were derived from mitochondrial genomes and there were few published polymerase chain reaction (PCR) primers. Reports of introns led to reservations among mycologists for using this gene as a phylogenetic marker or for DNA barcoding (Rossman 2007). If introns are frequent and of significant length, they would interfere with PCR by disrupting priming sites or increasing amplicon length. However, in the first comprehensive study of COX1 in a fungal group, Penicillium subgenus Penicillium (Seifert et al. 2007), introns were found in only about 1% of 360 strains. A subsequent study of a smaller genus in a different taxonomic order, Leohumicola, reported only 5% of sampled cultures with introns (Nguyen & Seifert 2008).

The Hypocreales (Fungi, Ascomycetes) include about 1000 species divided among many sexual (teleomorph) and asexual (anamorph) genera (Rossman et al. 1999). Fusarium is an anamorph genus of about 125 commonly accepted species (but undoubtedly much larger), widely distributed on plants and in the soil, which produce characteristic banana-shaped macroconidia (Leslie & Summerell 2006). It includes several economically important plant pathogens, such as Fusarium graminearum, F. verticillioides, F. oxysporum and F. solani. Several species produce mycotoxins, such as trichothecenes, zearalenone and fuminosins that are subject to international regulations (Desjardins 2006). Crops and other food commodities contaminated with these toxins affect human and animal health by chronic or acute toxicity, or long-term carcinogenic effects. Over the past five years, traditional morphological identification of Fusarium has been supplanted by sequencing of the translation elongation factor 1-alpha (TEF1) gene (Geiser et al. 2004).

A successful DNA barcoding system for identifying species of Fusarium would have great research value, and potential commercial value. Quarantine plant pathologists and regulators guarding the food system routinely deal with species of this genus. Private seed testing laboratories identify Fusarium species present in thousands of grain samples every year. Fusarium species are also increasingly isolated from human infections, or from medical products intended for human use (O'Donnell et al. 2007).

This study was designed to evaluate the utility of COX1 as a DNA barcode for identifying species of Fusarium, using newly designed primers. There is no comprehensive monograph or phylogenetic revision of Fusarium available. The atlas by Gerlach & Nirenberg (1982) provides our framework for the discussion of taxonomic sections, and O'Donnell et al. (2007) for several of the phylogenetic clades (species complexes) within the genus. The most recent phylogenetically based classification of the fungi (Hibbett et al. 2007) is used as the basis for discussion and nomenclature of higher taxa.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest statement
  9. References
  10. Supporting Information

Samples

Cultures were obtained from the Canadian Collection of Fungal Cultures (DAOM) and the Julius Kühn Institute, Institute of Epidemiology and Pathogen Diagnostics culture collection (BBA, Berlin/Braunschweig). Details of the origin of strains employed are provided in Table S1, Supporting information.

Genomic DNA was isolated from pure cultures grown in 9 cm polystyrene Petri dishes on potato dextrose agar (PDA, Difco), generally after 5–10 days incubation at room temperature. Aerial mycelium was scraped from the surface of the agar using a sterilized scalpel, and transferred to an extraction vial. A variety of DNA extraction kits were used, but most extracts were prepared with the Fast DNA standard DNA extraction kit (BIO-101, QBiogene) or the UltraClean Microbial DNA Isolation Kits (MO BIO Laboratories Inc.), following the manufacturers’ instructions.

All strains were identified by morphological analysis in comparison with Gerlach & Nirenberg (1982), by sequencing of the standard identification gene TEF1 and reference to GenBank, the online Fusarium identification database (isolate.fusariumdb.org, Geiser et al. 2004), and by comparison of TEF1 and other gene sequences generated during taxonomic studies in our laboratory.

Primer design

Primers for amplification of the COX1 barcode in Hypocreales were designed using an alignment of complete or partial mitochondrial genome sequences available in early 2007 for this fungal order, namely Fusarium oxysporum (GenBank accession AY874423, Cunnington 2007; AY945289, Pantou et al. 2008), Fusarium verticillioides (Brown et al. 2005), Metarhizium anisopliae (AY884128, Ghikas et al. 2006), Hypocrea jecorina (AF447590, Chambergo et al. 2002), and Lecanicillium muscarium (AF487277, Kouvelis et al. 2004). Of the sequences used for designing primers for Fusarium, those for Hypocrea jecorina and Metarhizium anisopliae had introns present in the COX1 gene; the others lacked introns. A survey of intron location and length using sequences from GenBank, revealed 11 potential intron sites in the 567-bp barcode region of the Pezizomycotina (including the Hypocreales) (Fig. 1, Table 1). Intron position and length were determined through alignments of both DNA and predicted amino acid sequences with samples known to contain no introns. At one of these sites (intron 4), two different, unalignable intron sequences occurred in the same location; these were labelled a and b. Introns were excluded from the alignment for the purposes of barcode primer design.

image

Figure 1. Map of COX1 barcode region in the Pezizomycotina. Boxes represent introns, and horizontal arrows indicate relative positions of primers designed in this study. Split boxes indicate sites where two nonhomologous introns have been reported. Not to scale.

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Table 1.  Positions and lengths of introns identified in the barcode region of the members of the fungal subphylum Pezizomycotina using publicly available mitochondrial genomes or COX1 sequences. The barcode region of the Fusarium oxysporum mitochondrial genome (AY874423) was used as the position numbering reference. The letters a and b distinguish between two nonorthologous, unalignable introns found at the same position. Examples are shown of different strains where introns were recovered, with the intron length (in bp) and position in the barcode where the intron was found. A+ sign indicates that only a partial sequence was obtained and the final length of the intron is greater than the indicated number. The species in the first eight rows are in the Hypocreales, the remainder are in other classes of the Pezizomycotina
Intron number1234a4b567891011a11bTotal bp
Barcode position of intron (bp)855689797138170253256350472566566567
Fusarium oxysporum AY874423               567
Fusarium delphinoides DAOM 235647               567
Fusarium verticillioides genome            1498~2000
Fusarium lateritium BBA 62244   512+          692+ ~3000
Fusarium solani DAOM 235651             861+ ~2000
Fusarium babinda DAOM 235678           633+  ~2000
Fusarium flocciferum BBA 64535  712+  229+         ~2000
Lanatonectria flocculenta DAOM 229273  609+  246+         ~2000
Hypocrea jecorina NC003388        1282 1138   2987
Podospora anserina X55026.12539  1238 126025001507 14562251 140714724
Aspergillus japonicus AF315722  842+  1239         2521 + 
Neurospora crassa X14669 2631   1345    1222   5765

One reverse primer and one forward primer were designed and tested for PCR and sequencing of the COX1 barcode region of species of Fusarium, and two of the frequently occurring introns (Fig. 1). To amplify the entire barcode region, the forward primer AHyFu-F (5′-CTTAGTGGGCCAGGAGTTCAATA-3′) was used together with the reverse primer AHyFu-R (5′-ACCTCAGGGTGTCCGAAGAAT-3′). Two additional forward primers were designed to amplify related genera; AHyLe-Fa (5′-TCAGGATTATTAGGTCAGCATTT-3′) for Lecanicillium and AHyMe-F (5′-TTAAGTGGCCCAGGAGTACAAT-3′) for Metarhizium, both of which paired with AHyFu-R.

To amplify templates within intron 3, forward primer Fus-I3-F (5′-TTAAAAGTATCGAAAAATCAAAAAGGTGT-3′) was used together with Fus-I3-R (5′-ATCTATCTCTTATTTCTTGGCTCATTGGTT-3′). A forward primer within intron 4b, Fus-I4b-F (5′-CCTTTAAAACTAGTACCGCAGAC-3′), was used in conjunction with the reverse barcode primer AHyFu-R.

We tested these primers on representatives of several genera and families of the order Hypocreales. To confirm that the sequences derived with the newly designed PCR primers amplified the correct gene, blast analyses were performed at National Center for Biotechnology Information to confirm the identity of putative COX1 sequences; the only alignable sequences recovered with these searches were COX1.

Degenerate primers were also designed to amplify COX1 from a broader range of taxa classified in the subdivision Pezizomycotina. To the alignment used for the Hypocreales described above, we added sequences from the Eurotiomycetes Aspergillus niger (DQ207726, AF177534) and Penicillium marneffei (NC005256), the Sordariomycetes Neurospora crassa (X01850, X14669, M36958), Podospora anserina (X55026), Torrubiella confragosa (DQ311640), and Verticillium dahliae (DQ351941), and the Leotiomycete Botrytis cinerea (AL110841), and focused on the barcode primer regions. Although these primers were not used in the main part of this study, they are briefly discussed in this paper because of their possible utility for other mycologists: Pez-F (5′-TCAGGRTTAYTAGGWACAGCATTT-3′) and at the same sequence position as AHyFu-R above, the degenerate primer Pez-R (5′-ACCTCAGGRTGYCCGAAGAAT-3′).

DNA amplification and sequencing protocols

PCRs were performed using, 0.1 mm dNTP (Invitrogen), 0.1 µm forward primer, 0.1 µm reverse primer, 3.5 µm MgCl2, 1× Taq buffer (Invitrogen), 0.5× Platinum Taq enzyme (Invitrogen), and 1.00 µL of DNA template (1–5 ng/µL) were mixed in sterile high-performance liquid chromatography (HPLC) grade water totalling 10 µL per reaction. The PCR was run in a Mastercycler epgradient S thermal cycler (Eppendorf). The following parameters were used to amplify COX1: 95 °C for 3 min, then 40 cycles at 95 °C for 30 s, 55 °C for 30 s, 72 °C for either 1 min or 3 min, then 72 °C for 5 min. For several templates, touchdown PCR was performed, beginning at 65 °C for 3 cycles dropping by 3 °C for 3 cycles at each temperature, ending with 25 cycles at 50 °C. For different barcode templates, annealing temperatures between 50–55 °C were employed. For intron amplifications, the touchdown programme described above was employed.

For screening of the Pez-F and Pez-R primers with outgroup species, PCRs were performed using: 0.1 mm dNTP, 0.08 µm forward primer, 0.08 µm reverse primer, 1× Titanium Taq and buffer 0.5× Titanium Taq enzyme (Clontech), 1.00 µL of DNA template (1–5 ng/µL) were mixed in sterile HPLC grade water totalling 20 µL per reaction. The following PCR profile was used: 95 °C for 3 min, then 35 cycles at 95 °C for 1 min, 56 °C for 45 s, 72 °C for either 1 or 3 min, then 72 °C for 10 min. PCRs were run either a Miometra T-gradient or Techne Genius thermocycler.

PCR products were separated by electrophoresis on a 1.5% agarose gel, stained with ethidium bromide and visualized under ultraviolet light.

Both forward and reverse strands were sequenced using BigDye Terminator (Applied Biosystems) using the protocol described by de Cock & Levesque (2004). The 10-µL sequencing reactions were run at 96 °C for 10 s, then 40 cycles at 96 °C for 20 s, 50 °C for 15 s, 60 °C for 4 min. The 20-µL sequencing reactions were run for testing of Pez-F and Pez-R primers were run at 95 °C for 3 min, then 30 cycles at 95 °C for 45 s, 56 °C for 30 s, 72 °C for 3 min. DNA sequences were determined using an ABI 3130XL Genetic Analyser (Applied Biosystems) following the manufacturer's instructions.

Contigs were assembled and edited using Sequencher version 4.8 (Gene Codes). All sequences are deposited in GenBank (accession nos FJ501222–FJ544543, FJ544547, FJ544549, FJ544551, FJ544553, FJ544554, FJ544556, FJ544558, FJ544560, FJ544561, FJ544563, FJ544565, FJ544567–FJ544570, FJ544572, FJ544573, FJ663051–FJ663057; for details see Table S1) and the Barcode of Life Database (Ratnasingham & Hebert 2007). Alignments were computed using ClustalW algorithm as implemented in BioEdit version 7 (Hall 1999).

Analyses of aligned sequences were performed using neighbour-joining and parsimony methods as implemented in mega version 4 (Tamura et al. 2007). Neighbour-joining methods used pairwise deletion and Kimura's 2-parameter model (Kimura 1980); parsimony methods used the default closest neighbour interchange search level with 10 random starts. Bootstrap analyses of 1000 replicates were undertaken for each analysis. Three separate phylogenetic analyses were performed. The partial barcode analysis from intron 4 to the end of the barcode included 411 bp of the 567 bp barcode region of COX1, including the barcode region from the F. oxysporum and F. verticillioides genomes used to design the primers. Separate analyses were run on the 290-bp alignment of intron 3 and 198-bp alignment of intron 4b.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest statement
  9. References
  10. Supporting Information

Primer design and introns in the barcode region

Four sets of barcode primers were designed and successfully tested during our experiments. The primer pair AHyFu-F and AHyFu-R was successful for amplifying and sequencing COX1 for 24 of 29 Fusarium strains tested. These primers were designed to amplify a 567-bp fragment of COX1 but yielded PCR fragments of three sizes: ~570 bp (no introns), ~2000 bp and ~3000 bp (introns present). Sequencing of Fusarium strains revealed introns at three of the known intron positions (3, 4, 11, see Fig. 1); at two positions two different sequences types were present (4a, 4b and 11a, 11b). In our initial survey, the COX1 of most Fusarium strains had either no introns present, or introns 3 and 4b (Figs 2–4, Table 2).

image

Figure 2. Gene tree of Fusarium COX1 from intron 4b to the end of the barcode only with Clonostachys rosea as outgroup. The neighbour-joining tree is on the left and the consensus maximum parsimony tree on the right (CI = 0.60; RI = 0.84; 138 most parsimonious trees length = 203). Roman numerals on the consensus tree indicate the main clades I–IV, each of which may represent a distinct COX1 copy. Names in bold text indicate species that occur in multiple clades. Side panels show heterozygous bases detected in barcode sequences of two species. An asterisk indicates heterozygous bases in the sequence, suggesting paralogous copies. Superscript codes on each strain refer to barcode type: 1 = 567 bp code obtained with the barcode primers AHyFu-F and AHyFu-R; a and b denote different sequences from different amplification replicates; 2 = intron 3 and 4b present but removed for this analysis, sequences obtained with barcode primers; 3 = Intron 4b present, obtained with primers Fus-I4b-F and AHyFu-R; 4: Intron 11a present, sequence obtained with barcode primers; 5 = Intron 11b present, sequence obtained barcode primers. The final gapless alignment was 411 bp, with 63 parsimony informative characters.

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image

Figure 3. Gene tree of intron 3 recovered from Fusarium species using primers Fus-I3-F and Fus-I3-R, rooted with Fusarium delphinoides. The neighbour-joining tree is on the left and the consensus maximum parsimony tree on the right (CI = 0.86; RI = 0.96; 208 most parsimonious trees length = 34). The side panel shows a heterozygous base pair detected in the intron of one strain. Although this intron was also recovered in some sequences using the barcode primers, it is unproven that all sequences in this tree actually occur in the COX1 gene; some could represent mobile mitochondrial introns. The alignment had 290-bp positions, of which 12 were parsimony informative.

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image

Figure 4. Gene tree of intron 4b recovered from Fusarium species using primers Fus-I4b-F and AHyFu-R, intron segment only, rooted with Fusarium delphinoides. The neighbour-joining tree is on the left and the consensus maximum parsimony tree on the right (CI = 0.84; RI = 0.93; 28 most parsimonious trees length = 27). Because the reverse primer is located in the COX1 barcode, these introns definitively occur in the COX1 gene. The alignment had 198-bp positions, of which 15 were parsimony informative.

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Table 2.  Summary of the number of different COX1 copies detected in individual strains of Fusarium sequenced in this study. Asterisks indicate the sequence had heterozygous bases, indicating the existence of additional multiple copies. Sequences which originally produced a barcode with no introns and later amplified intron 3 are labeled with a ^; or later amplified intron 3 and 4b are labeled with a °
 Barcode copiesNo intronsIntron
34b4a11a11b
Fusarium acuminatum BBA 621491 11   
Fusarium avenaceum BBA 717821 11   
Fusarium cf. avenaceum BBA 63784211   
Fusarium babinda DAOM 2356783 2*  1 
Fusarium boothii DAOM 235624211   
Fusarium circinatum DAOM 23575222     
F. circinatum DAOM 23575322     
F. circinatum DAOM 23575842*^2*    
Fusarium coeruleum BBA 6440011     
Fusarium concolor DAOM 2357311 11   
Fusarium delphinoides DAOM 235647211   
Fusarium diversisporum BBA 636061 ?1   
Fusarium equiseti BBA 677861 11   
Fusarium flocciferum BBA 645351 11   
Fusarium cf. heterosporum DAOM 2356431 1?   
Fusarium incarnatum BBA 684591 11   
Fusarium lateritium BBA 622442 1 1 1
F. lateritium var. buxi BBA 647981 11   
Fusarium pallidoroseum BBA 697921 11   
Fusarium sacchari DAOM 23579532*^1    
Fusarium solani DAOM 2356512 1   1
Fusarium torulosum BBA 6446521^1    
Fusarium cortaderiae DAOM 235621311   
Fusarium graminearum DAOM 23580021^1    

Fusarium circinatum and F. sacchari had heterozygous bases in the barcode sequences, indicating more than one COX1 copy in these putatively haploid strains (Fig. 2, Table 2). This, along with the presence of additional PCR products of sizes that would indicate the presence of introns (i.e. ~2000 or 3000 bp), led us to design intron-specific primers for the two most commonly found introns (3 and 4b). We used these to test for introns in Fusarium strains where they were not seen in the initial barcode amplifications. The intron primers targeted a 337-bp fragment of intron 3 (Fus-I3-F and Fus-I3-R) and a second primer (Fus-I4b-F) paired with the barcode reverse primer (AHyFu-R) to yield a 724-bp fragment starting in intron 4b. When these primers were tested on Fusarium genomic DNA that had previously yielded no introns, eight of the nine samples yielded intron-containing amplicons, suggesting the presence of multiple copies of COX1 (Table 2). It is possible that the intron 3 primers amplified copies of the intron outside of the COX1 gene. However, the primer designed for intron 4b, used in conjunction with the reverse barcode primer, would amplify only copies of this intron associated with COX1; four of nine samples yielded this product (Table 2).

We observed that when intron 3 was present, intron 4b was usually also present (Table 2). Although data were missing on intron 4b for some strains, none were confirmed as missing this intron when intron 3 was present. Some strains, such as Fusarium circinatum (DAOM 236752, 235753, 235758) may have additional copies, because multiple bands of variable length were seen in PCR amplifications. Two of these three strains (DAOM 235752, 235753) yielded different barcodes during replicate amplifications, while the final strain (DAOM 236758) had multiple heterozygous bases in the one isolated barcode (Table 2). Some strains had other COX1 copies with less common introns, Fusarium babinda (DAOM 235678) was the only strain to contain intron 11a; F. solani (DAOM 235651) and the F. verticilloides genome contained intron 11b. Fusarium lateritium (BBA 62244) had intron 11b as well as intron 4a, an intron otherwise only known in Podospora anserina (Tables 1 and 2). Some strains, such as F. babinda (DAOM 235678) and F. circinatum (DAOM 235758), had heterozygous bases in intron 3, suggesting multiple copies of that intron (Table 2, Fig. 3).

Outside the genus Fusarium, the forward primers AHyFu-F, AHyMe-F and AHyLe-Fa paired with AHyFu-R, were able to amplify Acremonium cf. chrysogenum (DAOM 226667), Metarhizium anisopliae (DAOM 237735), Monocillium mucidum (DAOM 226847), Beauveria bassiana (DAOM 233520, 210087), Lanatonectria flocculenta (DAOM 229273) Emericellopsis minima DAOM (226707), Hypocrea jecorina (DAOM 232048), and Neonectria ditissima (KAS 2832). All yielded a barcode with no introns, with the exception of L. flocculenta (DAOM 229273), which had introns 3 and 4b present (Table 1). The barcode sequence lacking introns for Hypocrea jecorina (DAOM 232048) confirmed that this species has two potential barcodes, because a second fragment with two introns was reported previously (Table 1; Chambergo et al. 2002).

The primer pair Pez-F and Pez-R was tested on a relatively small number of members of the fungal subdivision Pezizomycotina, but was successful at amplifying the barcode region of COX1 from the following member of the class Sordariomycetes: Acremonium murorum var. felina (DAOM 226750), Clonostachys rosea (DAOM 226795), C. compactiuscula (DAOM 226738; all Hypocreales, Bionectriaceae), Gliocladium viride (DAOM 226717; Hypocreales, Hypocreaceae), Stilbella aciculosa (DAOM 165520; Hypocreales, Nectriaceae), Arthro-botryum hyalospora (JCM 3809; Chaetosphaeriales), and Monilochaetes infuscans (CBS 379.77; Glomerellales). All yielded PCR products and barcode sequences of the expected length of c. 650 bp, none of which had introns.

Barcoding results

Neighbour-joining and maximum parsimony analyses of the barcode sequences obtained for the Fusarium species are shown in Fig. 2. This tree cannot be interpreted as a species tree, because individual strains occur repeatedly in different clades. There appear to be at least four major copies of COX1 in this tree (indicated by the Roman numerals I–IV), and possibly more. Our interpretation is that each major clade represents a distinct, homologous COX1 copy. This is supported by the fact that barcodes without introns (superscript 1 in Fig. 2) group together and barcodes with intron 4 (superscripts 2 and 3) group together on the tree in clades I and II. Oddly enough, clade III showed identical barcodes in both Fusarium cf. avenaceum (BBA 63784) and F. cortaderiae (DAOM 235621), one of which has no introns and the other has at least intron 4 present. It is difficult to interpret the significance of the smaller clades, whether they represent sporadically sampled copies, or phylogenetic signal from one copy overlaid on the multicopy tree.

The low barcoding utility of this data was evident by the relative lack of phylogenetic structure within each of the major clades. For example, clade I included four species with identical COX1 barcodes; they are classified in three different taxonomic sections (Gerlach & Nirenberg 1982), viz. Fusarium boothii and Fusarium graminearum in section Discolor (= F. graminearum species complex), Fusarium circinatum in section Liseola (= Gibberella fujikuroi species complex) and Fusarium oxysporum in section Elegans (= F. oxysporum species complex, O'Donnell et al. 2007). Clades II, III and IV included representatives of two different sections each.

The species resolution observed with intron 3 (Fig. 3, 304 bp) and intron 4b (Fig. 4, 198 bp) was similar to that seen with the exonic barcoding region (Fig. 2). With intron 3, the occurrence of two species from section Liseola (= Gibberella fujikuroi species complex), F. circinatum and F. sacchari, in two clades, and the relatively distant placement between the closely related species F. babinda and F. concolor, suggest the possible existence of paralogues of this intron. The distantly related species Fusarium equiseti and F. torulosum shared a common intron 3 sequence. Relatively few copies of intron 4b were recovered, but the inferred phylogeny was relatively congruent with the RPB2 phylogeny of O'Donnell et al. (2007), except that F. cortaderiae (DAOM 235621) was out of place. The phylogeny of intron 3 (Fig. 3) matched that of the partial barcode sequences with superscripts 2 and 3 (Fig. 2) for many strains but included additional strains for which the equivalent barcode was missing in Fig. 2. For example, the three members of the F. graminearum species complex, F. graminearum, F. cortaderiae and F. boothii formed a well-supported clade in Fig. 3. But F. cortaderiae occurred only in clade III in Fig. 2, suggesting that it may have barcodes yet to be isolated that would group in clades I and II.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest statement
  9. References
  10. Supporting Information

Multiple copies (paralogues) and a lack of species level resolution within homologous copies render COX1 an unsuitable barcode for identifying species of the commonly isolated and economically important fungal genus Fusarium. Although we designed effective primers for the amplification and sequencing of this gene in Fusarium, and the broader range of ascomycetes in subdivision Pezizomycotina, the presence of introns significantly affected amplification efficiency. As many as three different barcode sequences were detected in a single strain, and possibly four copies in F. circinatum (DAOM 235758), which included heterozygous bases in both the barcode copy that lacked introns and within intron 3. Multiple copies of COX1 confound the interpretation of the barcode tree of Fusarium (Fig. 2), with several strains and/or species occurring in multiple clades. These are not misidentified strains. The identity of all strains was confirmed with TEF1 sequencing (Geiser et al. 2004) and other genes where appropriate. We suspect that with comprehensive sampling, each main cluster within the barcode tree would reiterate the species phylogeny, as indicated by the presence of the same strain of the basal species Fusarium delphinioides both near the base of the tree in clade IV, and at the basal position of clade III. Even with a complete data set for each copy, the lack of resolution at the species level within homologous COX1 copies, with species from different sections of the genus having identical sequences, would render the sequences unsuitable as diagnostic barcodes.

This is in contrast to the study by Seifert et al. (2007) of Penicillium subgenus Penicillium (Eurotiomycetes, Pezizomycotina), where introns were rare and species resolution was intermediate between that provided by the nuclear ribosomal ITS and β-tubulin. However, they did not attempt to recover introns from genomic DNA using intron-specific primers. In Penicillium, and in a later study by Nguyen & Seifert (2008) of Leohumicola (Leotiomycetes, Pezizomycotina), COX1 functioned as an adequate barcode. However, in a study of the Aspergillus niger complex (Eurotiomycetes, Pezizomycotina), Geiser et al. (2007) found a similar situation to what we report here in Fusarium. They sequenced COX1 from 45 strains from the A. niger complex. Although introns were not detected, intraspecific and interspecific variation were similar, and species did not form exclusive monophyletic groups. Although their published paper does not discuss the possibility of different copies, it seems likely from their phylogram that paralogs of COX1 occur in A. niger.

From a barcoding perspective, the relevance of the paralogous copies of COX1 in Fusarium and possibly in Aspergillus could be questioned if sufficient species resolution was suspected in the expressed copy. Because our survey of Fusarium did not indicate that such resolution existed in any of the copies that we studied, we elected not to extend this research with reverse transcriptase and sequencing of expressed copies.

What do these different copies represent? If we assume that only one copy is actually functional, it is possible that the copies may be unexpressed pseudogenes, possibly resident in the nuclear genome (cf. Selosse et al. 2001). The recovery of sequences with heterozygous bases that could nevertheless both code for an identical functional protein calls even this assumption into question. Another possibility is that they might reflect hybridization events, either reflecting a heterogeneous populations of mitochondrial genome within one strain derived from several conspecific parental strains (D’Alessio 1998), or actual interspecific hybridization. Interspecific or interstrain hybridization could result not only in multiple copies of mitochondrial genes, but also multiple copies of entire genomes. There is evidence from fungal genome studies of a phenomenon resembling polyploidy among species of Aspergillus (Rokas et al. 2007), but this phenomenon has not been demonstrated in Fusarium.

One can wonder how many copies of COX1 could actually be recovered from individual strains of Fusarium if a concerted effort to locate them all was undertaken by cloning, and by selectively attempting to amplify all known introns. We were surprised in three instances when amplifying the barcode of Fusarium circinatum (DAOM 235752, 235753) and Fusarium cortaderiae (DAOM 235621) to recover apparently different copies in separate sequencing replicates from the same genomic DNA. It is also possible that this phenomenon occurs in genera other than Fusarium.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest statement
  9. References
  10. Supporting Information

We are grateful to the Canadian Collection of Fungal Cultures (DAOM, Ottawa) and the Julius-Kühn-Institute, Institute of Epidemiology and Pathogen Diagnostics culture collection (BBA, Berlin/Braunschweig for providing cultures and genomic DNA preps. We thank our colleague Dr B.D. Shenoy, Institute of Microbial Technology, Chandigarh, India, for his ideas on this project, and his preliminary experiments with reverse transcriptase. This research was funded by the Canadian Barcode of Life Network and Genome Canada through the Ontario Genomics Institute, NSERC, and other sponsors listed at http://www.bolnet.ca.

Conflict of interest statement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest statement
  9. References
  10. Supporting Information

The authors have no conflict of interest to declare and note that the funders of this research had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest statement
  9. References
  10. Supporting Information
  • Brown DW, Cheung F, Proctor RH et al . (2005) Comparative analysis of 87 000 expressed sequence tags from the fumonisin-producing fungus Fusarium verticillioides. Fungal Genetics and Biology, 42, 848861.
  • Chambergo FS, Bonaccorsi ED, Ferreira AJ et al . (2002) Elucidation of the metabolic fate of glucose in the filamentous fungus Trichoderma reesei using expressed sequence tag (EST) analysis and cDNA microarrays. Journal of Biological Chemistry, 277, 1398313988.
  • Cock AWAM De, Levesque CA (2004) New species of Pythium and Phytophthora. Studies in Mycology, 50, 481487.
  • Cunnington JH (2007) Organization of the mitochondrial genome of Fusarium oxysporum (anamorphic Hypocreales). Mycoscience, 48, 403406.
  • D’Alessio N (1998) Mitochondrial inheritance during a parasexual cycle in Fusarium oxysporum forma specialis cubense. PhD Thesis. Florida Internation University, Miami, Florida (ISBN 9780591808056, available on proquest.umi.com).
  • Desjardins AE (2006) Fusarium Mycotoxins: Chemistry, Genetics, and Biology. American Phytopathological Society Press, St. Paul, Minnesota.
  • Geiser DM, Jimenez-Gasco MM, Kang S et al . (2004) Fusarium-ID v.1.0: a DNA sequence database for identifying Fusarium. European Journal of Plant Pathology, 110, 473479.
  • Geiser DM, Klich MA, Frisvad JC, Peterson SW, Varga J, Samson RA (2007) The current status of species recognition and identification in Aspergillus. Studies in Mycology, 59, 110.
  • Gerlach W, Nirenberg HI (1982) The genus Fusarium— a pictorial atlas. Mitteilungen Aus der Biologischen Bundesanstalt für Land- und Forstwirtschaft Berlin-Dahlem, 209, 1406.
  • Ghikas DV, Kouvelis VN, Typas MA (2006) The complete mitochondrial genome of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae: Gene order and trn gene clusters reveal a common evolutionary course for all Sordariomycetes, while intergenic regions show variation. Archives of Microbiology, 185, 393401.
  • Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis programs for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 9598.
  • Hebert PDN, Cywinska A, Ball SL, DeWaard JR (2003) Biological identification through DNA barcodes. Proceedings of the Royal Society B: Biological Sciences, 270, 313322.
  • Hibbett DS, Binder M, Bischoff JF et al . (2007) A higher-level phylogenetic classification of the Fungi. Mycological Research, 111, 509547.
  • Kimura M (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16, 111120.
  • Kouvelis VN, Ghikas DV, Typas MA (2004) The analysis of the complete mitochondrial genome of Lecanicillium muscarium (synonym Verticillium lecanii) suggests a minimum common gene organization in mtDNAs of Sordariomycetes: phylogenetic implications. Fungal Genetics and Biology, 41, 930940.
  • Leslie JF, Summerell BA (2006) The Fusarium Laboratory Manual. Blackwell Publishing, Ames, Iowa.
  • Nguyen HDT, Seifert KA (2008) Description and DNA barcoding of three new species of Leohumicola from South Africa and the United States. Persoonia, 21, 5769.
  • O'Donnell K, Sarver BAJ, Brandt M et al . (2007) Phylogenetic diversity and microsphere array-based genotyping of human pathogenic Fusaria, including isolates from the multistate contact lens-associated U.S. keratitis outbreaks. Journal of Clinical Microbiology, 45, 22352248.
  • Pantou MP, Kouvelis VN, Typas MA (2008) The complete mitochondrial genome of Fusarium oxysporum: Insights into fungal mitochondrial evolution. Gene, 419, 715.
  • Ratnasingham S, Hebert PDN (2007) BOLD: The Barcode of Life Data System (http://www.barcodinglife.org). Molecular Ecology Notes, 7, 355364.
  • Rokas A, Payne G, Fedorova ND et al . (2007) What can comparative genomics tell us about species concepts in the genus Aspergillus? Studies in Mycology, 59, 1117.
  • Rossman A (2007) Report of the Planning Workshop for All Fungi DNA Barcoding. Inoculum, 58(6), 15.
  • Rossman AY, Samuels GJ, Rogerson CT, Lowen R (1999) Genera of Bionectriaceae, Hypocreaceae and Nectriaceae (Hypocreales, Ascomycetes). Studies in Mycology, 42, 1248.
  • Seifert KA, Samson RA, Dewaard JR et al . (2007) Prospects for fungus identification using CO1 DNA barcodes, with Penicillium as a test case. Proceedings of the National Academy of Sciences, USA, 104, 39013906.
  • Selosse M-A, Albert B, Godelle B (2001) Reducing the genome size of organelles favours gene transfer to the nucleus. Trends in Ecology & Evolution, 16, 135141.
  • Tamura K, Dudley J, Nei M, Kumar S (2007) mega 4: molecular evolutionary genetics analysis (mega) software version 4.0. Molecular Biology and Evolution, 24, 15961599.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest statement
  9. References
  10. Supporting Information

Table S1 Online supplementary material information on strain numbers, geographical origin and substrate or host of the fungal cultures used in this study. GenBank accession numbers for COX1 sequences generated in this research, and other sequences used to confirm the identity of these strains, are listed here.

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

FilenameFormatSizeDescription
MEN_2636_sm_TableS1.pdf10KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.