The CBS-KNAW Fungal Biodiversity Centre’s Fusarium MLST website (http://www.cbs.knaw.nl/Fusarium), and the corresponding Fusarium-ID
site hosted at the Pennsylvania State University (http://isolate.fusariumdb.org; Geiser et al. 2004, Park et al. 2010) were constructed
to facilitate identification of agriculturally and medically important fusaria by conducting nucleotide BLAST queries of these dedicated DNA sequence databases via the Internet. As
one of the world’s most important mycotoxin-producing plant pathogens and emergent human opportunistic pathogens (Balajee et al. 2009, Geiser et al. 2013), disease management and
infection control require that these economically destructive fungi be identified accurately and rapidly (Wingfield et al. 2012). However, numerous molecular phylogenetic studies over
the past two decades (see References) have established that morphological species recognition frequently fails to distinguish many fusaria that have been discovered employing genealogical
concordance phylogenetic species recognition (GCPSR sensu Taylor et al. 2000). When it was released in 2004 (Geiser et al. 2004), Fusarium-ID was populated initially with partial
translation elongation factor 1-α (TEF1) sequences, which can be used to identify most but not all fusaria. To address this shortcoming, a concerted effort has and will continue to be made to
populate the Fusarium-ID (Park et al. 2010) and Fusarium MLST databases with partial sequences from two additional phylogenetically informative loci, DNA-directed RNA polymerase II largest
(RPB1) and second largest subunit (RPB2). Work in progress is directed at populating these databases with portions of these three genes from the ~300 phylogenetically distinct fusaria discovered
to date via GCPSR. These three gene fragments can be amplified by PCR and sequenced using primers that are conserved across the phylogenetic breadth of Fusarium (O’Donnell et al. 2010) (see the
section on Searching and Identification below for the other loci currently available in Fusarium MLST).
Looking to the future, other phylogenetically informative, orthologous genes will be added to both databases that resolve at or near the species-level and can be used across the phylogenetic
breadth of Fusarium. A robust phylogeny of the genus inferred from partial RPB1+RPB2 sequence data resolved 20 monophyletic species complexes and nine monotypic lineages, which were named informally
to facilitate communication of an isolate’s clade membership and genetic diversity (Fig. 1, O’Donnell et al. 2013). Researchers interested in obtaining reference strains should contact the CBS-KNAW Biodiversity
Centre (http://www.cbs.knaw.nl/Collections/), which houses a large collection of phylogenetically diverse fusaria.
MLST typing schemes have been developed for the following agriculturally and medically important species complexes: F. solani (FSSC, O’Donnell et al. 2008, Zhang et al. 2006; ITS+LSU rDNA, TEF1 and RPB2), F. incarnatum-equiseti
(FIESC; O’Donnell et al. 2009b; ITS+LSU rDNA, TEF1 and RPB2), F. chlamydosporum (FCSC; O’Donnell et al. 2009b; ITS+LSU rDNA, TEF1 and RPB2), F. fujikuroi species complex (FFSC, O’Donnell et al. 1998; TEF1, β-tub, CaM, IGS rDNA),
. dimerum (FDSC; Schroers et al. 2009; TEF1, β-tub, ITS+LSU rDNA, RPB2), F. oxysporum (FOSC; O’Donnell et al. 2009a; TEF1 and IGS rDNA), and B-type trichothecene toxin-producing fusaria that comprises part of the F. sambucinum
species complex (FSAMSC; Sarver et al. 2011 and references therein; TEF1, benA, α-tub, mating-type (MAT) locus (O’Donnell et al. 2004), and several other genes). After we discovered that most of the phylogenetically distinct
pathogenic species within the FSSC, FIESC and FCSC lacked binomials, an ad hoc species/haplotype nomenclature was developed to facilitate accurate communication of their genetic diversity in which Arabic numerals and lowercase
roman letters were used, respectively, to distinguish species and multilocus haplotypes. In the future, similar ad hoc nomenclature will be developed for several species-rich species complexes that are poorly represented in Fusarium
MLST and Fusarium-ID (e.g., sambucinum, tricinctum and lateritium; see Fig. 1).
Single sequence and Multiple sequences options are
available for conducting queries of the Fusarium MLST database. The Single sequence alignment algorithm compares the sequence of an unknown against ones present in the Fusarium MLST reference database. It’s also possible to use the
unknown to query sequences deposited in GenBank and the CBS-KNAW sequence database. Using the Multiple sequences option, sequences from two or more loci from the unknown are queried against the Fusarium MLST
database using tools within the BioloMICS software. It is important to note that because the number of loci represented in the database can vary from strain to strain, queries using the Multiple sequences option sometimes return
contradictory best matches. This problem will be rectified as Fusarium MLST becomes populated with more and more sequences that sample the phylogenetic breadth of the genus. Where possible, we recommend multilocus sequence comparisons
because they typically return more reliable identifications.
CBS-KNAW Fungal Biodiversity Center, Utrecht, the Netherlands
Pedro W. Crous, Vincent A. R. G. Robert, Lorenzo Lombard, Alejandra Giraldo and Anne van Diepeningen
Bacterial Foodborne Pathogens and Mycology Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois
Kerry O’Donnell and Todd J. Ward
Department of Plant Pathology, The Pennsylvania State University, University Park, Pennsylvania
David M. Geiser and Seogchan Kang
How important is it that my query sequence be error free?
This step is critical for obtaining a reliable identification. Trimming either end of the sequence chromatograms should remove most errors, assuming ambiguous sites cannot be corrected, and the number of nucleotides within a homopolymer
should be examined carefully to avoid erroneous gaps. Checking every variable site in the alignments of two or more sequence chromatograms is highly recommended where possible. In addition, it is wise to recheck chromatograms when polymorphic
sites or gaps are present in the alignment of your query sequence and one or more of the top ‘hits’ that are displayed after a BLASTn search of Fusarium MLST has been conducted.
What locus or loci do you recommend that I use for species-level identifications within Fusarium?
If only one locus can be sampled, we recommend TEF1 because it frequently, but not always, resolves at the species-level and because of the dense sampling of this gene across the breadth of the genus currently represented in Fusarium MLST
and Fusarium-ID. Our next choice is RPB2 followed by RPB1. In contrast to TEF1, which can only be aligned reliably across a species complex or several closely related ones, the fragments of RPB1 and RPB2 PCR-amplified and sequenced can be
aligned easily across the breadth of the genus and beyond (see download of NEXUS file used in O’Donnell et al. 2013) and they are phylogenetically informative at or near the species level. Irrespective of what locus or loci are used to identify
unknowns, end users will find it highly beneficial to conduct phylogenetic analyses of the relevant published dataset (see links to NEXUS files in References) to which their unknown sequences have been added. The most robust identifications of
unknowns will be obtained by bootstrapping the dataset using any of a number of phylogenetic programs available from the Internet (e.g., MEGA6, http://www.megasoftware.net/).
What suggestions do you offer for analyzing a NEXUS file to which I have added my unknown sequences?
Once you have downloaded the NEXUS file of interest (see References), we recommend using a text editor such as TextPad for windows (available online at http://www.textpad.com/) for cutting and pasting your
sequences in the dataset followed by manual alignment. If the sequences are difficult to align, then download the free alignment program SeaView (http://doua.prabi.fr/software/seaview), and then save the file in NEXUS format. If you don’t
have access to the phylogenetics program PAUP* (version 4.0b10. Sunderland, Massachusetts: Sinauer Associates), then one option is to download the free software package MEGA 6.06 for Windows or Mac OS at http://www.megasoftware.net/ and then
take a few minutes to take the tutorial at http://www.megasoftware.net/tutorial.php. Note that MEGA can convert the NEXUS file into a format that MEGA can use, but sequences from different gene partitions cannot be interleaved. Many of the NEXUS files linked
to the References are interleaved, so you will need to use a text editor such as TextPad to place them all on one line before importing them into MEGA.
Why isn’t Fusarium MLST extensively populated with ITS rDNA sequences, the official fungal barcode?
Two reasons: ITS+LSU rDNA sequences frequently fail to distinguish closely related species, and highly divergent ITS2 rDNA paralogs or xenologs were discovered in several species complexes (O’Donnell and Cigelnik 1997, O’Donnell et al. 1998).
Nevertheless, this locus was included in MLST schemes for medically important fusaria within the FSSC, FIESC and FCSC (see References). In addition, ITS+LSU rDNA sequences of the 93 fusaria included in the RPB1+RPB2 phylogenetic analysis of Fusarium
(O’Donnell et al. 2013) will be deposited in Fusarium MLST to facilitate identification to species complex (Fig. 1), and in some instances to species.
Why do my BLASTn queries of Fusarium MLST and GenBank recover a different set of top ‘hits’?
The answer to this difficult question will depend on the results of each individual query. However, in most instances, it will likely be due to the large number of misidentified, uncurated sequences deposited in GenBank (Bidartondo et al. 2008).
Fusarium MLST and Fusarium-ID, by contrast, only house carefully curated sequences of isolates that are available from the CBS-KNAW, Fusarium Research Center (FRC, http://plantpath.psu.edu/directory/specialties/fusarium-research-center) or ARS
Culture Collection (NRRL, http://nrrl.ncaur.usda.gov/) where scientists have research focused on Fusarium molecular systematics and evolution. Thus, many of the sequences in Fusarium MLST and Fusarium-ID were generated in multilocus molecular
phylogenetics studies in which species were delimited based on genealogical concordance. It essential to carefully check the species names associated with the top ‘hits’ based on % identity and sequence coverage. If the top ‘hit’ is a member
of the FSSC, FIESC or FCSC, look for notes or comments in the accession record that identify the phylogenetic species/multilocus haplotype (e.g., FIESC 25-a, where 25 is the species and a is the haplotype within the phylogenetic species; see O’Donnell et al. 2009b).
When does a % similarity equate with conspecificity?
This difficult question will benefit from prior GCSPR-based studies of the particular species complex in which the unknown is nested and from bootstrap analyses of a published multilocus dataset that includes the unknown. In most but not all cases,
100% similarity to only one species within Fusarium MLST and 95% or more sequence coverage represents a definitive species identification (Geiser et al. 2004). However, it is important to note that recently evolved species in several species complexes
can share identical TEF1, RPB1 and RPB2 alleles (for examples, see Aoki et al. 2012, Kasson et al. 2013, O’Donnell et al. 2014). When the % similarity of a Single sequence query is at or below 99.4% (e.g., ≥4/680 bp differences in TEF1, ≥9/1600 bp in
RPB1, and ≥10/1760 bp in RPB2), assuming the query sequence is error free, we recommend conducting a multilocus GCPSR-based analysis because the unknown could represent a novel phylogenetically distinct species. Single sequence BLASTn queries that show
99.5 to 99.9% similarity will need to be interpreted on a case-by-case basis within the context of what sequences are present in Fusarium MLST. When assigning a species name to the unknown is in doubt, we strongly recommend: 1) sequencing at least one
other phylogenetically informative gene so that the Multiple sequences option can be implemented in Fusarium MLST, 2) conducting a molecular phylogenetic analysis as previously suggested, and 3) always err of the side of caution by reporting the unknown
as Fusarium sp., the species complex within which it is nested, including the BLASTn query results, and the most closely related species or strain number if it is also listed as Fusarium sp. (e.g., Fusarium sp. nested within FIESC with TEF1 showing 99.4%
identity to Fusarium sp. FIESC 13 NRRL 43635). Lastly, in some instances end users may find it useful to conduct BLASTn queries of GenBank for comparison, with the caveat that it is absolutely essential that the name associated with the top ‘hits’ be checked
for consistency because they rarely are, check to see who deposited the sequence and whether it was part of a GCPSR-based study (e.g., how critical was the identification made), and look for notes/comments in the accession records listed as Fusarium sp. to
see if the phylogenetic species is identified. For further discussion on DNA sequence-based identification, see Kang et al. (2010) and O’Donnell et al. (2015).
The overarching goal of Fusarium MLST is to provide a valuable resource to the global Fusarium community. Towards this end, we encourage end users to contact us in the space provided below to provide suggestions on how we can better serve you. In addition,
if your queries of Fusarium MLST indicate that you have discovered novel species diversity, we encourage you to contact Dr. Gerard Verkleij (firstname.lastname@example.org) and Dr. Vincent A. R. G. Robert (email@example.com), respectively, at the CBS-KNAW if you
are interested in depositing your strains and DNA sequence data in our database. Through such crowdsourcing, together we can grow Fusarium MLST so that it meets the needs of our highly interactive community.
Fig. 1. Fusarium phylogeny inferred from a RPB1 + RPB2 dataset (3383 bp) in which 20 species complexes (highlighted in gray) and 9 monotypic lineages were resolved (modified from Fig. 1 in O’Donnell et al. 2013). The approximate number of phylogenetically distinct species within each species complex based on more inclusive GCPSR-based analyses is indicated in parentheses. Sequences of Neonectria and Ilyonectria were used as outgroups.
Aoki, T., Tanaka, F., Suga, H., Hyakumachi, M., Scandiani, M. M., & O’Donnell, K. (2012). Fusarium azukicola sp. nov., an exotic azuki bean root-rot pathogen in Hokkaido, Japan. Mycologia 104, 1068–1084. Download
Balajee, S. A., Borman, A. M., Brandt, M. E., Cano, J., Cuenca-Estrella, M., Dannaoui, E., et al. (2009). Sequence-based identification of Aspergillus, Fusarium, and Mucorales species in the clinical mycology laboratory: Where are we and where should we go from here? Journal of Clinical Microbiology 47, 877–884. Download
Bidartondo, M. I., Bruns, T. D., Blackwell, M., Edwards, I., Taylor, A. F. S., Horton, T., et al. (2008). Preserving accuracy in GenBank. Science 319, 1616. Download
Geiser, D. M., Aoki, T., Bacon, C. W., Baker, S. E., Bhattacharyya, M. K., Brandt, M. E., et al. (2013). LETTER TO THE EDITOR: One fungus, one name: Defining the genus Fusarium in a scientifically robust way that preserves longstanding use. Phytopathology 103, 400–408. Download
Geiser, D. M., Jiménez-Gasco, M., Kang, S., Makalowska, I., Veeraraghavan, N., Ward, T. J., et al. (2004). FUSARIUM-ID v.1.0: A DNA sequence database for identifying Fusarium. European Journal of Plant Pathology 110, 473–479. Download
Kasson, M. T., O’Donnell, K., Rooney, A. P., Sink, S., Ploetz, R. C., Ploetz, J. N., et al. (2013). An inordinate fondness for Fusarium: Phylogenetic diversity of fusaria cultivated by ambrosia beetles in the genus Euwallacea on avocado and other plant hosts. Fungal Genetics and Biology 56, 147–157.Download
Kang, S., Mansfield, M. A., Park, B., Geiser, D. M., Ivors, K. L., Coffey, M. D., et al. (2010). The promise and pitfalls of sequence-based identification of plant pathogenic fungi and oomycetes. Phytopathology 100, 732–737. Download
O’Donnell, K., & Cigelnik, E. (1997). Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Molecular Phylogenetics and Evolution 7, 103–116. Download
O'Donnell, K., Cigelnik, E., & Nirenberg, H. I. (1998). Molecular systematics and phylogeography of the Gibberella fujikuroi species complex. Mycologia 90, 465–493. Download
O'Donnell, K., Gueidan, C., Sink, S., Johnston, P. R., Crous, P. W., Glenn, A., et al. (2009a). A two-locus DNA sequence database for typing plant and human pathogens within the Fusarium oxysporum species complex. Fungal Genetics and Biology 46, 936–948. Download
O’Donnell, K., Rooney, A. P., Proctor, R. H., Brown, D. W., McCormick, S. P., Ward, T. J., et al. (2013). Phylogenetic analyses of RPB1 and RPB2 support a middle Cretaceous origin for a clade comprising all agriculturally and medically important fusaria. Fungal Genetics and Biology 52, 20–31. Download
O’Donnell, K., Sink, S., Libeskind-Hadas, R., Hulcr, J., Kasson, M. T., Ploetz, R. C., et al. (2014). Discordant phylogenies suggest repeated host shifts in the Fusarium – Euwallacea ambrosia beetle mutualism. Fungal Genetics and Biology doi:10.1016/j.fgb.2014.10.014. Download
O’Donnell, K., Sutton, D. A., Fothergill, A., McCarthy, D., Rinaldi, M. G., Brandt, M. E., et al. (2008). Molecular phylogenetic diversity, multilocus haplotype nomenclature, and in vitro antifungal resistance within the Fusarium solani species complex. Journal of Clinical Microbiology 46, 2477–2490. Download
O’Donnell, K., Sutton, D. A., Rinaldi, M. G., Gueidan, C., Crous, P. W., Geiser, D. M. (2009b). Novel multilocus sequence typing scheme reveals high genetic diversity of human pathogenic members of the Fusarium incarnatum-F. equiseti and F. chlamydosporum species complexes within the United States. Journal of Clinical Microbiology 47, 3851–3861. Download
O'Donnell, K., Sutton, D. A., Rinaldi, M. G., Sarver, B. A. J., Balajee, S. A., Schroers, H.-J., et al. (2010). An Internet-accessible DNA sequence database for identifying fusaria from human and animal infections. Journal of Clinical Microbiology 48, 3708–3718. Download
O'Donnell, K., Ward, T. J., Geiser, D. M., Kistler, H. C., & Aoki, T. (2004). Genealogical concordance between the mating type locus and seven other nuclear genes supports formal recognition of nine phylogenetically distinct species within the Fusarium graminearum clade. Fungal Genetics and Biology 41, 600–623. Download
O’Donnell, K., Ward, T. J., Robert, V. A. R. G., Crous, P. W., Geiser, D. M., & Kang, S. (2015). DNA sequence-based identification of Fusarium: Current status and future directions. Phytoparasitica (in review).
Park, B., Park, J., Cheong, K.-C., Choi, J., Jung, K., Kim, D., et al. (2010). Cyber infrastructure for Fusarium: three integrated platforms supporting strain identification, phylogenetics, comparative genomics, and knowledge sharing. Nucleic Acids Research 39, D640–D646. Download
Sarver, B. A. J., Ward, T. J., Gale, L. R., Broz, K., Kistler, H. C., Aoki, T., et al. (2011). Novel Fusarium head blight pathogens from Nepal and Louisiana revealed by multilocus genealogical concordance. Fungal Genetics and Biology 48, 1096–1107. Download
Schroers, H.-J., O'Donnell, K., Lamprecht, S. C., Kammeyer, P. L., Johnson, S., Sutton, D. A., et al. (2009). Taxonomy and phylogeny of the Fusarium dimerum species group. Mycologia 101, 44–70. Download
Taylor, J. W., Jacobson, D. J., Kroken, S., Kasuga, T., Geiser, D. M., Hibbett, D. S., et al. (2000). Phylogenetic species recognition and species concepts in fungi. Fungal Genetics and Biology 31, 21–32. Download
Wingfield, M. J., de Beer, Z. W., Slippers, B., Wingfield, B. D., Groenewald, J. Z., Lombard, L., et al. (2012). One fungus, one name promotes progressive plant pathology. Molecular Plant Pathology 13, 604–613. Download
Zhang, N., O'Donnell, K., Sutton, D. A., Nalim, F. A., Summerbell, R. C., Padhye, A. A., et al. (2006). Members of the Fusarium solani species complex that cause infections in both humans and plants are common in the environment. Journal of Clinical Microbiology 44, 2186–2190. Download
Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 North University Street, Peoria, IL 61604-3999, USA
Phone: (309) 681-6383, Fax: (309) 681-6672
Vincent A. Robert
CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, Utrecht, The Netherlands