Internet-accessible DNA sequence database for identifying fusaria from human and animal infections

Abstract

Because less than one-third of clinically relevant fusaria can be accurately identified to species level using phenotypic data (i.e., morphological species recognition), we constructed a three-locus DNA sequence database to facilitate molecular identification of the 69 Fusarium species associated with human or animal mycoses encountered in clinical microbiology laboratories. The database comprises partial sequences from three nuclear genes: translation elongation factor 1α (EF-1α), the largest subunit of RNA polymerase (RPB1), and the second largest subunit of RNA polymerase (RPB2). These three gene fragments can be amplified by PCR and sequenced using primers that are conserved across the phylogenetic breadth of Fusarium. Phylogenetic analyses of the combined data set reveal that, with the exception of two monotypic lineages, all clinically relevant fusaria are nested in one of eight variously sized and strongly supported species complexes. The monophyletic lineages have been named informally to facilitate communication of an isolate's clade membership and genetic diversity. To identify isolates to the species included within the database, partial DNA sequence data from one or more of the three genes can be used as a BLAST query against the database which is Web accessible at FUSARIUM-ID (http://isolate.fusariumdb.org) and the Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Center (http://www.cbs.knaw.nl/fusarium). Alternatively, isolates can be identified via phylogenetic analysis by adding sequences of unknowns to the DNA sequence alignment, which can be downloaded from the two aforementioned websites. The utility of this database should increase significantly as members of the clinical microbiology community deposit in internationally accessible culture collections (e.g., CBS-KNAW or the Fusarium Research Center) cultures of novel mycosis-associated fusaria, along with associated, corrected sequence chromatograms and data, so that the sequence results can be verified and isolates are made available for future study.

FIG. 1.

Map of the RNA polymerase largest-subunit (RPB1) locus. Location and orientation of PCR and sequencing primers are indicated by half-arrows. PCR primers Fa and G2R (Table 2) successfully amplified an 1,894-bp fragment in all of the isolates except for NRRL 25197 Fusarium cf. lateritium, even though the PCR primer sites appear to be conserved in this isolate. Therefore, the Fa × G2R region was amplified in this isolate as two overlapping fragments using the PCR primer pairs Fa/R8 (1,127 bp) and F7/R9 (2,217 bp). Primers F5 and G2R flank the 1,607-bp RPB1 D-to-G region that was sequenced and analyzed. Sequences for 65 of the 71 isolates were generated using the F5, F7, and F8 sequencing primers. See Fig. 1 in Matheny et al. (41) for a detailed map of the entire RPB1 locus showing the position of the D-to-G region.

FIG. 2.

Best maximum likelihood tree inferred from the combined three-locus data set for 71 isolates representing 69 medically and veterinarily important Fusarium species. Because the branching order of the two most basal lineages, the F. solani and F. dimerum species complexes (FSSC and FDSC), was unresolved in more inclusive analyses (54), the phylogram was midpoint rooted. The Gibberella clade contains the six most derived, clinically relevant species complexes. Species and their multilocus haplotypes are identified by Arabic numbers and lowercase Roman letters, respectively, for members of the Fusarium incarnatum-F. equiseti species complex (FIESC), the F. chlamydosporum species complex (FCSC), and the FSSC as previously reported (56, 57). Numbers in parentheses by the three F. oxysporum species complex (FOSC) isolates refer to clades as reported by O'Donnell et al. (50). Note that Latin binomials can be applied with confidence to only 23 of the 69 species. Fusarium sporotrichioides and F. cf. lateritium are highlighted in gray to indicate that reports of these species causing human infections need to be confirmed. Numbers above internodes represent ML bootstrap values based on 1,000 pseudoreplicates of the data. MP bootstrap values are indicated below internodes only when they differed by ≥5% of the MP value. Af, African subclade; Am, American subclade; As, Asian subclade; FSAMSC, F. sambucinum species complex; FTSC, F. tricinctum species complex; and GFSC, Gibberella fujikuroi species complex.