Molecular phylogenetic diversity, multilocus haplotype nomenclature, and in vitro antifungal resistance within the Fusarium solani species complex

Abstract

Members of the species-rich Fusarium solani species complex (FSSC) are responsible for approximately two-thirds all fusarioses of humans and other animals. In addition, many economically important phytopathogenic species are nested within this complex. Due to their increasing clinical relevance and because most of the human pathogenic and plant pathogenic FSSC lack Latin binomials, we have extended the multilocus haplotype nomenclatural system introduced in a previous study (D. C. Chang, G. B. Grant, K. O'Donnell, K. A. Wannemuehler, J. Noble-Wang, C. Y. Rao, L. M. Jacobson, C. S. Crowell, R. S. Sneed, F. M. T. Lewis, J. K. Schaffzin, M. A. Kainer, C. A. Genese, E. C. Alfonso, D. B. Jones, A. Srinivasan, S. K. Fridkin, and B. J. Park, JAMA 296:953-963, 2006) to all 34 species within the medically important FSSC clade 3 to facilitate global epidemiological studies. The typing scheme is based on polymorphisms in portions of the following three genes: the internal transcribed spacer region and domains D1 plus D2 of the nuclear large-subunit rRNA, the translation elongation factor 1 alpha gene (EF-1alpha), and the second largest subunit of RNA polymerase II gene (RPB2). Of the 251 isolates subjected to multilocus DNA sequence typing, 191 sequence types were differentiated, and these were distributed among three strongly supported clades designated 1, 2, and 3. All of the mycosis-associated isolates were restricted to FSSC clade 3, as previously reported (N. Zhang, K. O'Donnell, D. A. Sutton, F. A Nalim, R. C. Summerbell, A. A. Padhye, and D. M. Geiser, J. Clin. Microbiol. 44:2186-2190, 2006), and these represent at least 20 phylogenetically distinct species. Analyses of the combined DNA sequence data by use of two separate phylogenetic methods yielded the most robust hypothesis of evolutionary relationships and genetic diversity within the FSSC to date. The in vitro activities of 10 antifungals tested against 19 isolates representing 18 species that span the breadth of the FSSC phylogeny show that members of this complex are broadly resistant to these drugs.

FIG. 1.

Bootstrapped ML out-group-rooted cladogram inferred from the combined DNA sequence data from three loci for 180 unique STs. Arabic numbers and lowercase roman letters identify the species and STs, respectively, within clade 3. Dark shading is used to identify isolates from nonclinical sources; light shading identifies clade 3. Note that only three of the clinically relevant species within clade 3 have Latin binomials. Eight putatively plant host-specific formae speciales of the polytypic morphospecies Fusarium solani are indicated with the f. sp. prefix. Numbers placed above and below the internodes represent the frequency (%) with which they were recovered, respectively, from 1,000 MP and 250 ML bootstrap pseudoreplicates of the data. An asterisk by a bootstrap value indicates that the ML and MP bootstrap support values are identical. A solid dot to the right of an NRRL number identifies 1 of 19 isolates subjected to antifungal susceptibility testing in vitro (Tables 4 and 5).

FIG. 2.

Histogram showing the sources of the 231 clade 3 FSSC isolates subjected to MLST. Twenty of the 34 phylogenetically distinct species within this clade appear to be clinically relevant, and these accounted for 135 of the 176 unique STs (Table 1). The height of each bar represents the number of STs within each species, except for 12 species, for which this number is given above the bars. The number below each bar identifies each species according to the haplotype nomenclature system of Chang et al. (11). FSSC 3+4 (Fusarium falciforme) was formerly known as FSSC groups 3 and 4 (63).