Gene Genealogies and AFLP Analyses in the Fusarium oxysporum Complex Identify Monophyletic and Nonmonophyletic Formae Speciales Causing Wilt and Rot Disease.

ABSTRACT The monophyletic origin of host-specific taxa in the plant-pathogenic Fusarium oxysporum complex was tested by constructing nuclear and mitochondrial gene genealogies and amplified fragment length polymorphism (AFLP)-based phylogenies for 89 strains representing the known genetic and pathogenic diversity in 8 formae speciales associated with wilt diseases and root and bulb rot. We included strains from clonal lineages of F. oxysporum f. spp. asparagi, dianthi, gladioli, lilii, lini, opuntiarum, spinaciae, and tulipae. Putatively nonpathogenic strains from carnation and lily were included and a reference strain from each of the three main clades identified previously in the F. oxysporum complex; sequences from related species were used as outgroups. DNA sequences from the nuclear translation elongation factor 1alpha and the mitochondrial small subunit (mtSSU) ribosomal RNA genes were combined for phylogenetic analysis. Strains in vegetative compatibility groups (VCGs) shared identical sequences and AFLP profiles, supporting the monophyly of the two single-VCG formae speciales, lilii and tulipae. Identical genotypes were also found for the three VCGs in F. oxysporum f. sp. spinaciae. In contrast, multiple evolutionary origins were apparent for F. oxysporum f. spp. asparagi, dianthi, gladioli, lini, and opuntiarum, although different VCGs within each of these formae speciales often clustered close together or shared identical EF-1alpha and mtSSU rDNA haplotypes. Kishino-Hasegawa analyses of constraints forcing the monophyly of these formae speciales supported the exclusive origin of F. oxysporum f. sp. opuntiarum but not the monophyly of F. oxysporum f. spp. asparagi, dianthi, gladioli, and lini. Most of the putatively nonpathogenic strains from carnation and lily, representing unique VCGs, were unrelated to F. oxysporum f. spp. dianthi and lilii, respectively. Putatively nonpathogenic or rot-inducing strains did not form exclusive groups within the molecular phylogeny. Parsimony analyses of AFLP fingerprint data supported the gene genealogy-based phylogram; however, AFLP-based phylogenies were considerably more homoplasious than the gene genealogies. The predictive value of the forma specialis naming system within the F. oxysporum complex is questioned.

Phylogeny and PCR identification of clinically important Zygomycetes based on nuclear ribosomal-DNA sequence data.

A molecular database for all clinically important Zygomycetes was constructed from nucleotide sequences from the nuclear small-subunit (18S) ribosomal DNA and domains D1 and D2 of the nuclear large-subunit (28S) ribosomal DNA. Parsimony analysis of the aligned 18S and 28S DNA sequences was used to investigate phylogenetic relationships among 42 isolates representing species of Zygomycetes reported to cause infections in humans and other animals, together with commonly cultured contaminants, with emphasis on members of the Mucorales. The molecular phylogeny provided strong support for the monophyly of the Mucorales, exclusive of Echinosporangium transversale and Mortierella spp., which are currently misclassified within the Mucorales. Micromucor ramannianus, traditionally classified within Mortierella, and Syncephalastrum racemosum represent the basal divergences within the Mucorales. Based on the 18S gene tree topology, Absidia corymbifera and Rhizomucor variabilis appear to be misplaced taxonomically. A. corymbifera is strongly supported as a sister group of the Rhizomucor miehei-Rhizomucor pusillus clade, while R. variabilis is nested within Mucor. The aligned 28S sequences were used to design 13 taxon-specific PCR primer pairs for those taxa most commonly implicated in infections. All of the primers specifically amplified DNA of the size predicted based on the DNA sequence data from the target taxa; however, they did not cross-react with phylogenetically related species. These primers have the potential to be used in a PCR assay for the rapid and accurate identification of the etiological agents of mucormycoses and entomophthoromycoses.

Multiple evolutionary origins of the fungus causing Panama disease of banana: concordant evidence from nuclear and mitochondrial gene genealogies.

Panama disease of banana, caused by the fungus Fusarium oxysporum f. sp. cubense, is a serious constraint both to the commercial production of banana and cultivation for subsistence agriculture. Previous work has indicated that F. oxysporum f. sp. cubense consists of several clonal lineages that may be genetically distant. In this study we tested whether lineages of the Panama disease pathogen have a monophyletic origin by comparing DNA sequences of nuclear and mitochondrial genes. DNA sequences were obtained for translation elongation factor 1alpha and the mitochondrial small subunit ribosomal RNA genes for F. oxysporum strains from banana, pathogenic strains from other hosts and putatively nonpathogenic isolates of F. oxysporum. Cladograms for the two genes were highly concordant and a partition-homogeneity test indicated the two datasets could be combined. The tree inferred from the combined dataset resolved five lineages corresponding to "F. oxysporum f. sp. cubense" with a large dichotomy between two taxa represented by strains most commonly isolated from bananas with Panama disease. The results also demonstrate that the latter two taxa have significantly different chromosome numbers. F. oxysporum isolates collected as nonpathogenic or pathogenic to other hosts that have very similar or identical elongation factor 1alpha and mitochondrial small subunit genotypes as banana pathogens were shown to cause little or no disease on banana. Taken together, these results indicate Panama disease of banana is caused by fungi with independent evolutionary origins.

Molecular phylogenetic, morphological, and mycotoxin data support reidentification of the Quorn mycoprotein fungus as Fusarium venenatum.

Molecular phylogenetic, morphological, and mycotoxin data were obtained in order to investigate the relationships and identity of the Quorn mycoprotein fungus within Fusarium and to examine Quorn strains and commercial Quorn food products for trichothecene mycotoxins. Phylogenetic analyses of aligned DNA sequences obtained via the polymerase chain reaction from the nuclear 28S ribosomal DNA, nuclear ribosomal internal transcribed spacer region, and beta-tubulin gene exons and introns indicate that the Quorn fungus is Fusarium venenatum, rather than F. graminearum as previously reported. All of the Quorn strains examined were morphologically degenerate aconidial colonial mutants except for NRRL 25139, which produced chlamydospores in recurved terminal chains together with mostly 5-septate sporodochial conidia on doliform monophialides diagnostic of F. venenatum. Bootstrap and decay analyses provide strong support for a monophyletic lineage containing F. venenatum and several other type A trichothecene-producing species, while reference strains of F. graminearum were nested in a separate clade of species that produce type B trichothecenes and/or zearalenone. Analysis of mycotoxins from rice cultures inoculated with Quorn strain NRRL 25416 revealed that four type A trichothecenes are produced, but at low levels relative to strain NRRL 22198 of F. venenatum. No trichothecene mycotoxins, however, were detected from the analysis of three commercial Quorn products marketed for human consumption in England.

Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous.

The evolutionary history of the phytopathogenic Gibberella fujikuroi complex of Fusarium and related species was investigated by cladistic analysis of DNA sequences obtained from multiple unlinked loci. Gene phylogenies inferred from the mitochondrial small subunit (mtSSU) rDNA, nuclear 28S rDNA, and beta-tubulin gene were generally concordant, providing strong support for a fully resolved phylogeny of all biological and most morphological species. Discordance of the nuclear rDNA internal transcribed spacer 2 (ITS2) gene tree is due to paralogous or xenologous ITS2 sequences. PCR and sequence analysis demonstrated that every strain of the ingroup species tested possesses two highly divergent nonorthologous ITS2 types designated type I and type II. Only the major ITS2 type, however, is discernable when PCR products are amplified and sequenced directly with conserved primers. The minor ITS2 type was recovered using ITS2 type-specific PCR primers. Distribution of the major ITS2 type within the species lineages exhibits a homoplastic pattern of evolution, thus obscuring true phylogenetic relationships. The results suggest that the ancestral ITS2 types may have arisen following an ancient interspecific hybridization or gene duplication which occurred prior to the evolutionary radiation of the Gibberella fujikuroi complex and related species of Fusarium. The results also indicate that current morphological-based taxonomic schemes for these fungi are unnatural and a new classification is required.