Todd J. Ward

FUSARIUM-ID v. 1.0: a DNA sequence database for identifying Fusarium

One of the greatest impediments to the study of Fusarium has been the incorrect and confused application of species names to toxigenic and pathogenic isolates, owing in large part to intrinsic limitations of morphological species recognition and its application. To address this problem, we have created FUSARIUM-ID v. 1.0, a publicly available database of partial translation elongation factor 1-alpha (TEF) DNA sequences, presently representing a selected sample of the diversity of the genus diversity, with excellent representation of Type-B trichothecene toxin producers, and the Gibberella fujikuroi, Fusarium oxysporum and F. solani species complexes. Users can generate sequences using primers that are conserved across the genus, and use the sequence as a query to BLAST the database, which can be accessed at http://fusarium.cbio.psu.edu, or in a phylogenetic analysis. Correct identification of a known species in these groups often can be performed using this gene region alone. This growing database will contain only vouchered sequences attached to publicly available cultures. In the future, FUSARIUM-ID will be expanded to include additional sequences, including multiple sequences from the same species, sequences from new and revised species, and information from additional genes.

The Fusarium graminearum Genome Reveals a Link Between Localized Polymorphism and Pathogen Specialization

We sequenced and annotated the genome of the filamentous fungus Fusarium graminearum, a major pathogen of cultivated cereals. Very few repetitive sequences were detected, and the process of repeat-induced point mutation, in which duplicated sequences are subject to extensive mutation, may partially account for the reduced repeat content and apparent low number of paralogous (ancestrally duplicated) genes. A second strain of F. graminearum contained more than 10,000 single-nucleotide polymorphisms, which were frequently located near telomeres and within other discrete chromosomal segments. Many highly polymorphic regions contained sets of genes implicated in plant-fungus interactions and were unusually divergent, with higher rates of recombination. These regions of genome innovation may result from selection due to interactions of F. graminearum with its plant hosts.

Ancestral polymorphism and adaptive evolution in the trichothecene mycotoxin gene cluster of phytopathogenic Fusarium

Filamentous fungi within the Fusarium graminearum species complex (Fg complex) are the primary etiological agents of Fusarium head blight (scab) of wheat and barley. Scab is an economically devastating plant disease that greatly limits grain yield and quality. In addition, scabby grain is often contaminated with trichothecene mycotoxins that act as virulence factors on some hosts, and pose a serious threat to animal health and food safety. Strain-specific differences in trichothecene metabolite profiles (chemotypes) are not well correlated with the Fg complex phylogeny based on genealogical concordance at six single-copy nuclear genes. To examine the basis for this discord between species and toxin evolution, a 19-kb region of the trichothecene gene cluster was sequenced in 39 strains chosen to represent the global genetic diversity of species in the Fg complex and four related species of Fusarium. Phylogenetic analyses demonstrated that polymorphism within these virulence-associated genes is transspecific and appears to have been maintained by balancing selection acting on chemotype differences that originated in the ancestor of this important group of plant pathogens. Chemotype-specific differences in selective constraint and evidence of adaptive evolution within trichothecene genes are also reported.

Phylogenetic Diversity and Microsphere Array-Based Genotyping of Human Pathogenic Fusaria, Including Isolates from the Multistate Contact Lens-Associated U.S. Keratitis Outbreaks of 2005 and 2006

ABSTRACT In 2005 and 2006, outbreaks of Fusarium keratitis associated with soft contact lens use occurred in multiple U.S. states and Puerto Rico. A case-control study conducted by the Centers for Disease Control and Prevention (CDC) showed a significant association between infections and the use of one particular brand of lens solution. To characterize the full spectrum of the causal agents involved and their potential sources, partial DNA sequences from three loci (RPB2, EF-1α, and nuclear ribosomal rRNA) totaling 3.48 kb were obtained from 91 corneal and 100 isolates from the patients environment (e.g., contact lens and lens cases). We also sequenced a 1.8-kb region encoding the RNA polymerase II second largest subunit (RPB2) from 126 additional pathogenic isolates to better understand how the keratitis outbreak isolates fit within the full phylogenetic spectrum of clinically important fusaria. These analyses resulted in the most robust phylogenetic framework for Fusarium to date. In addition, RPB2 nucleotide variation within a 72-isolate panel was used to design 34 allele-specific probes to identify representatives of all medically important species complexes and 10 of the most important human pathogenic Fusarium in a single-well diagnostic assay, using flow cytometry and fluorescent microsphere technology. The multilocus data revealed that one haplotype from each of the three most common species comprised 55% of CDCs corneal and environmental isolates and that the corneal isolates comprised 29 haplotypes distributed among 16 species. The high degree of phylogenetic diversity represented among the corneal isolates is consistent with multiple sources of contamination.

Multilocus genotyping and molecular phylogenetics resolve a novel head blight pathogen within the Fusarium graminearum species complex from Ethiopia

A survey of Fusarium head blight (FHB)-contaminated wheat in Ethiopia recovered 31 isolates resembling members of the Fusarium graminearum species complex. Results of a multilocus genotyping (MLGT) assay for FHB species and trichothecene chemotype determination suggested that 22 of these isolates might represent a new species within the Fg complex. Phylogenetic analyses of multilocus DNA sequence data resolved the 22 Ethiopian isolates as a novel, phylogenetically distinct species. The new species also appears to be novel in that MLGT probe data and sequence analysis of both ends of the TRI-cluster identified 15ADON and NIV recombination blocks, documenting inter-chemotype recombination involving the chemotype-determining genes near the ends of the TRI-cluster. Results of pathogenicity experiments and analyses of trichothecene mycotoxins demonstrated that this novel Fg complex species could induce FHB on wheat and elaborate 15ADON in planta. Herein the FHB pathogen from Ethiopia is formally described as a novel species.

The trichothecene biosynthesis gene cluster of Fusarium graminearum F15 contains a limited number of essential pathway genes and expressed non-essential genes

We report for the first time the complete structure and sequence of the trichothecene biosynthesis gene cluster (i.e. Tri5‐cluster) from Fusarium graminearum F15, a strain that produces 3‐acetyldeoxynivalenol (3‐ADON). A putative tyrosinase and polysaccharide deacetylase gene flank the Tri5‐cluster: the number of pathway genes between them is less than half the total number of steps necessary for 3‐ADON biosynthesis. In comparison with partial Tri5‐cluster sequences of strains with 15‐acetyldeoxynivalenol and 4‐acetylnivalenol chemotypes, the Tri5‐cluster from strain F15 contains three genes that are apparently unnecessary for the biosynthesis of 3‐ADON (i.e. Tri8 and Tri3, which are expressed, and pseudo‐Tri13, which is not expressed). In addition, the Tri7 gene was missing from the cluster. Recombinant TRI3 protein showed limited trichothecene C‐15 acetylase activity. In contrast, recombinant TRI8 protein displayed no C‐3 deacetylase activity, suggesting that the loss or alteration of function contribute directly to the chemotype difference.

A novel Asian clade within the Fusarium graminearum species complex includes a newly discovered cereal head blight pathogen from the Russian Far East

We investigated Fusarium graminearum complex (Fg complex) species diversity and toxin potential in European and Asian regions of the Russian Federation and adjoining regions northwest to Finland and south near Harbin, Heilongjiang Province, China, to expand our knowledge of the host range and geographic distribution of these economically devastating cereal head blight pathogens. Results of a recently described multilocus genotyping (MLGT) assay revealed that F. graminearum was the sole Fg complex pathogen in northern Europe and the predominant one in Asia (90.5%). Even though isolates of F. graminearum were segregating for 3-acetyldeoxynivalenol (3ADON) and 15-acetyldeoxynivalenol (15ADON) chemotype in nearly equal frequencies in the regions sampled on both continents, significant differences in the geographic distribution of isolates producing these acetyl ester derivatives of deoxynivalenol (DON) were observed in Europe. While 93.5% of the isolates in southern Russia (n = 43 of 46) possessed the 15ADON chemotype, isolates of F. graminearum recovered in Finland and northwestern Russia (n = 40) were exclusively 3ADON producers. Based on results of the MLGT assay, species identity of 10 genetically novel Fg complex isolates from the Russian Far East was investigated further via molecular phylogenetic analyses of multi-locus DNA sequence data. Results of these analyses resolved these isolates as a phylogenetically distinct, reciprocally monophyletic sister lineage of F. asiaticum, which together with F. vorosii form a newly discovered Asian clade within the Fg complex. Because this novel lineage fulfills the highly conservative criterion of genealogical exclusivity under phylogenetic species recognition it is formally described herein as F. ussurianum. In addition to morphologically characterizing isolates of F. ussurianum, experiments were conducted to assess pathogenicity to wheat and trichothecene toxin potential in planta.

Multilocus Genotyping Assays for Single Nucleotide Polymorphism-Based Subtyping of Listeria monocytogenes Isolates

ABSTRACT Listeria monocytogenes is responsible for serious invasive illness associated with consumption of contaminated food and places a significant burden on public health and the agricultural economy. We recently developed a multilocus genotyping (MLGT) assay for high-throughput subtype determination of L. monocytogenes lineage I isolates based on interrogation of single nucleotide polymorphisms (SNPs) via multiplexed primer extension reactions. Here we report the development and validation of two additional MLGT assays that address the need for comprehensive DNA sequence-based subtyping of L. monocytogenes. The first of these novel MLGT assays targeted variation segregating within lineage II, while the second assay combined probes for lineage III strains with probes for strains representing a recently characterized fourth evolutionary lineage (IV) of L. monocytogenes. These assays were based on nucleotide variation identified in >3.8 Mb of comparative DNA sequence and consisted of 115 total probes that differentiated 93% of the 100 haplotypes defined by the multilocus sequence data. MLGT reproducibly typed the 173 isolates used in SNP discovery, and the 10,448 genotypes derived from MLGT analysis of these isolates were consistent with DNA sequence data. Application of the MLGT assays to assess subtype prevalence among isolates from ready-to-eat foods and food-processing facilities indicated a low frequency (6.3%) of epidemic clone subtypes and a substantial population of isolates (>30%) harboring mutations in inlA associated with attenuated virulence in cell culture and animal models. These mutations were restricted to serogroup 1/2 isolates, which may explain the overrepresentation of serotype 4b isolates in human listeriosis cases.

Population Subdivision of Fusarium graminearum Sensu Stricto in the Upper Midwestern United States.

ABSTRACT A collection of 712 Fusarium graminearum sensu stricto (s.s.) strains, predominantly gathered between 1999 and 2000 from nine states within the United States, was examined for population structure and polymerase chain reaction-based trichothecene type. Most strains belonged to a cohesive genetic population characterized by a 15-acetyldeoxynivalenol (15ADON) trichothecene type. However, using a Bayesian model-based clustering method, we also identified genetically divergent groups of strains in some sampled locations of Minnesota and North Dakota. Strains of the major group of divergent populations were of a 3ADON trichothecene type and formed a distinct cluster with a collection of previously gathered strains from Italy, which displayed all three trichothecene types (15ADON, 3ADON, and nivalenol). The co-existence of genetically divergent populations of F. graminearum s.s. in the Upper Midwest allows for the rejection of the hypothesis that F. graminearum s.s. in the United States consists of a single population. These results also suggest that recombination has been insufficiently frequent in this homothallic (selfing) fungal species to homogenize the divergent populations observed in the Upper Midwest.

Novel Fusarium head blight pathogens from Nepal and Louisiana revealed by multilocus genealogical concordance

This study was conducted to assess evolutionary relationships, species diversity and trichothecene toxin potential of five Fusarium graminearum complex (FGSC) isolates identified as genetically novel during prior Fusarium head blight (FHB) surveys in Nepal and Louisiana. Results of a multilocus genotyping (MLGT) assay for B-trichothecene species determination indicated these isolates might represent novel species within the FGSC. GCPSR-based phylogenetic analyses of a 12-gene dataset, comprising portions of seven loci totaling 13.1 kb of aligned DNA sequence data, provided strong support for the genealogical exclusivity of the Nepalese and Louisianan isolates. Accordingly, both species are formally recognized herein as novel FGSC species. Fusarium nepalense was resolved as the sister lineage of Fusarium ussurianum+Fusarium asiaticum within an Asian subclade of the FGSC. Fusarium louisianense was strongly supported as a reciprocally monophyletic sister of Fusarium gerlachii+F. graminearum, suggesting that this subclade might be endemic to North America. Multilocus Bayesian species tree analyses augment these results and provide evidence for a distinct lineage within F. graminearum predominately from the Gulf Coast of Louisiana. As predicted by the MLGT assay, mycotoxin analyses demonstrated that F. nepalense and F. louisianense could produce 15ADON and nivalenol, respectively, in planta. In addition, both species were only able to induce mild FHB symptoms on wheat in pathogenicity experiments.