Liane R. Gale

Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium

Fusarium species are among the most important phytopathogenic and toxigenic fungi. To understand the molecular underpinnings of pathogenicity in the genus Fusarium, we compared the genomes of three phenotypically diverse species: Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp. lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. LS regions are rich in transposons and genes with distinct evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition. Experimentally, we demonstrate the transfer of two LS chromosomes between strains of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS chromosomes between otherwise genetically isolated strains explains the polyphyletic origin of host specificity and the emergence of new pathogenic lineages in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new perspective.

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.

Molecular Characterization of the Fusarium graminearum Species Complex in Japan

Members of the Fusarium graminearum species complex are important cereal pathogens worldwide and belong to one of at least nine phylogenetically distinct species. We examined 298 strains of the F. graminearum species complex collected from wheat or barley in Japan to determine the species and trichothecene chemotype. Phylogenetic analyses and species-diagnostic polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLPs) revealed the presence and differential distribution of F. graminearum sensu stricto (s. str.) and F. asiaticum in Japan. F. graminearum s. str. is predominant in the north, especially in the Hokkaido area, while F. asiaticum is predominant in southern regions. In the Tohoku area, these species co-occurred. Trichothecene chemotyping of all strains by multiplex PCR revealed significantly different chemotype compositions of these species. All 50 strains of F. graminearum s. str. were of a 15- or 3-acetyl deoxynivalenol type, while 173 (70%) out of 246 strains of F. asiaticum were of a nivalenol type. The possibility of gene flow between the two species was investigated by use of 15 PCR-RFLP markers developed in this study. However, no obvious hybrids were detected from 98 strains examined, including strains collected from regions where both species co-occur.

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.

Nivalenol-type populations of Fusarium graminearum and F. asiaticum are prevalent on wheat in southern Louisiana.

U.S. populations of the Fusarium graminearum clade cause head blight on wheat and barley and usually contaminate grain with the trichothecene mycotoxin deoxynivalenol (DON). Recently, however, individual nivalenol (NIV)-type isolates from the United States were described that belonged to either the newly described species F. gerlachii or the genetically distinct Gulf Coast population of F. graminearum sensu stricto (s.s.). Here, we describe the discovery of NIV-type F. graminearum s.s. populations that were found in high proportion (79%) among isolates from small-grain-growing regions of Louisiana. We genotyped 237 isolates from Louisiana with newly developed polymerase chain reaction (PCR) restriction fragment length polymorphism markers and multiplex PCR primers that distinguish among the three trichothecene types: the two DON types (15ADON and 3ADON) and NIV. These isolates were compared with 297 isolates from 11 other U.S. states, predominantly from the Midwest. Using Bayesian-model-based clustering, we discovered a southern Louisiana population of F. graminearum s.s. that was genetically distinct from the previously recognized pathogen population in the Midwest (MW15ADON population). Population membership was correlated with trichothecene type. Most isolates from the southern Louisiana population were of the NIV type, while the majority of the isolates from the Midwest were of the 15ADON type. A smaller proportion of isolates from Louisiana belonged to the previously described Gulf Coast population that was mostly of the 3ADON type. The NIV type was also identified in collections from Arkansas (12%), North Carolina (40%), and Missouri (2%), with the collections from Arkansas and North Carolina being small and unrepresentative. F. asiaticum was detected from the two southern Louisiana parishes Acadia and Alexandria. All identified 41 F. asiaticum isolates were of the NIV type. Greenhouse tests indicated that U.S. NIV types accumulated four times less trichothecene toxin than DON types on inoculated wheat. This is the first report of NIV-type populations of F. graminearum s. s. and F. asiaticum in the United States.

Chromosome Complement of the Fungal Plant Pathogen Fusarium graminearum Based on Genetic and Physical Mapping and Cytological Observations

A genetic map of the filamentous fungus Fusarium graminearum (teleomorph: Gibberella zeae) was constructed to both validate and augment the draft whole-genome sequence assembly of strain PH-1. A mapping population was created from a cross between mutants of the sequenced strain (PH-1, NRRL 31084, originally isolated from Michigan) and a field strain from Minnesota (00-676, NRRL 34097). A total of 111 ascospore progeny were analyzed for segregation at 235 loci. Genetic markers consisted of sequence-tagged sites, primarily detected as dCAPS or CAPS (n = 131) and VNTRs (n = 31), in addition to AFLPs (n = 66) and 7 other markers. While most markers exhibited Mendelian inheritance, segregation distortion was observed for 25 predominantly clustered markers. A linkage map was generated using the Kosambi mapping function, using a LOD threshold value of 3.5. Nine linkage groups were detected, covering 1234 cM and anchoring 99.83% of the draft sequence assembly. The nine linkage groups and the 22 anchored scaffolds from the sequence assembly could be assembled into four chromosomes, leaving only five smaller scaffolds (59,630 bp total) of the nuclear DNA unanchored. A chromosome number of four was confirmed by cytological karyotyping. Further analysis of the genetic map data identified variation in recombination rate in different genomic regions that often spanned several hundred kilobases.

Population structure of the Fusarium graminearum species complex from a single Japanese wheat field sampled in two consecutive years.

The Fusarium graminearum species complex (Fg complex) that consists of at least 11 phylogenetically distinct species contains important Fusarium head blight (FHB) pathogens of wheat and barley worldwide. We obtained members of the Fg complex by sampling from a 500-m2 experimental wheat field in Kumamoto Prefecture, Japan in two consecutive years and assessed them for species identity and trichothecene chemotype. Haplotype diversity was estimated by using 11 variable numbers of tandem repeat (VNTR) markers. In addition to these two samples (group 03W in 2003 and group 04W in 2004), pathogen populations from seed that was harvested in Fukuoka Prefecture and planted in the experimental field in 2002 (group 02WSC) and pathogen populations from seed that was harvested in Nagasaki Prefecture and planted in 2003 (group 03WSC) were analyzed for this study. Forty-six isolates were collected in each group. Most isolates from wheat heads were classified as F. asiaticum; only four isolates were classified as F. graminearum sensu stricto (s. str.). Out of a total of 183 Fg complex strains, 80 isolates (44%) were of the NIV type, while 103 isolates (56%), including all four F. graminearum s. str. isolates, were of the 3ADON type. No 15ADON type isolate was detected in this study. Trichothecene chemotype compositions of 03W and 04W were nearly identical. High gene diversity of F. asiaticum was observed in all groups. Based on the observed low level of fixation index (FST) and high level of effective number of migrants (Nm), distinctive population subdivision of F. asiaticum was not inferred among the four groups. These results suggest that FHB in the experimental wheat field in both 2003 and 2004 was caused by a genetically similar population, which prevails in Kumamoto, Fukuoka, and Nagasaki prefectures.

The Probable Center of Origin of Fusarium oxysporum f. sp. lycopersici VCG 0033

Isolates of the tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici, predominantly from commercial tomato fields in Florida and southwestern Georgia, were characterized using vegetative compatibility grouping (VCG), nuclear restriction fragment length polymorphism (RFLP), and virulence. All field isolates that could be grouped into VCG belonged to VCG 0033. This VCG was first described by Marlatt et al. in 1996 for isolates from northern Florida, Arkansas, and North Carolina. This study demonstrates that VCG 0033 is also widespread in central and southern Florida, in addition to southwestern Georgia, and also was found to be present in Puerto Rico. Population genetic and phylogenetic analyses of 121 isolates indicated that molecular diversity among VCG 0033 isolates was by far the highest in Manatee County, FL, suggesting it to be the probable center of origin of this relatively newly described VCG. Virulence tests with a subset of isolates identified all VCG 0033 isolates as race 3, although differences in aggressiveness were observed among tested isolates, independent of resistance genes in the differential cultivars. The widespread VCG 0030 of F. oxysporum f. sp. lycopersici was not present in our field collections. This was unexpected, as strains from Florida isolated prior to 1990 were predominantly VCG 0030. This would suggest that VCG 0033 has replaced VCG 0030 in recent years in commercial tomato fields of Florida and southwestern Georgia.

Detecting migrants in populations of Rhizoctonia solani anastomosis group 3 from potato in North Carolina using multilocus genotype probabilities

ABSTRACT The relative contribution of migration of Rhizoctonia solani anastomosis group 3 (AG-3) on infested potato seed tubers originating from production areas in Canada, Maine, and Wisconsin (source population) to the genetic diversity and structure of populations of R. solani AG-3 in North Carolina (NC) soil (recipient population) was examined. The frequency of alleles detected by multilocus polymerase chain reaction-restriction fragment length polymorphisms, heterozygosity at individual loci, and gametic phase disequilibrium between all pairs of loci were determined for subpopulations of R. solani AG-3 from eight sources of potato seed tubers and from five soils in NC. Analysis of molecular variation revealed little variation between seed source and NC recipient soil populations or between subpopulations within each region. Analysis of population data with a Bayesian-based statistical method previously developed for detecting migration in human populations suggested that six multilocus genotypes from the NC soil population had a statistically significant probability of being migrants from the northern source population. The one-way (unidirectional) migration of genotypes of R. solani AG-3 into NC on infested potato seed tubers from Canada, Maine, and Wisconsin provides a plausible explanation for the lack of genetic subdivision (differentiation) between populations of the pathogen in NC soils or between the northern source and the NC recipient soil populations.