Temporal dynamics and population genetic structure of Fusarium graminearum in the upper Midwestern United States.

Fusarium graminearum sensu stricto causes Fusarium head blight (FHB) in wheat and barley, and contaminates grains with several trichothecene mycotoxins, causing destructive yield losses and economic impact in the United States. Recently, a F. graminearum strain collected from Minnesota (MN) was determined to produce a novel trichothecene toxin, called NX-2. In order to determine the spatial and temporal dynamics of NX-2 producing strains in MN, North Dakota (ND) and South Dakota (SD), a total of 463 F. graminearum strains were collected from three sampling periods, 1999-2000, 2006-2007 and 2011-2013. A PCR-RFLP based diagnostic test was developed and validated for NX-2 producing strains based on polymorphisms in the Tri1 gene. Trichothecene biosynthesis gene (Tri gene)-based polymerase chain reaction (PCR) assays and ten PCR-restriction fragment length polymorphism (RFLP) markers were used to genotype all strains. NX-2 strains were detected in each sampling period but with a very low overall frequency (2.8%) and were mainly collected near the borders of MN, ND and SD. Strains with the 3ADON chemotype were relatively infrequent in 1999-2000 (4.5%) but increased to 29.4% in 2006-2007 and 17.2% in 2011-2013. The distribution of 3ADON producing strains also expanded from a few border counties between ND and MN in 1999-2000, southward toward the border between SD and MN in 2006-2007 and westward in 2011-2013. Genetic differentiation between 2006-2007 and 2011-2013 populations (3%) was much lower than that between 1999-2000 and 2006-2007 (22%) or 1999-2000 and 2011-2013 (20%) suggesting that most change to population genetic structure of F. graminearum occurred between 1999-2000 and 2006-2007. This change was associated with the emergence of a new population consisting largely of individuals with a 3ADON chemotype. A Bayesian clustering analysis suggested that NX-2 chemotype strains are part of a previously described Upper Midwestern population. However, these analyses also suggest that the NX-2 isolates could represent a distinct population, but that interpretations of population assignment are influenced by the small number of NX-2 strains available for analysis.

A SIX1 homolog in Fusarium oxysporum f.sp. cubense tropical race 4 contributes to virulence towards Cavendish banana.

The fungus Fusarium oxysporum f.sp. cubense (Focub) causes Fusarium wilt of banana. Focub strains are divided into races according to their host specificity, but which virulence factors underlie these interactions is currently unknown. In the F. oxysporum f.sp. lycopersici (Fol)-tomato system, small secreted fungal proteins, called Six proteins, were identified in the xylem sap of infected plants. The Fol Six1 protein contributes to virulence and has an avirulence function by activating the I-3 immune receptor of tomato. The Focub tropical race 4 (TR4) genome harbors three SIX1 homologs: SIX1a, b and c. In this study, the role of Focub-SIX1a in pathogenicity was evaluated since this homolog is present in not only TR4 but also in other races. A deletion mutant of the SIX1a gene from Focub TR4 strain II5 was generated (FocubΔSIX1a) and tested in planta. Mutants were found to be severely compromised in their virulence. Ectopic integration of the Focub-SIX1a gene in the FocubΔSIX1a strain restored virulence to wild type levels. We conclude that Focub-SIX1a is required for full virulence of Focub TR4 towards Cavendish banana.

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.

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.

Nrs1, a repetitive element linked to pisatin demethylase genes on a dispensable chromosome of Nectria haematococca.

We have identified a repetitive DNA element in Nectria haematococca mating population VI, isolate T-2. This repetitive sequence has been called Nrs1. DNA hybridization analysis indicates the sequence is found in several isolates of the fungus pathogenic to Pisum sativum. A 2,027-bp clone containing the Nrs1-2 allele contains a long polyA sequence, imperfect RNA polymerase III promoter sequences, multiple inverted repeats, and the potential for extensive secondary structure similar to known RNA polymerase III transcripts and related retroelements. Ten of the 11 HindIII restriction fragments from isolate T-2 DNA that hybridize to Nrs1-2 segregate in a manner consistent with a 1:1 ratio for random ascospore progeny. The 10 restriction fragment length polymorphism (RFLP) loci define three linkage groups and correspond to three chromosome-sized DNAs from T-2 separated by pulsed field gel electrophoresis. Three RFLP loci defined by hybridization to the gene for pisatin demethylase and localized on the 1.6 million base pair (Mb) chromosome were genetically linked to each other and to several Nrs1 loci. These sequences recombined despite the fact that no obvious homolog exists for the 1.6-Mb chromosome in one parent strain. Allelic RFLPs corresponding to the gene sequence of cutinase were unlinked to Nrs1 loci.

Autonomously replicating plasmids and chromosome rearrangement during transformation of Nectria haematococca.

A previously described, autonomously replicating plasmid was examined for its ability to replicate in the plant pathogenic fungus, Nectria haematococca (Nh). The plasmid, pFOLT4R4, replicates as a linear molecule, contains a subterminal inverted repeat, as well as the repeated hexanucleotide telomere consensus sequence, TTAGGG, at both ends, and increases frequency of fungal transformation approximately 100-fold compared to a similar integrative plasmid, pHRC. Transformation of Nh occurs by way of autonomous replication; the transformed, hygromycin B-resistant (HyR) phenotype is unstable without selection and in most cases pFOLT4R4 is maintained in the fungus, separate from chromosome-sized DNAs. Surprisingly, a non-autonomously replicating derivative of pFOLT4R4 (called pLD), lacking the subterminal inverted repeat and having the 5'-TTAGGG repeat in only one direction on the plasmid, transformed Nh at a rate as high as pFOLT4R4. Therefore, autonomous replication and high-frequency transformation are separable phenomena in Nh. In pLD transformants, plasmid sequences are integrated into chromosome-sized DNAs of Nh and these cultures generally have a stable HyR phenotype. Treatments involving ligation of Nh genomic DNA to pLD result in a lower frequency of transformation. In many cultures transformed with pLD plus genomic DNA, one wild-type chromosome-sized band is not visible, but another smaller chromosome-sized band is found. Mobility changes in some cases are consistent with deletions of over 1000 kb. Some HyS revertants of transformants appear to lack the entire chromosome into which integration had occurred. These results indicate that the Nh genome is extremely malleable and large portions may be non-essential for growth in culture.

Fusarium oxysporum f. sp. cubense Consists of a Small Number of Divergent and Globally Distributed Clonal Lineages.

ABSTRACT A worldwide collection of Fusarium oxysporum f. sp. cubense was analyzed using anonymous, single-copy, restriction fragment length polymorphism (RFLP) loci. Several lines of evidence indicated that this pathogen has a clonal population structure. Of the 165 isolates examined, only 72 RFLP haplotypes were identified, and nearly half the isolates were represented by the five most common haplotypes. Individuals with identical haplotypes were geographically dispersed, and clone-corrected tests of gametic disequilibrium indicated significant nonrandom association among pairs of alleles for 34 of 36 loci tested. Parsimony analysis divided haplotypes into two major branches (bootstrap value = 99%) that together contained eight clades supported by significant bootstrap values. With the exception of two isolates, all isolates within a vegetative compatibility group were in the same clade and clonal lineage. Clonal lineages were defined by isolates having coefficients of similarity between 0.94 and 1.00. Ten clonal lineages were identified, and the two largest lineages had pantropical distribution. Minor lineages were found only in limited geographical regions. Isolates composing one lineage (FOC VII) may represent either an ancient genetic exchange between individuals in the two largest lineages or an ancestral group. The two largest lineages (FOC I and FOC II) and a lineage from East Africa (FOC V) are genetically distinct; each may have acquired the ability to be pathogenic on banana independently.

Population Genetic Analysis Corroborates Dispersal of Fusarium oxysporum f. sp. radicis-lycopersici from Florida to Europe.

ABSTRACT Fusarium oxysporum isolates from tomato plants displaying crown and root rot symptoms were collected in central and southern Florida and analyzed using vegetative compatibility grouping (VCG) and nuclear restriction fragment length polymorphism (RFLP) data. VCG 0094 of F. oxysporum f. sp. radicis-lycopersici, previously known only from northwestern Europe, was predominant among 387 isolates assessed. In addition, two newly described VCGs (0098 and 0099) were detected at low frequencies. Floridian VCG 0094 isolates displayed a continuum of compatibilities, which is in contrast to the three distinct subgroups previously identified among European VCG 0094 isolates. RFLP haplotypes were constructed using one repetitive and three low-copy probes. Population subdivision of VCG 0094 from various Floridian counties and from northwestern Europe (Belgium, the Netherlands, and the United Kingdom) was evaluated by analysis of molecular variance. A "natural" population structure was revealed, differentiating populations from the east and west coasts of Florida. In addition, isolates from Europe were statistically indistinguishable from the Palm Beach County, FL, population. Furthermore, gene diversity among Palm Beach County VCG 0094 isolates was more than five times greater than among European isolates. Results from both VCG and RFLP analyses strongly support the inference that the European VCG 0094 constitutes a founder population that resulted from intercontinental migration of a few isolates from Palm Beach County, FL.

Rapid Detection of the Fusarium oxysporum Lineage Containing the Canary Island Date Palm Wilt Pathogen.

ABSTRACT Fusarium oxysporum f. sp. canariensis causes Fusarium wilt disease on the Canary Island date palm (Phoenix canariensis). To facilitate disease management, a polymerase chain reaction diagnostic method has been developed to rapidly detect the pathogen. A partial genomic library of F. oxysporum f. sp. canariensis isolate 95-913 was used to identify a DNA sequence diagnostic for a lineage containing all tested isolates of F. oxysporum f. sp. canariensis. Two oligonucleotide primers were designed and used to amplify a 567-bp fragment with F. oxysporum f. sp. canariensis DNAs. DNA from 61 outgroup isolates did not amplify using these primers. Once the primers were shown to amplify a 0.567-kb fragment from DNA of all the F. oxysporum f. sp. canariensis isolates tested, a rapid DNA extraction procedure was developed that led to the correct identification of 98% of the tested F. oxysporum f. sp. canariensis isolates.

Population Analysis of Fusarium graminearum from Wheat Fields in Eastern China.

ABSTRACT Wheat heads showing symptoms of Fusarium head blight were collected from four commercial fields in Zhejiang Province, China, an area where epidemics occur regularly. A total of 225 isolates were subjected to population-level analyses using restriction fragment length polymorphism (RFLP) as markers. Diagnostic RFLP markers established that all isolates belonged to Fusarium graminearum lineage 6. Nine polymorphic probes were hybridized to all isolates, resulting in 65 multilocus RFLP haplotypes (MRH). Probing with the telomeric clone pNla17, which reveals differences among isolates in the hypervariable subtelomeric region, differentiated the 65 MRH further into 144 clones. Mean gene diversity for the four field populations was similar, ranging from H = 0.306 - 0.364 over the nine RFLP loci for clone-corrected data. High levels of gene flow were inferred from a low level of population subdivision among all field populations, indicating that they were part of the same population. Pairwise linkage disequilibrium measures did not unequivocally support a random mating population, because one-third of locus pairs were significantly different from the null hypothesis of no-association between alleles. We speculate therefore that sexual recombination may not be frequent and that high levels of genotypic diversity may be maintained by relatively low selection pressure acting on a highly diverse population.

Origin of Race 3 of Fusarium oxysporum f. sp. lycopersici at a Single Site in California.

ABSTRACT Thirty-nine isolates of Fusarium oxysporum were collected from tomato plants displaying wilt symptoms in a field in California 2 years after F. oxysporum f. sp. lycopersici race 3 was first observed at that location. These and other isolates of F. oxysporum f. sp. lycopersici were characterized by pathogenicity, race, and vegetative compatibility group (VCG). Of the 39 California isolates, 22 were in VCG 0030, 11 in VCG 0031, and six in the newly described VCG 0035. Among the isolates in VCG 0030, 13 were race 3, and nine were race 2. Of the isolates in VCG 0031, seven were race 2, one was race 1, and three were nonpathogenic to tomato. All six isolates in VCG 0035 were race 2. Restriction fragment length polymorphisms (RFLPs) and sequencing of the intergenic spacer (IGS) region of rDNA identified five IGS RFLP haplotypes, which coincided with VCGs, among 60 isolates of F. oxysporum from tomato. Five race 3 isolates from California were of the same genomic DNA RFLP haplotype as a race 2 isolate from the same location, and all 13 race 3 isolates clustered together into a subgroup in the neighbor joining tree. Collective evidence suggests that race 3 in California originated from the local race 2 population.

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.

First Report of Sphaeropsis tumefaciens on an Endangered St. John's-Wort in Florida.

Edison's St. John's-Wort (Hypericum edisonianum (Small) Adams & Robson) is a state-endangered, clonal, endemic shrub found in seasonal ponds in DeSoto, Glades, Highlands, and Polk counties in Florida. In 1998, a population of H. edisonianum with large (2 to 6 cm long), abnormal growths on stems was observed. Initial gall symptoms were rounded swellings beneath undisturbed bark. With age, these galls became irregular in shape, and the bark became fissured with occasional witches' broom. Galls were collected, and surface-sterilized fragments were cultured on acidified potato dextrose agar. Cultures were uniformly black with pycnidia and oblong, nonseptate, or one-septate, hyaline conidia. The fungus was identified as Sphaeropsis tumefaciens Hedges. Shallow stem cuts were made in plants with a sterile scalpel, and small pieces of the fungal culture were inserted and wrapped with Parafilm. Small swellings at the wound site were observed within 8 weeks on fungus-inoculated plants. Maximum growth of stem galls was 1 cm in length on stems after 1 year. S. tumefaciens was consistently reisolated from galls. There were seven replicate plants (with controls) in each of three experiments with similar results. To our knowledge, this is a new host record for S. tumefaciens, a widespread fungal pathogen in Florida. Under the drought conditions of 2000, wild plant populations without infection had as much as 48% mortality, while the population infected with S. tumefaciens had 83% mortality.

Linear plasmidlike DNA in the plant pathogenic fungus Fusarium oxysporum f. sp. conglutinans.

Double-stranded, 1.9-kilobase-pair (kbp) DNA molecules were found in 18 strains representing three pathogenic races of Fusarium oxysporum f. sp. conglutinans. The DNA element (pFOXC1) from a race 1 strain and the DNA element (pFOXC2) from a race 2 strain were shown by restriction endonuclease mapping to be linear. pFOXC2 was found in mitochondrial preparations and appears to have blocked 5' termini, as it was sensitive to 3'----5' exonuclease III but insensitive to 5'----3' lambda exonuclease. The major 1.8-kbp BglII restriction endonuclease fragment of pFOXC2 was cloned in plasmid pUC12. The recombinant plasmid (pCK1) was not homologous to the mitochondrial or nuclear genomes from F. oxysporum f. sp. conglutinans. This suggests that pFOXC2 is self-replicating. pCK1 was homologous to all 1.9-kbp DNA elements of race 2 but was not homologous to those of race 1 or race 5. All race 1 and 5 elements were also shown to share common DNA sequences.

The mitochondrial genome of Fusarium oxysporum.

Physical and genetic maps have been constructed for mtDNA from strains of the fungus Fusarium oxysporum representing three pathogenically specialized forms. All three mtDNA maps are circular. Their sizes are 45 kb for F. oxysporum f.sp. raphani and 52 kb for both F. oxysporum f.sp. conglutinans and F. oxysporum f.sp. matthioli. The genetic loci for cytochrome b, the mitochondrial 25S ribosomal RNA and cytochrome oxidase subunit II, have been identified and are similarly arranged on the three genomes.

In vivo rearrangement of foreign DNA by Fusarium oxysporum produces linear self-replicating plasmids.

Particular combinations of fungal strains and transformation vectors allow for fungal rearrangement of normally integrative plasmids, resulting in the creation of linear self-replicating plasmids in Fusarium oxysporum. The rearrangement results in the addition of fungal DNA, including telomere consensus sequences, to plasmid termini. The mechanism by which this rearrangement occurs is unclear, but it has similarities to extrachromosomal gene amplification. A DNA fragment which allows for linear autonomous replication upon reintroduction to the fungus was subcloned and sequenced. This DNA sequence contains the repeated telomeric sequence TTAGGG flanked by a region of twofold symmetry consisting primarily of pUC12 DNA. Isolation and identification of this sequence is the first step toward development of vectors that function as artificial chromosomes in filamentous fungi. This sequence was shown to promote autonomous replication and enhance transformation in several strains of F. oxysporum, Nectria haematococca, and Cryphonectria parasitica.

Linear mitochondrial plasmids of Fusarium oxysporum contain genes with sequence similarity to genes encoding a reverse transcriptase from Neurospora spp.

Two linear mitochondrial plasmids called pFOXC1 and pFOXC2 from the fungus Fusarium oxysporum were previously described. DNA sequence comparisons indicated that the derived amino acid sequences of both plasmids exhibit similarity to the reverse transcriptase of the Mauriceville and Varkud plasmids of Neurospora spp. The derived amino acid sequence of pFOXC2 has 51% similarity and 32% identity to the Neurospora reverse transcriptase; sequence similarity was greatest for seven blocks of amino acids that are conserved in reverse transcriptases from a wide range of biological sources. Northern analysis suggests that full-length RNAs corresponding to the plasmids are found in representative isolates.