NX Trichothecenes Are Required for Fusarium graminearum Infection of Wheat.

Fusarium graminearum causes Fusarium head blight (FHB) on wheat and barley and contaminates grains with various mycotoxins that are toxic to humans and animals. Deoxynivalenol (DON), a type B trichothecene, is an essential virulence factor that is required for F. graminearum to spread within a wheat head. Recently, novel type A trichothecenes NX-2 and NX-3 (NX) have been found in F. graminearum. NX trichothecenes lack a keto group at the C8 position. To determine if NX trichothecenes play a role similar to that of DON during F. graminearum infection, deletion mutants of TRI5, the first gene for trichothecene biosynthesis, were generated from strains PH-1, NRRL46422, and NRRL44211 (hereafter 44211) representing the 15-acetyl-DON, 3-acetyl-DON, and NX chemotypes. No trichothecene production was detected in any of the Δtri5 mutants in cultures or inoculated wheat heads. FHB symptoms were restricted to the inoculated wheat spikelets when point-inoculated with the Δtri5 mutants, confirming the necessity of NX and DON for FHB spread. Furthermore, whole-head dip inoculations revealed significant reductions in disease and fungal biomass in wheat heads inoculated with 44211Δtri5 compared with 44211. Introduction of the native 44211 TRI5 and a Trichoderma arundinaceum TRI5 ortholog in the 44211Δtri5 mutant complemented trichothecene production in vitro; however, introducing both TRI5 partially restored wild-type levels of NX in infected heads. Our results demonstrate that NX trichothecenes play an important role in Fusarium graminearum initial infection as well as FHB spread. Thus, TRI5 may serve as an ideal target to control plant infection, FHB spread, and mycotoxin production simultaneously. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Insights into the Aggressiveness of the Emerging North American Population 3 (NA3) of Fusarium graminearum.

In the United States and Canada, Fusarium graminearum (Fg) is the predominant etiological agent of Fusarium head blight (FHB), an economically devastating fungal disease of wheat and other small grains. Besides yield losses, FHB leads to grain contamination with trichothecene mycotoxins that are harmful to plant, human, and livestock health. Three genetic North American populations of Fg, differing in their predominant trichothecene chemotype (i.e., NA1/15ADON, NA2/3ADON, and NA3/NX-2), have been identified. To improve our understanding of the newly discovered population NA3 and how population-level diversity influences FHB outcomes, we inoculated heads of the moderately resistant wheat cultivar Alsen with 15 representative strains from each population and evaluated disease progression, mycotoxin accumulation, and mycotoxin production per unit Fg biomass. Additionally, we evaluated population-specific differences in induced host defense responses. The NA3 population was significantly less aggressive than the NA1 and NA2 populations but posed a similar mycotoxigenic potential. Multiomics analyses revealed patterns in mycotoxin production per unit Fg biomass, expression of Fg aggressiveness-associated genes, and host defense responses that did not always correlate with the NA3-specific severity difference. Our comparative disease assay of NA3/NX-2 and admixed NA1/NX-2 strains indicated that the reduced NA3 aggressiveness is not due solely to the NX-2 chemotype. Notably, the NA1 and NA2 populations did not show a significant advantage over NA3 in perithecia production, a fitness-related trait. Together, our data highlight that the disease outcomes were not due to mycotoxin production or host defense alone, indicating that other virulence factors and/or host defense mechanisms are likely involved.

Fusarium graminearum Species Complex: A Bibliographic Analysis and Web-Accessible Database for Global Mapping of Species and Trichothecene Toxin Chemotypes.

Fusarium graminearum is ranked among the five most destructive fungal pathogens that affect agroecosystems. It causes floral diseases in small grain cereals including wheat, barley, and oats, as well as maize and rice. We conducted a systematic review of peer-reviewed studies reporting species within the F. graminearum species complex (FGSC) and created two main data tables. The first contained summarized data from the articles including bibliographic, geographic, methodological (ID methods), host of origin and species, while the second data table contains information about the described strains such as publication, isolate code(s), host/substrate, year of isolation, geographical coordinates, species and trichothecene genotype. Analyses of the bibliographic data obtained from 123 publications from 2000 to 2021 by 498 unique authors and published in 40 journals are summarized. We describe the frequency of species and chemotypes for 16,274 strains for which geographical information was available, either provided as raw data or extracted from the publications, and sampled across six continents and 32 countries. The database and interactive interface are publicly available, allowing for searches, summarization, and mapping of strains according to several criteria including article, country, host, species and trichothecene genotype. The database will be updated as new articles are published and should be useful for guiding future surveys and exploring factors associated with species distribution such as climate and land use. Authors are encouraged to submit data at the strain level to the database, which is accessible at https://fgsc.netlify.app.

Intrapopulation Antagonism Can Reduce the Growth and Aggressiveness of the Wheat Head Blight Pathogen Fusarium graminearum.

Fusarium graminearum is a causal agent of Fusarium head blight (FHB), a disease that reduces yield and quality of cereal crops and contaminates grain with mycotoxins that pose health risks to humans and livestock. Interpopulation antagonistic interactions between isolates that produce different trichothecene mycotoxins can reduce FHB in wheat, but it is not known if interactions between isolates with a shared population identity that produce the same trichothecenes have a similar effect. Using isolates from the predominant F. graminearum populations in North America (NA1 and NA2), we examined intrapopulation interactions by comparing growth, disease progression, and toxin production of individual isolates with multi-isolate mixes. In vitro, mycelial growth was significantly greater when most NA1 and NA2 isolates were cultured individually versus when cultured as a mixture of isolates from the same population. In susceptible wheat Norm, FHB generally progressed faster in heads inoculated with an individual isolate versus a multi-isolate mixture, but the antagonistic effect of intrapopulation interactions was more pronounced for NA1 than NA2 isolates. By contrast, in moderately resistant wheat Alsen, mixtures of isolates from either population caused obvious reductions in FHB development. Mycotoxin contamination was not consistently affected by intrapopulation interactions and varied depending on the interacting isolates from either population. Our results indicate that antagonistic intrapopulation interactions can influence FHB in controlled environmental conditions. Understanding if the regional composition of pathogen populations similarly influences FHB in the field could improve disease forecasting and management practices.

Fusarium graminearum arabinanase (Arb93B) Enhances Wheat Head Blight Susceptibility by Suppressing Plant Immunity.

Fusarium head blight (FHB) of wheat and barley caused by the fungus Fusarium graminearum reduces crop yield and contaminates grain with mycotoxins. In this study, we investigated two exo-1,5-α-L-arabinanases (Arb93A and Arb93B) secreted by F. graminearum and their effect on wheat head blight development. Arabinan is an important component of plant cell walls but it was not known whether these arabinanases play a role in FHB. Both ARB93A and ARB93B were induced during the early stages of infection. arb93A mutants did not exhibit a detectable change in ability to cause FHB, whereas arb93B mutants caused lower levels of FHB symptoms and deoxynivalenol contamination compared with the wild type. Furthermore, virulence and deoxynivalenol contamination were restored to wild-type levels in ARB93B complemented mutants. Fusion proteins of green fluorescent protein (GFP) with the predicted chloroplast peptide or the mature protein of Arb93B were not observed in the chloroplast. Reactive oxygen species (ROS) production was reduced in the infiltrated zones of Nicotiana benthamiana leaves expressing ARB93B-GFP. Coexpression of ARB93B-GFP and Bax in N. benthamiana leaves significantly suppressed Bax-programmed cell death. Our results indicate that Arb93B enhances plant disease susceptibility by suppressing ROS-associated plant defense responses.

Four new species of Metschnikowia and the transfer of seven Candida species to Metschnikowia and Clavispora as new combinations.

From comparisons of ITS1-5.8S-ITS2 and gene sequences for nuclear D1/D2 LSU rRNA, nuclear SSU (18S) rRNA, translation elongation factor 1-α (EF1-α) and RNA polymerase II subunit 2 (RPB2), the following four new ascosporogenous yeast species were resolved and are described as Metschnikowia anglica (NRRL Y-7298T [type strain], CBS 15342, MycoBank MB 823167), Metschnikowia leonuri (NRRL Y-6546T, CBS 15341, MB 823166), Metschnikowia peoriensis (NRRL Y-5942T, CBS 15345, MB 823164) and Metschnikowia rubicola (NRRL Y-6064T, CBS 15344, MB 823165). The following six species of Candida are members of the Metschnikowia clade and are proposed for transfer to Metschnikowia as new combinations: Candida chrysomelidarum (NRRL Y-27749T, CBS 9904, MB 823223), Candida gelsemii (NRRL Y-48212T, CBS 10509, MB 823192), Candida kofuensis (NRRL Y-27226T, CBS 8058, MB 823195), Candida picachoensis (NRRL Y-27607T, CBS 9804, MB 823197), Candida pimensis (NRRL Y-27619T, CBS 9805, MB 823205) and Candida rancensis (NRRL Y-48702T, CBS 8174, MB 823224). Candida fructus (NRRL Y-17072T, CBS 6380, MB 823206) is transferred to Clavispora as a new combination, and Candida musae is shown to be a synonym of C. fructus. Apparent multiple alleles for ITS, D1/D2, EF1-α and RPB2 were detected in strains of some species.

Development of a PCR-RFLP method based on the transcription elongation factor 1-α gene to differentiate Fusarium graminearum from other species within the Fusarium graminearum species complex.

Fusarium head blight (FHB) is a destructive disease of cereals crops worldwide and a major food safety concern due to grain contamination with trichothecenes and other mycotoxins. Fusarium graminearum, a member of the Fusarium graminearum species complex (FGSC) is the dominant FHB pathogen in many parts of the world. However, a number of other Fusarium species, including other members of the FGSC, may also be present for example in Argentina, New Zealand, Ethiopia, Nepal, Unites States in cereals such as wheat and barley. Proper species identification is critical to research aimed at improving disease and mycotoxin control programs. Identification of Fusarium species is are often unreliable by traditional, as many species are morphologically cryptic. DNA sequence-based methods offer a reliable means of species identification, but can be expensive when applied to the analyses of population samples. To facilitate identification of the major causative agent of FHB, this work describes an easy and inexpensive method to differentiate F. graminearum from the remaining species within the FGSC and from the other common Fusarium species causing FHB in cereals. The developed method is based on a PCR-RFLP of the transcription elongation factor (TEF 1-α) gene using the restriction enzyme BsaHI.

Species composition, toxigenic potential and aggressiveness of Fusarium isolates causing Head Blight of barley in Uruguay.

Fusarium Head Blight (FHB) is a major constraint to barley production that substantially reduces yield and grain quality. FHB is also a major food safety concern because FHB pathogens contaminate grain with trichothecenes and other mycotoxins. DNA sequence-based analyses and in-vitro toxin assessments were used to characterize the species and trichothecene chemotype composition of FHB pathogens on barley in Uruguay. F. graminearum was the dominant species (89.7%), and three other members of the F. graminearum species complex (FGSC) were identified as FHB pathogens of barley in Uruguay for the first time. Other minor contributors to FHB species diversity included F. poae, F. avenaceum, F. pseudograminearum and an unnamed species from the F. incarnatum-equiseti species complex (FIESC). Most isolates (89.7%) had the 15-acetyldeoxynivalenol (15-ADON) trichothecene type. However, the results expanded the known area of occurrence within Uruguay for the nivalenol (NIV) toxin type, which was observed among isolates from three species of the FGSC, F. pseudograminearum, and F. poae. Isolates with the 3-acetyldeoxynivalenol (3-ADON) or NX-2 toxin types were not observed, although a previously published multilocus genotyping assay was updated to identify NX-2 strains. Analyses of population structure and comparisons with FHB isolates from wheat in Uruguay indicated that F. graminearum constitutes a single genetic population with no evidence of population differentiation related to the sampled hosts. Inter and intraspecific differences were observed in aggressiveness toward four barley genotypes with different levels of resistance to FHB, and in general nivalenol producers were the least aggressive isolates. Sensitivity to metconazole was approximately 10 times higher than was detected for tebuconazole. This is the first report regarding tebuconazole and metconazole sensitivity for Fusarium species causing FHB in barley in Uruguay, and constitutes an important starting point for monitoring temporal or spatial changes in FGSC sensitivity, which is critical to define FHB management practices.

Population genetic structure and mycotoxin potential of the wheat crown rot and head blight pathogen Fusarium culmorum in Algeria.

Surveys for crown rot (FCR) and head blight (FHB) of Algerian wheat conducted during 2014 and 2015 revealed that Fusarium culmorum strains producing 3-acetyl-deoxynivalenol (3ADON) or nivalenol (NIV) were the causal agents of these important diseases. Morphological identification of the isolates (n FCR=110, n FHB=30) was confirmed by sequencing a portion of TEF1. To assess mating type idiomorph, trichothecene chemotype potential and global population structure, the Algerian strains were compared with preliminary sample of F. culmorum from Italy (n=27), Australia (n=30) and the United States (n=28). A PCR assay for MAT idiomorph revealed that MAT1-1 and MAT1-2 strains were segregating in nearly equal proportions, except within Algeria where two-thirds of the strains were MAT1-2. An allele-specific PCR assay indicated that the 3ADON trichothecene genotype was predominant globally (83.8% 3ADON) and in each of the four countries sampled. In vitro toxin analyses confirmed trichothecene genotype PCR data and demonstrated that most of the strains tested (77%) produced culmorin. Global population genetic structure of 191 strains was assessed using nine microsatellite markers (SSRs). AMOVA of the clone corrected data indicated that 89% of the variation was within populations. Bayesian analysis of the SSR data identified two globally distributed, sympatric populations within which both trichothecene chemotypes and mating types were represented.

The Arsenic Resistance-Associated Listeria Genomic Island LGI2 Exhibits Sequence and Integration Site Diversity and a Propensity for Three Listeria monocytogenes Clones with Enhanced Virulence.

In the foodborne pathogen Listeria monocytogenes, arsenic resistance is encountered primarily in serotype 4b clones considered to have enhanced virulence and is associated with an arsenic resistance gene cluster within a 35-kb chromosomal region, Listeria genomic island 2 (LGI2). LGI2 was first identified in strain Scott A and includes genes putatively involved in arsenic and cadmium resistance, DNA integration, conjugation, and pathogenicity. However, the genomic localization and sequence content of LGI2 remain poorly characterized. Here we investigated 85 arsenic-resistant L. monocytogenes strains, mostly of serotype 4b. All but one of the 70 serotype 4b strains belonged to clonal complex 1 (CC1), CC2, and CC4, three major clones associated with enhanced virulence. PCR analysis suggested that 53 strains (62.4%) harbored an island highly similar to LGI2 of Scott A, frequently (42/53) in the same location as Scott A (LMOf2365_2257 homolog). Random-primed PCR and whole-genome sequencing revealed seven novel insertion sites, mostly internal to chromosomal coding sequences, among strains harboring LGI2 outside the LMOf2365_2257 homolog. Interestingly, many CC1 strains harbored a noticeably diversified LGI2 (LGI2-1) in a unique location (LMOf2365_0902 homolog) and with a novel additional gene. With few exceptions, the tested LGI2 genes were not detected in arsenic-resistant strains of serogroup 1/2, which instead often harbored a Tn554-associated arsenic resistance determinant not encountered in serotype 4b. These findings indicate that in L. monocytogenes, LGI2 has a propensity for certain serotype 4b clones, exhibits content diversity, and is highly promiscuous, suggesting an ability to mobilize various accessory genes into diverse chromosomal loci.IMPORTANCEListeria monocytogenes is widely distributed in the environment and causes listeriosis, a foodborne disease with high mortality and morbidity. Arsenic and other heavy metals can powerfully shape the populations of human pathogens with pronounced environmental lifestyles such as L. monocytogenes Arsenic resistance is encountered primarily in certain serotype 4b clones considered to have enhanced virulence and is associated with a large chromosomal island, Listeria genomic island 2 (LGI2). LGI2 also harbors a cadmium resistance cassette and genes putatively involved in DNA integration, conjugation, and pathogenicity. Our findings indicate that LGI2 exhibits pronounced content plasticity and is capable of transferring various accessory genes into diverse chromosomal locations. LGI2 may serve as a paradigm on how exposure to a potent environmental toxicant such as arsenic may have dynamically selected for arsenic-resistant subpopulations in certain clones of L. monocytogenes which also contribute significantly to disease.

The geographic distribution and complex evolutionary history of the NX-2 trichothecene chemotype from Fusarium graminearum.

Fusarium graminearum and 21 related species comprising the F. sambucinum species complex lineage 1 (FSAMSC-1) are the most important Fusarium Head Blight pathogens of cereal crops world-wide. FSAMSC-1 species typically produce type B trichothecenes. However, some F. graminearum strains were recently found to produce a novel type A trichothecene (NX-2) resulting from functional variation in the trichothecene biosynthetic enzyme Tri1. We used a PCR-RFLP assay targeting the TRI1 gene to identify the NX-2 allele among a global collection of 2515 F. graminearum. NX-2 isolates were only found in southern Canada and the northern U.S., where they were observed at low frequency (1.8%), but over a broader geographic range and set of cereal hosts than previously recognized. Phylogenetic analyses of TRI1 and adjacent genes produced gene trees that were incongruent with the history of species divergence within FSAMSC-1, indicating trans-species evolution of ancestral polymorphism. In addition, placement of NX-2 strains in the TRI1 gene tree was influenced by the accumulation of nonsynonymous substitutions associated with the evolution of the NX-2 chemotype, and a significant (P<0.001) change in selection pressure was observed along the NX-2 branch (ω=1.16) in comparison to other branches (ω=0.17) in the TRI1 phylogeny. Parameter estimates were consistent with positive selection for specific amino-acid changes during the evolution of NX-2, but direct tests of positive selection were not significant. Phylogenetic analyses of fourfold degenerate sites and intron sequences in TRI1 indicated the NX-2 chemotype had a single evolutionary origin and evolved recently from a type B ancestor. Our results indicate the NX-2 chemotype may be indigenous, and possibly endemic, to southern Canada and the northern U.S. In addition, we demonstrate that the evolution of TRI1 within FSAMSC-1 has been complex, with evidence of trans-species evolution and chemotype-specific shifts in selective constraint.

Determination of Evolutionary Relationships of Outbreak-Associated Listeria monocytogenes Strains of Serotypes 1/2a and 1/2b by Whole-Genome Sequencing.

We used whole-genome sequencing to determine evolutionary relationships among 20 outbreak-associated clinical isolates of Listeria monocytogenes serotypes 1/2a and 1/2b. Isolates from 6 of 11 outbreaks fell outside the clonal groups or "epidemic clones" that have been previously associated with outbreaks, suggesting that epidemic potential may be widespread in L. monocytogenes and is not limited to the recognized epidemic clones. Pairwise comparisons between epidemiologically related isolates within clonal complexes showed that genome-level variation differed by 2 orders of magnitude between different comparisons, and the distribution of point mutations (core versus accessory genome) also varied. In addition, genetic divergence between one closely related pair of isolates from a single outbreak was driven primarily by changes in phage regions. The evolutionary analysis showed that the changes could be attributed to horizontal gene transfer; members of the diverse bacterial community found in the production facility could have served as the source of novel genetic material at some point in the production chain. The results raise the question of how to best utilize information contained within the accessory genome in outbreak investigations. The full magnitude and complexity of genetic changes revealed by genome sequencing could not be discerned from traditional subtyping methods, and the results demonstrate the challenges of interpreting genetic variation among isolates recovered from a single outbreak. Epidemiological information remains critical for proper interpretation of nucleotide and structural diversity among isolates recovered during outbreaks and will remain so until we understand more about how various population histories influence genetic variation.

Fusarium praegraminearum sp. nov., a novel nivalenol mycotoxin-producing pathogen from New Zealand can induce head blight on wheat.

We report on the molecular and morphological characterization of a novel type B trichothecene toxin-producing species (i.e. B clade) recovered from litter in a maize field near Wellington, New Zealand, which is described as Fusarium praegraminearum sp. nov. This species was initially identified as F. acuminatum based on morphological characters. However, it differs from this species by producing longer, slightly asymmetrically curved macroconidia in which the apical cell is not as pointed and by its much faster colony growth rate on agar. Molecular phylogenetic analyses of portions of 13 genes resolved F. praegraminearum as the most basal species within the B clade. Mycotoxin analyses demonstrated that it was able to produce 4-acetylnivalenol and 4,15-diacetylnivalenol trichothecenes, the nontrichothecene sesquiterpenes culmorin and hydroxy-culmorins, and the estrogen zearalenone in vitro. Results of a pathogenicity experiment revealed that F. praegraminearum induced moderate head blight on wheat.

New tricks of an old enemy: isolates of Fusarium graminearum produce a type A trichothecene mycotoxin.

The ubiquitous filamentous fungus Fusarium graminearum causes the important disease Fusarium head blight on various species of cereals, leading to contamination of grains with mycotoxins. In a survey of F. graminearum (sensu stricto) on wheat in North America several novel strains were isolated, which produced none of the known trichothecene mycotoxins despite causing normal disease symptoms. In rice cultures, a new trichothecene mycotoxin (named NX-2) was characterized by liquid chromatography-tandem mass spectrometry. Nuclear magnetic resonance measurements identified NX-2 as 3α-acetoxy-7α,15-dihydroxy-12,13-epoxytrichothec-9-ene. Compared with the well-known 3-acetyl-deoxynivalenol (3-ADON), it lacks the keto group at C-8 and hence is a type A trichothecene. Wheat ears inoculated with the isolated strains revealed a 10-fold higher contamination with its deacetylated form, named NX-3, (up to 540 mg kg(-1) ) compared with NX-2. The toxicities of the novel mycotoxins were evaluated utilizing two in vitro translation assays and the alga Chlamydomonas reinhardtii. NX-3 inhibits protein biosynthesis to almost the same extent as the prominent mycotoxin deoxynivalenol, while NX-2 is far less toxic, similar to 3-ADON. Genetic analysis revealed a different TRI1 allele in the N-isolates, which was verified to be responsible for the difference in hydroxylation at C-8.

Diversity of Fusarium head blight populations and trichothecene toxin types reveals regional differences in pathogen composition and temporal dynamics.

Analyses of genetic diversity, trichothecene genotype composition, and population structure were conducted using 4086 Fusarium graminearum isolates collected from wheat in eight Canadian provinces over a three year period between 2005 and 2007. The results revealed substantial regional differences in Fusarium head blight pathogen composition and temporal population dynamics. The 3ADON trichothecene type consistently predominated in Maritime provinces (91%) over the sampled years, and increased significantly (P<0.05) between 2005 and 2007 in western Canada, accounting for 66% of the isolates in Manitoba by the end of the sampling period. In contrast, 3ADON frequency was lower (22%, P<0.001) in the eastern Canadian provinces of Ontario and Québec and did not change significantly between 2005 and 2007, resulting in two distinct longitudinal clines in 3ADON frequency across Canada. Overall, genetic structure was correlated with toxin type, as the endemic population (NA1) was dominated by 15ADON isolates (86%), whereas a second population (NA2) consisted largely of 3ADON isolates (88%). However, the percentage of isolates with trichothecene genotypes that were not predictive of their genetic population assignment (recombinant genotypes) increased from 10% in 2005 to 17% in 2007, indicating that trichothecene type became an increasingly unreliable marker of population identity over time. In addition, there were substantial regional differences in the composition of recombinant genotypes. In western and maritime provinces, NA2 isolates with 15ADON genotypes were significantly more common than NA1 isolates with 3ADON genotypes (P<0.001), and the reverse was true in the eastern provinces of Québec and Ontario. Temporal trends in recombinant genotype composition also varied regionally, as the percentage of 15ADON isolates with NA2 genetic backgrounds increased approximately three fold in western and Maritime provinces, while the opposite trends were observed in Québec and Ontario. The results indicate that F. graminearum population dynamics in Canada have been influenced by a complex adaptive landscape comprising different regional selective pressures, and do not reflect a simple model of dispersal and integration following the introduction of a novel pathogen population. In addition, we identified F. graminearum strains that produce the recently discovered A-trichothecene mycotoxin (NX-2) for the first time in Canada, representing a significant expansion of the known range of NX-2 producing strains in North America.

Population Subdivision of Fusarium graminearum from Barley and Wheat in the Upper Midwestern United States at the Turn of the Century.

Fusarium graminearum, the causal agent of Fusarium head blight (FHB) in wheat and barley, is one of the most economically destructive pathogens of these grains worldwide. Recent population genetic studies of the pathogen obtained from wheat in North America supported population subdivision in part correlated with the spectrum of trichothecene mycotoxins (chemotype) produced by individuals within each population. In contrast, a recent study of F. graminearum obtained from diseased barley in the upper Midwestern United States concluded that only a single population was present, consisting of individuals with various chemotypes. To test whether strains derived from different hosts potentially have different population dynamics, we obtained the barley strains used in the previous study and compared them with wheat strains isolated at a similar time and geographic origin. A total of 247 F. graminearum isolates from barley were assigned firmly into two clusters using a Bayesian clustering method. Subdivision within the barley population corresponded to the previously described NA1 (correlated with the 15ADON chemotype) and NA2 (correlated with the 3ADON chemotype) populations from wheat. However, in both sampling periods the barley population exhibited a higher level of genetic differentiation between NA1 and NA2 populations, fewer admixed individuals and evidence of unidirectional gene introgression (15ADON strains with NA2 genetic backgrounds). These results suggest less recombination between NA1 and NA2 populations on barley compared with wheat. The frequency of 3ADON chemotype strains in the most recently surveyed barley population suggests a latitudinal cline from the northern (49%), central (40%) to the southern (29%) sampling area. The potential to produce a novel trichothecene, 3α-acetoxy,7α,15-dihydroxy-12,13-epoxytrichothe-9-ene (NX-2), was not detected in the barley population but occurred at a low rate (2.4%) in the wheat population.

Regional and field-specific factors affect the composition of fusarium head blight pathogens in subtropical no-till wheat agroecosystem of Brazil.

A multiyear survey of >200 wheat fields in Paraná (PR) and Rio Grande do Sul (RS) states was conducted to assess the extent and distribution of Fusarium graminearum species complex (FGSC) diversity in the southern Brazilian wheat agroecosystem. Five species and three trichothecene genotypes were found among 671 FGSC isolates from Fusarium head blight (FHB)-infected wheat heads: F. graminearum (83%) of the 15-acetyldeoxynivalenol (15-ADON) genotype, F. meridionale (12.8%) and F. asiaticum (0.4%) of the nivalenol (NIV) genotype, and F. cortaderiae (2.5%) and F. austroamericanum (0.9%) with either the NIV or the 3-ADON genotype. Regional differences in FGSC composition were observed, with F. meridionale and the NIV type being significantly (P<0.001) more prevalent in PR (>28%) than in RS (≤9%). Within RS, F. graminearum was overrepresented in fields below 600 m in elevation and in fields with higher levels of FHB incidence (P<0.05). Species composition was not significantly influenced by previous crop or the stage of grain development at sampling. Habitat-specific differences in FGSC composition were evaluated in three fields by characterizing a total of 189 isolates collected from corn stubble, air above the wheat canopy, and symptomatic wheat kernels. Significant differences in FGSC composition were observed among these habitats (P<0.001). Most strikingly, F. meridionale and F. cortaderiae of the NIV genotype accounted for the vast majority (>96%) of isolates from corn stubble, whereas F. graminearum with the 15-ADON genotype was dominant (>84%) among isolates from diseased wheat kernels. Potential differences in pathogenic fitness on wheat were also suggested by a greenhouse competitiveness assay in which F. graminearum was recovered at much higher frequency (>90%) than F. meridionale from four wheat varieties inoculated with an equal mixture of F. graminearum and F. meridionale isolates. Taken together, the data presented here suggest that FGSC composition and, consequently, the trichothecene contamination in wheat grown in southern Brazil is influenced by host adaptation and pathogenic fitness. Evidence that F. meridionale and F. cortaderiae with the NIV genotype are regionally significant contributors to FHB may have significant implications for food safety and the economics of cereal production.

Fusarium dactylidis sp. nov., a novel nivalenol toxin-producing species sister to F. pseudograminearum isolated from orchard grass (Dactylis glomerata) in Oregon and New Zealand.

The B trichothecene toxin-producing clade (B clade) of Fusarium includes the etiological agents of Fusarium head blight, crown rot of wheat and barley and stem and ear rot of maize. B clade isolates also have been recovered from several wild and cultivated grasses, including Dactylis glomerata (orchard grass or cock's foot), one of the world's most important forage grasses. Two isolates from the latter host are formally described here as F. dactylidis. Phenotypically F. dactylidis most closely resembles F. ussurianum from the Russian Far East. Both species produce symmetrical sporodochial conidia that are similar in size and curved toward both ends. However, conidia of F. ussurianum typically end in a narrow apical beak while the apical cell of F. dactylidis is acute. Fusarium dactylidis produced nivalenol mycotoxin in planta as well as low but detectable amounts of the estrogenic mycotoxin zearalenone in vitro. Results of a pathogenicity test revealed that F. dactylidis induced mild head blight on wheat.

Birth, death and horizontal transfer of the fumonisin biosynthetic gene cluster during the evolutionary diversification of Fusarium.

Fumonisins are a family of carcinogenic secondary metabolites produced by members of the Fusarium fujikuroi species complex (FFSC) and rare strains of Fusarium oxysporum. In Fusarium, fumonisin biosynthetic genes (FUM) are clustered, and the cluster is uniform in gene organization. Here, sequence analyses indicated that the cluster exists in five different genomic contexts, defining five cluster types. In FUM gene genealogies, evolutionary relationships between fusaria with different cluster types were largely incongruent with species relationships inferred from primary-metabolism (PM) gene genealogies, and FUM cluster types are not trans-specific. In addition, synonymous site divergence analyses indicated that three FUM cluster types predate diversification of FFSC. The data are not consistent with balancing selection or interspecific hybridization, but they are consistent with two competing hypotheses: (i) multiple horizontal transfers of the cluster from unknown donors to FFSC recipients and (ii) cluster duplication and loss (birth and death). Furthermore, low levels of FUM gene divergence in F. bulbicola, an FFSC species, and F. oxysporum provide evidence for horizontal transfer of the cluster from the former, or a closely related species, to the latter. Thus, uniform gene organization within the FUM cluster belies a complex evolutionary history that has not always paralleled the evolution of Fusarium.

Population structure of Listeria monocytogenes serotype 4b isolates from sporadic human listeriosis cases in the United States from 2003 to 2008.

Listeria monocytogenes can cause severe food-borne disease (listeriosis). Numerous outbreaks have involved three serotype 4b epidemic clones (ECs): ECI, ECII, and ECIa. However, little is known about the population structure of L. monocytogenes serotype 4b from sporadic listeriosis in the United States, even though most cases of human listeriosis are in fact sporadic. Here we analyzed 136 serotype 4b isolates from sporadic cases in the United States, 2003 to 2008, utilizing multiple tools including multilocus genotyping, pulsed-field gel electrophoresis, and sequence analysis of the inlAB locus. ECI, ECII, and ECIa were frequently encountered (32, 17, and 7%, respectively). However, annually 30 to 68% of isolates were outside these ECs, and several novel clonal groups were identified. An estimated 33 and 17% of the isolates, mostly among the ECs, were resistant to cadmium and arsenic, respectively, but resistance to benzalkonium chloride was uncommon (3%) among the sporadic isolates. The frequency of clonal groups fluctuated within the 6-year study period, without consistent trends. However, on several occasions, temporal clusters of isolates with indistinguishable genotypes were detected, suggesting the possibility of hidden multistate outbreaks. Our analysis suggests a complex population structure of serotype 4b L. monocytogenes from sporadic disease, with important contributions by ECs and several novel clonal groups. Continuous monitoring will be needed to assess long-term trends in clonality patterns and population structure of L. monocytogenes from sporadic listeriosis.