The identification of a key gene highlights macrocyclic ring's role in trichothecene toxicity.

Trichothecenes are toxins produced by certain species from several fungal genera, including Aspergillus, Fusarium, Isaria, Paramyrothecium, Stachybotrys, Trichoderma, and Trichothecium. These toxins are of interest because they contribute to the toxigenicity, plant pathogenicity, and/or biological control activities of some fungi. All trichothecenes have the same core (12,13-epoxytrichothec-9-ene or EPT) structure but can differ from one another by the presence or absence of a macrocyclic ring formed from polyketide and isoprenoid substituents esterified to carbon atoms 4 and 15 of EPT, respectively. Genes required for formation and some modifications of EPT have been elucidated, but almost nothing is known about genes specific to the formation of the macrocyclic ring. Therefore, we used genomic, transcriptomic, metabolomic, and gene deletion analyses to identify genes that are required specifically for the formation of the macrocyclic ring. These analyses identified one gene, TRI24, that is predicted to encode an acyltransferase and that is required for macrocyclic ring formation during biosynthesis of macrocyclic trichothecenes by the fungus Paramyrothecium roridum. In addition, a TRI24 deletion mutant of P. roridum caused less severe disease symptoms on common bean and had less antifungal activity than its wild-type progenitor strain. We propose that the reduced aggressiveness and antifungal activity of the mutant resulted from its inability to produce trichothecenes with a macrocyclic ring. To our knowledge, this is the first report of a gene required specifically for the formation of the macrocyclic ring of trichothecenes and that loss of the macrocyclic ring of trichothecenes can alter the biological activities of a fungus. KEY POINTS: • TRI24 gene is found in all known macrocyclic trichothecene-producing fungi. • A tri24-deletion mutant exhibits a reduction in antifungal and plant disease activities. • TRI24 is the first described gene specific to macrocyclic trichothecene biosynthesis.

Analysis of substrate specificity of cytochrome P450 monooxygenases involved in trichothecene toxin biosynthesis.

Trichothecenes are a structurally diverse family of toxic secondary metabolites produced by certain species of multiple fungal genera. All trichothecene analogs share a core 12,13-epoxytrichothec-9-ene (EPT) structure but differ in presence, absence and types of substituents attached to various positions of EPT. Formation of some of the structural diversity begins early in the biosynthetic pathway such that some producing species have few trichothecene biosynthetic intermediates in common. Cytochrome P450 monooxygenases (P450s) play critical roles in formation of trichothecene structural diversity. Within some species, relaxed substrate specificities of P450s allow individual orthologs of the enzymes to modify multiple trichothecene biosynthetic intermediates. It is not clear, however, whether the relaxed specificity extends to biosynthetic intermediates that are not produced by the species in which the orthologs originate. To address this knowledge gap, we used a mutant complementation-heterologous expression analysis to assess whether orthologs of three trichothecene biosynthetic P450s (TRI11, TRI13 and TRI22) from Fusarium sporotrichioides, Trichoderma arundinaceum, and Paramyrothecium roridum can modify trichothecene biosynthetic intermediates that they do not encounter in the organism in which they originated. The results indicate that TRI13 and TRI22 could not modify the intermediates that they do not normally encounter, whereas TRI11 could modify an intermediate that it does not normally encounter. These findings indicate that substrate promiscuity varies among trichothecene biosynthetic P450s. One structural feature that likely impacts the ability of the P450s to use biosynthetic intermediates as substrates is the presence and absence of an oxygen atom attached to carbon atom 3 of EPT.

Phylogenetic Diversity and Mycotoxin Potential of Emergent Phytopathogens Within the Fusarium tricinctum Species Complex.

Recent studies on multiple continents indicate members of the Fusarium tricinctum species complex (FTSC) are emerging as prevalent pathogens of small-grain cereals, pulses, and other economically important crops. These understudied fusaria produce structurally diverse mycotoxins, among which enniatins (ENNs) and moniliformin (MON) are the most frequent and of greatest concern to food and feed safety. Herein a large survey of fusaria in the Fusarium Research Center and Agricultural Research Service culture collections was undertaken to assess species diversity and mycotoxin potential within the FTSC. A 151-strain collection originating from diverse hosts and substrates from different agroclimatic regions throughout the world was selected from 460 FTSC strains to represent the breadth of FTSC phylogenetic diversity. Evolutionary relationships inferred from a five-locus dataset, using maximum likelihood and parsimony, resolved the 151 strains as 24 phylogenetically distinct species, including nine that are new to science. Of the five genes analyzed, nearly full-length phosphate permease sequences contained the most phylogenetically informative characters, establishing its suitability for species-level phylogenetics within the FTSC. Fifteen of the species produced ENNs, MON, the sphingosine analog 2-amino-14,16-dimethyloctadecan-3-ol (AOD), and the toxic pigment aurofusarin (AUR) on a cracked corn kernel substrate. Interestingly, the five earliest diverging species in the FTSC phylogeny (i.e., F. iranicum, F. flocciferum, F. torulosum, and Fusarium spp. FTSC 8 and 24) failed to produce AOD and MON, but synthesized ENNs and/or AUR. Moreover, our reassessment of nine published phylogenetic studies on the FTSC identified 11 additional novel taxa, suggesting this complex comprises at least 36 species.

DNA Sequence-Based Identification of Fusarium: A Work in Progress.

Accurate species-level identification of an etiological agent is crucial for disease diagnosis and management because knowing the agent's identity connects it with what is known about its host range, geographic distribution, and toxin production potential. This is particularly true in publishing peer-reviewed disease reports, where imprecise and/or incorrect identifications weaken the public knowledge base. This can be a daunting task for phytopathologists and other applied biologists that need to identify Fusarium in particular, because published and ongoing multilocus molecular systematic studies have highlighted several confounding issues. Paramount among these are: (i) this agriculturally and clinically important genus is currently estimated to comprise more than 400 phylogenetically distinct species (i.e., phylospecies), with more than 80% of these discovered within the past 25 years; (ii) approximately one-third of the phylospecies have not been formally described; (iii) morphology alone is inadequate to distinguish most of these species from one another; and (iv) the current rapid discovery of novel fusaria from pathogen surveys and accompanying impact on the taxonomic landscape is expected to continue well into the foreseeable future. To address the critical need for accurate pathogen identification, our research groups are focused on populating two web-accessible databases (FUSARIUM-ID v.3.0 and the nonredundant National Center for Biotechnology Information nucleotide collection that includes GenBank) with portions of three phylogenetically informative genes (i.e., TEF1, RPB1, and RPB2) that resolve at or near the species level in every Fusarium species. The objectives of this Special Report, and its companion in this issue (Torres-Cruz et al. 2022), are to provide a progress report on our efforts to populate these databases and to outline a set of best practices for DNA sequence-based identification of fusaria.

Weeds Harbor Fusarium Species that Cause Malformation Disease of Economically Important Trees in Western Mexico.

Mango malformation disease (MMD) caused by Fusarium spp. is an important limiting factor in most production areas worldwide. Fusarium mexicanum and F. pseudocircinatum have been reported as causing MMD in Mexico. These two pathogens also cause a similar disease in Swietenia macrophylla (big-leaf mahogany malformation disease) in central western Mexico, and F. pseudocircinatum was recently reported as causing malformation disease in Tabebuia rosea (rosy trumpet) in the same region. These studies suggest that additional plant species, including weeds, might be hosts of these pathogens. The role that weed hosts might have in the disease cycle is unknown. The objectives of this work were to recover Fusarium isolates from understory vegetation in mango orchards with MMD, identify the Fusarium isolates through DNA sequence data, and determine whether F. mexicanum is capable of inducing disease in the weedy legume Senna uniflora (oneleaf senna). Additional objectives in this work were to compare Fusarium isolates recovered from weeds and mango trees in the same orchards by characterizing their phylogenetic relationships, assessing in vitro production of mycotoxins, and identifying their mating type idiomorph. A total of 59 Fusarium isolates from five species complexes were recovered from apical and lateral buds from four weed species. Two of the species within the F. fujikuroi species complex are known to cause MMD in Mexico. Trichothecene production was detected in five isolates, including F. sulawense and F. irregulare in the F. incarnatum-equiseti species complex and F. boothii in the F. sambucinum species complex. Both mating types were present among mango and weed isolates. This is the first report of herbaceous hosts harboring Fusarium species that cause mango malformation in Mexico. The information provided should prove valuable for further study of the epidemiological role of weeds in MMD and help manage the disease.

Phylogenomic Analysis of a 55.1-kb 19-Gene Dataset Resolves a Monophyletic Fusarium that Includes the Fusarium solani Species Complex.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option available.

Malformation Disease in Tabebuia rosea (Rosy Trumpet) Caused by Fusarium pseudocircinatum in Mexico.

Tabebuia rosea (rosy trumpet) is an economically important neotropical tree in Mexico that is highly valued for the quality of its wood, which is used for furniture, crafts, and packing, and for its use as an ornamental and shade tree in parks and gardens. During surveys conducted in the lower Balsas River Basin region in the states of Guerrero and Michoacán, symptoms of floral malformation were detected in T. rosea trees. The main objectives of this study were to describe this new disease, to determine its causal agent, and to identify it using DNA sequence data. A second set of objectives was to analyze the phylogenetic relationship of the causal agent to Fusarium spp. associated with Swietenia macrophylla trees with malformation surveyed in the same region and to compare mycotoxin production and the mating type idiomorphs of fusaria recovered from T. rosea and S. macrophylla. Tabebuia rosea showed malformed inflorescences with multiple tightly curled shoots and shortened internodes. A total of 31 Fusarium isolates recovered from symptomatic T. rosea (n = 20) and S. macrophylla (n = 11) trees were identified by molecular analysis as Fusarium pseudocircinatum. Pathogenicity tests showed that isolates of F. pseudocircinatum recovered from T. rosea induced malformation in inoculated T. rosea seedlings. Eighteen F. pseudocircinatum isolates were tested for their ability to produce mycotoxins and other secondary metabolites. Moniliformin, fusaric acid, bikaverin, beauvericin, aurofusarin. and 8-O-methylbostrycoidin were produced by at least one strain of the 18 isolates tested. A multiplex PCR assay for mating type idiomorph revealed that 22 F. pseudocircinatum isolates were MAT1-1 and that 9 were MAT1-2. Here, we report a new disease of T. rosea in Mexico caused by F. pseudocircinatum.

Identification and distribution of gene clusters required for synthesis of sphingolipid metabolism inhibitors in diverse species of the filamentous fungus Fusarium.

BACKGROUND: Sphingolipids are structural components and signaling molecules in eukaryotic membranes, and many organisms produce compounds that inhibit sphingolipid metabolism. Some of the inhibitors are structurally similar to the sphingolipid biosynthetic intermediate sphinganine and are referred to as sphinganine-analog metabolites (SAMs). The mycotoxins fumonisins, which are frequent contaminants in maize, are one family of SAMs. Due to food and feed safety concerns, fumonisin biosynthesis has been investigated extensively, including characterization of the fumonisin biosynthetic gene cluster in the agriculturally important fungi Aspergillus and Fusarium. Production of several other SAMs has also been reported in fungi, but there is almost no information on their biosynthesis. There is also little information on how widely SAM production occurs in fungi or on the extent of structural variation of fungal SAMs. RESULTS: Using fumonisin biosynthesis as a model, we predicted that SAM biosynthetic gene clusters in fungi should include a polyketide synthase (PKS), an aminotransferase and a dehydrogenase gene. Surveys of genome sequences identified five putative clusters with this three-gene combination in 92 of 186 Fusarium species examined. Collectively, the putative SAM clusters were distributed widely but discontinuously among the species. We propose that the SAM5 cluster confers production of a previously reported Fusarium SAM, 2-amino-14,16-dimethyloctadecan-3-ol (AOD), based on the occurrence of AOD production only in species with the cluster and on deletion analysis of the SAM5 cluster PKS gene. We also identified SAM clusters in 24 species of other fungal genera, and propose that one of the clusters confers production of sphingofungin, a previously reported Aspergillus SAM. CONCLUSION: Our results provide a genomics approach to identify novel SAM biosynthetic gene clusters in fungi, which should in turn contribute to identification of novel SAMs with applications in medicine and other fields. Information about novel SAMs could also provide insights into the role of SAMs in the ecology of fungi. Such insights have potential to contribute to strategies to reduce fumonisin contamination in crops and to control crop diseases caused by SAM-producing fungi.

Correction to: Identification and distribution of gene clusters required for synthesis of sphingolipid metabolism inhibitors in diverse species of the filamentous fungus Fusarium.

An amendment to this paper has been published and can be accessed via the original article.

Pseudoflowers produced by Fusarium xyrophilum on yellow-eyed grass (Xyris spp.) in Guyana: A novel floral mimicry system?

Pseudoflower formation is arguably the rarest outcome of a plant-fungus interaction. Here we report on a novel putative floral mimicry system in which the pseudoflowers are composed entirely of fungal tissues in contrast to modified leaves documented in previous mimicry systems. Pseudoflowers on two perennial Xyris species (yellow-eyed grass, X. setigera and X. surinamensis) collected from savannas in Guyana were produced by Fusarium xyrophilum, a novel Fusarium species. These pseudoflowers mimic Xyris flowers in gross morphology and are ultraviolet reflective. Axenic cultures of F. xyrophilum produced two pigments that had fluorescence emission maxima in light ranges that trichromatic insects are sensitive to and volatiles known to attract insect pollinators. One of the volatiles emitted by F. xyrophilum cultures (i.e., 2-ethylhexanol) was also detected in the head space of X. laxifolia var. iridifolia flowers, a perennial species native to the New World. Results of microscopic and PCR analyses, combined with examination of gross morphology of the pseudoflowers, provide evidence that the fungus had established a systemic infection in both Xyris species, sterilized them and formed fungal pseudoflowers containing both mating type idiomorphs. Fusarium xyrophilum cultures also produced the auxin indole-3-acetic acid (IAA) and the cytokinin isopentenyl adenosine (iPR). Field observations revealed that pseudoflowers and Xyris flowers were both visited by bees. Together, the results suggest that F. xyrophilum pseudoflowers are a novel floral mimicry system that attracts insect pollinators, via visual and olfactory cues, into vectoring its conidia, which might facilitate outcrossing of this putatively heterothallic fungus and infection of previously uninfected plants.

Evolution of structural diversity of trichothecenes, a family of toxins produced by plant pathogenic and entomopathogenic fungi.

Trichothecenes are a family of terpenoid toxins produced by multiple genera of fungi, including plant and insect pathogens. Some trichothecenes produced by the fungus Fusarium are among the mycotoxins of greatest concern to food and feed safety because of their toxicity and frequent occurrence in cereal crops, and trichothecene production contributes to pathogenesis of some Fusarium species on plants. Collectively, fungi produce over 150 trichothecene analogs: i.e., molecules that share the same core structure but differ in patterns of substituents attached to the core structure. Here, we carried out genomic, phylogenetic, gene-function, and analytical chemistry studies of strains from nine fungal genera to identify genetic variation responsible for trichothecene structural diversity and to gain insight into evolutionary processes that have contributed to the variation. The results indicate that structural diversity has resulted from gain, loss, and functional changes of trichothecene biosynthetic (TRI) genes. The results also indicate that the presence of some substituents has arisen independently in different fungi by gain of different genes with the same function. Variation in TRI gene duplication and number of TRI loci was also observed among the fungi examined, but there was no evidence that such genetic differences have contributed to trichothecene structural variation. We also inferred ancestral states of the TRI cluster and trichothecene biosynthetic pathway, and proposed scenarios for changes in trichothecene structures during divergence of TRI cluster homologs. Together, our findings provide insight into evolutionary processes responsible for structural diversification of toxins produced by pathogenic fungi.

Intestinal hydrolysis and microbial biotransformation of diacetoxyscirpenol-α-glucoside, HT-2-ß-glucoside and N-(1-deoxy-d-fructos-1-yl) fumonisin B1 by human gut microbiota in vitro.

Fusarium mycotoxins are common contaminants in cereals and often co-occur with plant-derived mycotoxin sugar conjugates. Several of these modified mycotoxins are not degraded in the small intestine and hence carried through to the large intestine where microbial transformation may occur. This study aims to assess the gastrointestinal stability of the trichothecenes HT-2 toxin (HT-2), HT-2-ß-glucoside (HT-2-Glc), diacetoxyscirpenol (DAS), DAS-α-glucoside (DAS-Glc) and fumonisin B1 (FB1), N-(1-deoxy-d-fructos-1-yl) fumonisin-B1 (NDF-FB1). All tested modified mycotoxins were stable under upper gastrointestinal (GI) conditions. In faecal batch culture experiments, HT-2-Glc was hydrolysed efficiently and no further microbial biotransformation of HT-2 was observed. DAS-Glc hydrolysis was slow and DAS was de-acetylated to 15-monoacetoxyscripenol. NDF-FB1 was hydrolysed at the slowest rate and FB1 accumulation varied between donor samples. Our results demonstrate that all tested modified mycotoxins are stable in the upper GI tract and efficiently hydrolysed by human gut microbiota, thus potentially contributing to colonic toxicity. Hence the microbial biotransformation of any novel modified mycotoxins needs to be carefully evaluated.

Immunoassay utilizing imaging surface plasmon resonance for the detection of cyclopiazonic acid (CPA) in maize and cheese.

α-Cyclopiazonic acid (CPA) is a tremorgenic mycotoxin produced by Aspergillus and Penicillium fungal species, commonly found on agricultural commodities or fermented food products. A sensitive and rapid imaging surface plasmon resonance (iSPR) assay was developed to detect CPA in maize and cheese by combining an indirect competitive immunoassay and signal amplification based upon a secondary antibody (Ab2) conjugated with gold nanoparticles. Matrix-matched calibration curves were used to determine CPA content in maize and cheese samples. Recoveries, at two spiking levels in maize and cheese, were 89 to 126%, with standard deviations of repeatability (RSDr) of less than 16%. The limits of detection were 17 and 6 µg/kg in maize and cheese, respectively. To separate the CPA-contaminated samples from uncontaminated samples, a cutoff validation level of 40 µg/kg was introduced. The assay was applied to samples of naturally contaminated maize and was compared with competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA). This is the first report to detect CPA using an immuno-biosensor iSPR format.

Determinants and Expansion of Specificity in a Trichothecene UDP-Glucosyltransferase from Oryza sativa.

Family 1 UDP-glycosyltransferases (UGTs) in plants primarily form glucose conjugates of small molecules and, besides other functions, play a role in detoxification of xenobiotics. Indeed, overexpression of a barley UGT in wheat has been shown to control Fusarium head blight, which is a plant disease of global significance that leads to reduced crop yields and contamination with trichothecene mycotoxins such as deoxynivalenol (DON), T-2 toxin, and many other structural variants. The UGT Os79 from rice has emerged as a promising candidate for inactivation of mycotoxins because of its ability to glycosylate DON, nivalenol, and hydrolyzed T-2 toxin (HT-2). However, Os79 is unable to modify T-2 toxin (T-2), produced by pathogens such as Fusarium sporotrichioides and Fusarium langsethii. Activity toward T-2 is desirable because it would allow a single UGT to inactivate co-occurring mycotoxins. Here, the structure of Os79 in complex with the products UDP and deoxynivalenol 3-O-glucoside is reported together with a kinetic analysis of a broad range of trichothecene mycotoxins. Residues associated with the trichothecene binding pocket were examined by site-directed mutagenesis that revealed that trichothecenes substituted at the C4 position, which are not glycosylated by wild-type Os79, can be accommodated in the binding pocket by increasing its volume. The H122A/L123A/Q202L triple mutation, which increases the volume of the active site and attenuates polar contacts, led to strong and equivalent activity toward trichothecenes with C4 acetyl groups. This mutant enzyme provides the broad specificity required to control multiple toxins produced by different Fusarium species and chemotypes.

Crystal Structure of Os79 (Os04g0206600) from Oryza sativa: A UDP-glucosyltransferase Involved in the Detoxification of Deoxynivalenol.

Fusarium head blight is a plant disease with significant agricultural and health impact which affects cereal crops such as wheat, barley, and maize and is characterized by reduced grain yield and the accumulation of trichothecene mycotoxins such as deoxynivalenol (DON). Studies have identified trichothecene production as a virulence factor in Fusarium graminearum and have linked DON resistance to the ability to form DON-3-O-glucoside in wheat. Here, the structures of a deoxynivalenol:UDP-glucosyltransferase (Os79) from Oryza sativa are reported in complex with UDP in an open conformation, in complex with UDP in a closed conformation, and in complex with UDP-2-fluoro-2-deoxy-d-glucose and trichothecene at 1.8, 2.3, and 2.2 Å resolution, respectively. The active site of Os79 lies in a groove between the N-terminal acceptor and the C-terminal donor-binding domains. Structural alignments reveal that Os79 likely utilizes a catalytic mechanism similar to those of other plant UGTs, with His 27 activating the trichothecene O3 hydroxyl for nucleophilic attack at C1' of the UDP-glucose donor. Kinetic analysis of mutant Os79 revealed that Thr 291 plays a critical role in catalysis as a catalytic acid or to position the UDP moiety during the nucleophilic attack. Steady-state kinetic analysis demonstrated that Os79 conjugates multiple trichothecene substrates such as DON, nivalenol, isotrichodermol, and HT-2 toxin, but not T-2 toxin. These data establish a foundation for understanding substrate specificity and activity in this enzyme and can be used to guide future efforts to increase DON resistance in cereal crops.

Fusarium agapanthi sp. nov., a novel bikaverin and fusarubin-producing leaf and stem spot pathogen of Agapanthus praecox (African lily) from Australia and Italy.

This study was conducted to characterize a novel Fusarium species that caused leaf and stem spot on Agapanthus praecox (Agapanthus, African lily) in northern Italy and leaf rot and spot on the same host in Melbourne, Australia. Formally described as Fusarium agapanthi, this pathogen was analyzed using phenotypic, phytopathogenic, secondary metabolite, molecular phylogenetic and genomic data. Five strains were characterized, including one isolated in 1999 from symptomatic A. praecox in Saluzzo, Italy, and four in 2010 from diseased leaf tissue from the same host exhibiting leaf rot and spot symptoms in the Melbourne Gardens, Royal Botanic Gardens Victoria, Australia. Maximum parsimony and maximum likelihood molecular phylogenetic analyses of portions of six individual genes and the combined dataset all strongly supported F. agapanthi either as the earliest diverging genealogically exclusive lineage in the American Clade of the F. fujikuroi species complex, or alternatively a novel monotypic lineage sister to the American Clade. Koch's postulates were completed on dwarf blue- and large white-flowering varieties of A. praecox, where two isolates of F. agapanthi produced slowly spreading necrotic lesions when inoculated onto leaves and flower stems. Fusarium agapanthi is distinguished from other fusaria by the production of densely branched aerial conidiophores with polyphialides throughout the aerial mycelium on synthetic nutrient-poor agar. BLASTn searches of the F. agapanthi NRRL 31653 and NRRL 54464 (= VPRI 41787) genome sequences were conducted to predict sexual reproductive mode and mycotoxin potential. Results indicated that they possessed MAT1-2 and MAT1-1 idiomorphs, respectively, indicating that this species might be heterothallic. Furthermore, based on the presence of homologs of the bikaverin and fusarubin biosynthetic gene clusters in the F. agapanthi genomes, liquid chromatography-mass spectrometry analysis was conducted and confirmed production of these secondary metabolites in rice and corn kernel cultures of the fungus.

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.

Identification of a 12-gene Fusaric Acid Biosynthetic Gene Cluster in Fusarium Species Through Comparative and Functional Genomics.

In fungi, genes involved in biosynthesis of a secondary metabolite (SM) are often located adjacent to one another in the genome and are coordinately regulated. These SM biosynthetic gene clusters typically encode enzymes, one or more transcription factors, and a transport protein. Fusaric acid is a polyketide-derived SM produced by multiple species of the fungal genus Fusarium. This SM is of concern because it is toxic to animals and, therefore, is considered a mycotoxin and may contribute to plant pathogenesis. Preliminary descriptions of the fusaric acid (FA) biosynthetic gene (FUB) cluster have been reported in two Fusarium species, the maize pathogen F. verticillioides and the rice pathogen F. fujikuroi. The cluster consisted of five genes and did not include a transcription factor or transporter gene. Here, analysis of the FUB region in F. verticillioides, F. fujikuroi, and F. oxysporum, a plant pathogen with multiple hosts, indicates the FUB cluster consists of at least 12 genes (FUB1 to FUB12). Deletion analysis confirmed that nine FUB genes, including two Zn(II)2Cys6 transcription factor genes, are required for production of wild-type levels of FA. Comparisons of FUB cluster homologs across multiple Fusarium isolates and species revealed insertion of non-FUB genes at one or two locations in some homologs. Although the ability to produce FA contributed to the phytotoxicity of F. oxysporum culture extracts, lack of production did not affect virulence of F. oxysporum on cactus or F. verticillioides on maize seedlings. These findings provide new insights into the genetic and biochemical processes required for FA production.

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.

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.