Fusarium Head Blight In Cereals Research Articles

Functional Characterization of Aldehyde Dehydrogenase in Fusarium graminearum.

Aldehyde dehydrogenase (ALDH), a common oxidoreductase in organisms, is an aldehyde scavenger involved in various metabolic processes. However, its function in different pathogenic fungi remains unknown. Fusarium graminearum causes Fusarium head blight in cereals, which reduces grain yield and quality and is an important global food security problem. To elucidate the pathogenic mechanism of F. graminearum, seven genes encoding ALDH were knocked out and then studied for their function. Single deletions of seven ALDH genes caused a decrease in spore production and weakened the pathogenicity. Furthermore, these deletions altered susceptibility to various abiotic stresses. FGSG_04194 is associated with a number of functions, including mycelial growth and development, stress sensitivity, pathogenicity, toxin production, and energy metabolism. FGSG_00139 and FGSG_11482 are involved in sporulation, pathogenicity, and SDH activity, while the other five genes are multifunctional. Notably, we found that FGSG_04194 has an inhibitory impact on ALDH activity, whereas FGSG_00979 has a positive impact. RNA sequencing and subcellular location analysis revealed that FGSG_04194 is responsible for biological process regulation, including glucose and lipid metabolism. Our results suggest that ALDH contributes to growth, stress responses, pathogenicity, deoxynivalenol synthesis, and mitochondrial energy metabolism in F. graminearum. Finally, ALDH presents a potential target and theoretical basis for fungicide development.

Ubiquitination of acetyltransferase Gcn5 contributes to fungal virulence in Fusarium graminearum.

The histone acetyltransferase general control non-depressible 5 (Gcn5) plays a critical role in the epigenetic landscape and chromatin modification for regulating a wide variety of biological events. However, the post-translational regulation of Gcn5 itself is poorly understood. Here, we found that Gcn5 was ubiquitinated and deubiquitinated by E3 ligase Tom1 and deubiquitinating enzyme Ubp14, respectively, in the important plant pathogenic fungus Fusarium graminearum. Tom1 interacted with Gcn5 in the nucleus and subsequently ubiquitinated Gcn5 mainly at K252 to accelerate protein degradation. Conversely, Ubp14 deubiquitinated Gcn5 and enhanced its stability. In the deletion mutant Δubp14, protein level of Gcn5 was significantly reduced and resulted in attenuated virulence in the fungus by affecting the mycotoxin production, autophagy process, and the penetration ability. Our findings indicate that Tom1 and Ubp14 show antagonistic functions in the control of the protein stability of Gcn5 via post-translational modification and highlight the importance of Tom1-Gcn5-Ubp14 circuit in the fungal virulence. IMPORTANCE Post-translational modification (PTM) enzymes have been reported to be involved in regulating numerous cellular processes. However, the modification of these PTM enzymes themselves is largely unknown. In this study, we found that the E3 ligase Tom1 and deubiquitinating enzyme Ubp14 contributed to the regulation of ubiquitination and deubiquitination of acetyltransferase Gcn5, respectively, in Fusarium graminearum, the causal agent of Fusarium head blight of cereals. Our findings provide deep insights into the modification of acetyltransferase Gcn5 and its dynamic regulation via ubiquitination and deubiquitination. To our knowledge, this work is the most comprehensive analysis of a regulatory network of ubiquitination that impinges on acetyltransferase in filamentous pathogens. Moreover, our findings are important because we present the novel roles of the Tom1-Gcn5-Ubp14 circuit in fungal virulence, providing novel possibilities and targets to control fungal diseases.

CRISPR-Cas9 Gene Editing and Secondary Metabolite Screening Confirm Fusarium graminearum C16 Biosynthetic Gene Cluster Products as Decalin-Containing Diterpenoid Pyrones.

Fusarium graminearum is a causal organism of Fusarium head blight in cereals and maize. Although a few secondary metabolites produced by F. graminearum are considered disease virulence factors, many molecular products of biosynthetic gene clusters expressed by F. graminearum during infection and their associated role in the disease are unknown. In particular, the predicted meroterpenoid products of the biosynthetic gene cluster historically designated as "C16" are likely associated with pathogenicity. Presented here are the results of CRISPR-Cas9 gene-editing experiments disrupting the polyketide synthase and terpene synthase genes associated with the C16 biosynthetic gene cluster in F. graminearum. Culture medium screening experiments using transformant strains were profiled by UHPLC-HRMS and targeted MS2 experiments to confirm the associated secondary metabolite products of the C16 biosynthetic gene cluster as the decalin-containing diterpenoid pyrones, FDDP-D and FDDP-E. Both decalin-containing diterpenoid pyrones were confirmed to be produced in wheat heads challenged with F. graminearum in growth chamber trials. The extent to which the F. graminearum C16 biosynthetic gene cluster is dispersed within the genus Fusarium is discussed along with a proposed role of the FDDPs as pathogen virulence factors.

Design and characterization of an artificial two-strain bacterial consortium for the efficient biodegradation of deoxynivalenol

Deoxynivalenol (DON) is a trichothecene mycotoxin mainly produced by various Fusarium species which cause Fusarium head blight in cereals. DON accumulation in grains not only poses a serious health threat to human and animals but also causes economic loss in the food and livestock industries. Here we reported the construction of an artificial bacterial consortium comprising of Devosia sp. A8 and Paracoccus yeei A9 isolated from an enriched bacterial culture, which shows effective degradation capability towards DON. The optimal conditions for temperature, pH and initial inoculum volume for DON degradation were 35 °C, pH 8–9 and 0.5 %, respectively. The degradation product was identified as 3-keto-DON by LC-MS/MS and 1H NMR spectra. Devosia sp. A8 played the major role in DON degradation, but the enhancement of the DON degradation activity by adding NADPH or riboflavin suggested that Paracoccus yeei A9 provided electrons for Devosia sp. A8 to degrade DON. Moreover, other trichothecenes including 15-acetyl DON, 3-acetyl DON and T-2 toxin were also degraded by the consortium. To the best of our knowledge, this is the first report about a Devosia species capable of degrading DON when co-cultured with Paracoccus yeei. The results demonstrated that the artificial bacterial consortium is a potential candidate for DON biodegradation as well as other trichothecene mycotoxins in a safe and eco-friendly manner.

Fusarium fruiting body microbiome member Pantoea agglomerans inhibits fungal pathogenesis by targeting lipid rafts

Plant-pathogenic fungi form intimate interactions with their associated bacterial microbiota during their entire life cycle. However, little is known about the structure, functions and interaction mechanisms of bacterial communities associated with fungal fruiting bodies (perithecia). Here we examined the bacterial microbiome of perithecia formed by Fusarium graminearum, the major pathogenic fungus causing Fusarium head blight in cereals. A total of 111 shared bacterial taxa were identified in the microbiome of 65 perithecium samples collected from 13 geographic locations. Within a representative culture collection, 113 isolates exhibited antagonistic activity against F. graminearum, with Pantoea agglomerans ZJU23 being the most efficient in reducing fungal growth and infectivity. Herbicolin A was identified as the key antifungal compound secreted by ZJU23. Genetic and chemical approaches led to the discovery of its biosynthetic gene cluster. Herbicolin A showed potent in vitro and in planta efficacy towards various fungal pathogens and fungicide-resistant isolates, and exerted a fungus-specific mode of action by directly binding and disrupting ergosterol-containing lipid rafts. Furthermore, herbicolin A exhibited substantially higher activity (between 5- and 141-fold higher) against the human opportunistic fungal pathogens Aspergillus fumigatus and Candida albicans in comparison with the clinically used fungicides amphotericin B and fluconazole. Its mode of action, which is distinct from that of other antifungal drugs, and its efficacy make herbicolin A a promising antifungal drug to combat devastating fungal pathogens, both in agricultural and clinical settings.

Comparative assessment of spray nozzles efficacy in the control of fusarium head blight in the barley crops using developed quantitative PCR assay

Fusarium species infect cereal spikes during anthesis and cause Fusarium head blight (FHB), a destructive disease of cereal crops with worldwide economic relevance. The necessity for these phytopathogenic fungi effective control becomes increasingly important for the production of both cultivated plants and those plants seeds. Fungicide application is a key methodology for controlling the disease development and mycotoxin contamination in cereals. Polymerase chain reaction (PCR) is currently the most commonly admitted DNA-based technology for specific, rapid and precise Fusarium detection. We have developed and patented the method for detection and quantitative determination of phytopathogenic fungi F. avenaceum and F. graminearum in plant seeds using Real-Time PCR with a pair of primers, designed to amplify sequences of the internal transcribed spacer at the ribosomal RNA gene cluster of those phytopathogenic fungi. This study was aimed to perform a comparative assessment of the efficacy of different spray nozzles for antifungal treatment to control F. avenaceum and F. graminearum infection of barley grains using a developed qPCR diagnostic system. A single application of a fungicide (active ingredient's content: 250 g/l propiconazole, 80 g/l cyproconazole) at BBCH 65 (middle of flowering) was carried out. For this purpose, four spray nozzles with different technical characteristics were used: Flat Fan 030, Amistar 030, Defy 3D 030 and Vegetable 060 (Pentair, USA). DNA-based fungi detection and identification was performed using conventional PCR and developed qPCR. The level of mycotoxins in barley grain was determined using enzyme-linked immunosorbent assay (ELISA). Grain count in the ear of barley and thousand seed weight (TSW) were also examined.
 A single application of the fungicide inhibited the development of FHB and is accompanied by the slight increase of TSW values in treated plants. It was found, that the most effective fungicide was against F. avenaceum and F. graminearum. The inhibitory effect depended on sprayer type. According to qPCR results, the best performance was achieved when using Amistar 030 and Flat Fan (FF) 030 sprayers. The average concentration of deoxynivalenol (DON) content in all barley grain samples were up to 4 times higher than the permissible level. Overall, because of the high contamination levels, found in tested samples, it is possible to state that a single application of the fungicide at the flowering phase was not able to effectively reduce DON contamination in barley samples.
 The developed test-system for qPCR provides new important information in the study of the effectiveness of fungicides and development of strategies to control FHB in cereals, not achievable with conventional PCR.

Transcriptional Responses of Fusarium graminearum Interacted with Soybean to Cause Root Rot.

Fusarium graminearum is the most devastating pathogen of Fusarium head blight of cereals, stalk and ear of maize, and it has recently become a potential threat for soybean as maize-soybean strip relay intercropping is widely practiced in China. To elucidate the pathogenesis mechanism of F. graminearum on intercropped soybean which causes root rot, transcriptional profiling of F. graminearum at 12, 24, and 48 h post-inoculation (hpi) on soybean hypocotyl tissues was conducted. In total, 2313 differentially expressed genes (DEGs) of F. graminearum were annotated by both KEGG pathway and Gene Ontology (GO) analysis. Among them, 128 DEGs were commonly expressed at three inoculation time points while the maximum DEGs were induced at 24 hpi. In addition, DEGs were also rich in carbon metabolism, ribosome and peroxisome pathways which might contribute to carbon source utilization, sexual reproduction, virulence and survival of F. graminearum when infected on soybean. Hence, this study will provide some basis for the deep understanding the pathogenesis mechanism of F. graminearum on different hosts and its effective control in maize-soybean strip relay intercropping systems.

Moving from qPCR to Chip Digital PCR Assays for Tracking of some Fusarium Species Causing Fusarium Head Blight in Cereals.

Fusarium Head Blight (FHB) is one of the major diseases affecting small-grain cereals, worldwide spread and responsible for severe yield and quality losses annually. Diagnostic tools, able to track Fusarium species even in the early stages of infection, can contribute to mycotoxins’ risk control. Among DNA-based technologies for Fusarium detection, qPCR (single and multiplex assays) is currently the most applied method. However, pathogen diagnostics is now enforced by digital PCR (dPCR), a breakthrough technology that provides ultrasensitive and absolute nucleic acid quantification. In our work, a panel of chip digital PCR assays was developed to quantify Fusarium graminearum, F.culmorum, F. sporotrichioides, F. poae and F. avenaceum. The primers/probes combinations were evaluated on pure fungal samples with cdPCR technique, in comparison with the qPCR approach. Moreover, the cdPCR assays were applied to quantify Fusarium in durum wheat and oat samples, naturally contaminated or spiked with fungal DNA. For a better evaluation of infection level in plants, duplex assays were developed, able to co-amplify both plant and fungal DNA. To the best of our knowledge, this is the first study directed to the application of digital PCR to Fusarium diagnosis in plants.

Transcriptomic and Exometabolomic Profiling Reveals Antagonistic and Defensive Modes of Clonostachys rosea Action Against Fusarium graminearum.

The mycoparasite Clonostachys rosea ACM941 is under development as a biocontrol organism against Fusarium graminearum, the causative agent of Fusarium head blight in cereals. To identify molecular factors associated with this interaction, the transcriptomic and exometabolomic profiles of C. rosea and F. graminearum GZ3639 were compared during coculture. Prior to physical contact, the antagonistic activity of C. rosea correlated with a response heavily dominated by upregulation of polyketide synthase gene clusters, consistent with the detected accumulation of corresponding secondary metabolite products. Similarly, prior to contact, trichothecene gene clusters were upregulated in F. graminearum, while those responsible for fusarielin and fusarin biosynthesis were downregulated, correlating with an accumulation of trichothecene products in the interaction zone over time. A concomitant increase in 15-acetyl deoxynivalenol-3-glucoside in the interaction zone was also detected, with C. rosea established as the source of this detoxified mycotoxin. After hyphal contact, C. rosea was found to predominantly transcribe genes encoding cell wall-degradation enzymes, major facilitator superfamily sugar transporters, anion:cation symporters, as well as alternative carbon source utilization pathways, together indicative of a transition to necrotropism at this stage. F. graminearum notably activated the transcription of phosphate starvation pathway signature genes at this time. Overall, a number of signature molecular mechanisms likely contributing to antagonistic activity by C. rosea against F. graminearum, as well as its mycotoxin tolerance, are identified in this report, yielding several new testable hypotheses toward understanding the basis of C. rosea as a biocontrol agent for continued agronomic development and application.

Specific Detection and Identification of Fusarium graminearum Sensu Stricto Using a PCR-RFLP Tool and Specific Primers Targeting the Translational Elongation Factor 1α Gene.

Fusarium graminearum is a toxigenic plant pathogen that causes Fusarium head blight (FHB) disease on cereal crops. It has recently shown to have cross-pathogenicity on noncereals (i.e., Fusarium root rot [FRR] on soybean) in Canada and elsewhere. Specific detection and differentiation of this potent toxigenic, trichothecene-producing pathogen among other closely related species is extremely important for disease control and mycotoxin monitoring. Here, we designed a PCR restriction fragment length polymorphism protocol based on the DNA sequence of the translational elongation factor 1α (TEF1α) gene. A unique restriction site to the enzyme HpaII is only found in F. graminearum sensu stricto strains among different Fusarium strains in the F. graminearum species complex (FGSC) and other Fusarium spp. associated with FHB in cereals and FRR in soybean. Partial amplification of the TEF1α gene with newly designed primers mh1/mh2 generated a 459-bp PCR fragment. Restriction digestion of the generated fragments with the HpaII enzyme generated a unique restriction pattern that can rapidly and accurately differentiate F. graminearum sensu stricto among all other Fusarium spp. A primer pair (FgssF/FgssR) specific to F. graminearum sensu stricto also was designed and can distinguish F. graminearum sensu stricto from all other Fusarium spp. in the FGSC and other closely related Fusarium spp. involved in FHB and FRR. This finding will be very useful for the specific detection of F. graminearum sensu stricto for diagnostic purposes as well as for the accurate detection of this pathogen in breeding and other research purposes.

Evaluation of resistance to Fusarium graminearum in oats

Fusarium head blight (FHB) is a serious threat for the production of high-quality oats, a crop known for its health beneficial traits. The fungal pathogen, Fusarium graminearum, which is the main causal agent of FHB in cereals has become more common in the main oat production areas. FHB reduces yield quality due to the accumulation of several mycotoxins such as deoxynivalenol. Strong environmental dependence of infection and variable causal agents make FHB only partly controllable by agricultural or manufacturing practices. This underlines the need for resistant cultivars. In recent years various improvements in the FHB resistance breeding of oats have been reported including improved inoculation and screening methods as well as resistance and association studies. This review demonstrates the current techniques used in the phenotyping of oats resistance to FHB. Resistance found in breeding material and gene bank accessions is described and several resistance related traits are discussed. In addition, an outlook to the future prospects of FHB resistance breeding in oats is provided.

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.

Challenges facing the biological control strategies for the management of Fusarium Head Blight of cereals caused by F. graminearum

Fusarium Head Blight (FHB) is one of the most devastating diseases in the world, threatening both crop productions by affecting kernel development, and human and animal health by contaminating heads and seeds with mycotoxins. FHB is caused mainly by a Fusarium graminearum species complex, with F. graminearum sensu stricto (lineage 7) being the most prevalently isolated species from disease kernels. Recently it has been designated as the fourth fungal plant pathogen based on its economic impacts. To face the lack of effective strategies for management of FHB and the rising concerns of mycotoxin contamination, new control strategies must be developed, evaluated and applied. The use of biocontrol agents to mitigate or control plant diseases, such as FHB, has received an increase in interest. Even with a proven efficacy of many biocontrol agents, however, few are available on the market as developed products. Regulatory complexity further compounds the improvement of biocontrol strategies. This review offers an insight of the current biocontrol strategies against F. graminearum, from selection to field-testing. The steps needed to ensure the effectiveness of the candidates, as well as the assessing and monitoring of their efficacy and environmental innocuousness are also discussed.

The potential of antagonistic fungi for control of Fusarium graminearum and Fusarium crookwellense varies depending on the experimental approach.

To investigate the potential of fungal antagonists to control Fusarium head blight (FHB) causing pathogens (Fusarium graminearum and F.crookwellense) with two different experimental approaches. Using two invitro tests, Clonostachys rosea, Cladosporium cladosporioides and 10 Trichoderma strains were screened. In a co-culture assay, all Trichoderma strains significantly reduced the colony area of F.graminearum and F.crookwellense by 45-93%, whereas C.rosea and C.cladosporioides were not effective. In another assay, all antagonists from a chosen subset reduced the number of perithecia and ascospores on wheat straw by 88-100% when inoculated before the pathogen. Only C.rosea, a weak antagonist in the co-culture assay, was effective when inoculated after the pathogen, reducing perithecia and ascospore production by 73 and 100%, respectively. For screening antagonists and to avoid sorting out highly effective strains, it is crucial to consider different experimental approaches since the efficacy might differ substantially depending on the incubation conditions. By using two distinct experimental set-ups, we identified promising biological control agents. FHB is one of the most devastating fungal cereal diseases worldwide. As the pathogen overwinters on crop residues, application of antagonists on residues of the previous crop during harvest could be a promising approach to efficiently control FHB in cereals as an essential part of an integrated disease management.

Review article: Artificial inoculum and inoculation techniques commonly used in the investigation of Fusarium head blight in cereals

Fusarium head blight (FHB) is a disease of small-grain cereals that has a proven negative impact on crop yield, quality and food safety. In this regard, it is one of the most studied diseases of small-grain cereals worldwide. This paper reports the commonly used artificial sources of inoculum and inoculation techniques employed in the study of FHB epidemiology. Spore suspensions and grain spawn are the most popular forms of artificial inoculums. Spray inoculation technique is more commonly used than the point inoculation technique for delivering the inocula to the target sites. Spray inoculation has an advantage over point inoculation in that it can be used to detect both Type I and II resistances to FHB, point inoculation can only detect Type II resistance to FHB. Grain spawn is the commonest soil-surface inoculum used in the study of FHB. Its advantage lies in the fact that it is capable of propagating inoculum over a long period of time compared to other methods. It can also detect the five types of resistances reported (Types I, II, III, IV and V). In order to gain further information regarding the epidemiology of FHB, researchers need to explore other potential sources of artificial inoculum.

Fusarium graminearum Possesses Virulence Factors Common to Fusarium Head Blight of Wheat and Seedling Rot of Soybean but Differing in Their Impact on Disease Severity.

Fusarium graminearum is a toxigenic fungal pathogen that causes Fusarium head blight (FHB) and crown rot on cereal crops worldwide. This fungus also causes damping-off and crown and root rots at the early stage of crop development in soybean cultivated in North and South America. Several F. graminearum genes were investigated for their contribution to FHB in cereals but no inherent study is reported for the dicotyledonous soybean host. In this study we determined the disease severity on soybean seedlings of five single gene disrupted mutants of F. graminearum, previously characterized in wheat spike infection. Three of these mutants are impaired on a specific function as the production of deoxynivalenol (DON, Δtri5), lipase (ΔFgl1), and xylanase (Δxyl03624), while the remaining two are MAP kinase mutants (ΔFgOS-2, Δgpmk1), which are altered in signaling pathways. The mutants that were reduced in virulence (Δtri5, ΔFgl1, and ΔFgOS-2) or are avirulent (Δgpmk1) on wheat were correspondently less virulent or avirulent in soybean seedlings, as shown by the extension of lesions and seedling lengths. The Δxyl03624 mutant was as virulent as the wild type mirroring the behavior observed in wheat. However, a different ranking of symptom severity occurred in the two hosts: the ΔFgOS-2 mutant, that infects wheat spikelets similarly to Δtri5 and ΔFgl1 mutants, provided much reduced symptoms in soybean. Differently from the other mutants, we observed that the ΔFgOS-2 mutant was several fold more sensitive to the glyceollin phytoalexin suggesting that its reduced virulence may be due to its hypersensitivity to this phytoalexin. In conclusion, lipase and DON seem important for full disease symptom development in soybean seedlings, OS-2 and Gpmk1 MAP kinases are essential for virulence, and OS-2 is involved in conferring resistance to the soybean phytoalexin.

Development of a Selective Medium for the Fungal Pathogen Fusarium graminearum Using Toxoflavin Produced by the Bacterial Pathogen Burkholderia glumae.

The ascomycete fungus Fusarium graminearum is a major causal agent for Fusarium head blight in cereals and produces mycotoxins such as trichothecenes and zearalenone. Isolation of the fungal strains from air or cereals can be hampered by various other airborne fungal pathogens and saprophytic fungi. In this study, we developed a selective medium specific to F. graminearum using toxoflavin produced by the bacterial pathogen Burkholderia glumae. F. graminearum was resistant to toxoflavin, while other fungi were sensitive to this toxin. Supplementing toxoflavin into medium enhanced the isolation of F. graminearum from rice grains by suppressing the growth of saprophytic fungal species. In addition, a medium with or without toxoflavin exposed to wheat fields for 1 h had 84% or 25%, respectively, of colonies identified as F. graminearum. This selection medium provides an efficient tool for isolating F. graminearum, and can be adopted by research groups working on genetics and disease forecasting.

Phylogenetic analyses of RPB1 and RPB2 support a middle Cretaceous origin for a clade comprising all agriculturally and medically important fusaria

Fusarium (Hypocreales, Nectriaceae) is one of the most economically important and systematically challenging groups of mycotoxigenic phytopathogens and emergent human pathogens. We conducted maximum likelihood (ML), maximum parsimony (MP) and Bayesian (B) analyses on partial DNA-directed RNA polymerase II largest (RPB1) and second largest subunit (RPB2) nucleotide sequences of 93 fusaria to infer the first comprehensive and well-supported phylogenetic hypothesis of evolutionary relationships within the genus and 20 of its near relatives. Our analyses revealed that Cylindrocarpon formed a basal monophyletic sister to a ‘terminal Fusarium clade’ (TFC) comprising 20 strongly supported species complexes and nine monotypic lineages, which we provisionally recognize as Fusarium (hypothesis F1). The basal-most divergences within the TFC were only significantly supported by Bayesian posterior probabilities (B-PP 0.99–1). An internode of the remaining TFC, however, was strongly supported by MP and ML bootstrapping and B-PP (hypothesis F2). Analysis of seven Fusarium genome sequences and Southern analysis of fusaria elucidated the distribution of genes required for synthesis of 26 families of secondary metabolites within the phylogenetic framework. Diversification time estimates date the origin of the TFC to the middle Cretaceous 91.3million years ago. We also dated the origin of several agriculturally important secondary metabolites as well as the lineage responsible for Fusarium head blight of cereals. Dating of several plant-associated species complexes suggests their evolution may have been driven by angiosperm diversification during the Miocene. Our results support two competing hypotheses for the circumscription of Fusarium and provide a framework for future comparative phylogenetic and genomic analyses of this agronomically and medically important genus.

FgVelB globally regulates sexual reproduction, mycotoxin production and pathogenicity in the cereal pathogen Fusarium graminearum

The velvet genes are conserved in ascomycetous fungi and function as global regulators of differentiation and secondary metabolism. Here, we characterized one of the velvet genes, designated FgVelB, in the plant-pathogenic fungus Fusarium graminearum, which causes fusarium head blight in cereals and produces mycotoxins within plants. FgVelB-deleted (ΔFgVelB) strains produced fewer aerial mycelia with less pigmentation than those of the wild-type (WT) during vegetative growth. Under sexual development conditions, the ΔFgVelB strains produced no fruiting bodies but retained male fertility, and conidiation was threefold higher compared with the WT strain. Production of trichothecene and zearalenone was dramatically reduced compared with the WT strain. In addition, the ΔFgVelB strains were incapable of colonizing host plant tissues. Transcript analyses revealed that FgVelB was highly expressed during the sexual development stage, and may be regulated by a mitogen-activated protein kinase cascade. Microarray analysis showed that FgVelB affects regulatory pathways mediated by the mating-type loci and a G-protein alpha subunit, as well as primary and secondary metabolism. These results suggest that FgVelB has diverse biological functions, probably by acting as a member of a possible velvet protein complex, although identification of the FgVelB-FgVeA complex and the determination of its roles require further investigation.

Fusarium Head Blight of Cereals in Denmark: Species Complex and Related Mycotoxins

Quantitative real-time polymerase chain reaction differentiating 10 Fusarium spp. and Microdochium nivale or M. majus was applied to a total of 396 grain samples of wheat, barley, triticale, oat, and rye sampled across Denmark from 2003 to 2007, along with selected samples of wheat and barley from 1957 to 2000, to determine incidence and abundance of individual Fusarium spp. The mycotoxins deoxynivalenol (DON), nivalenol, zearalenone, T-2, and HT-2 were quantified using liquid chromatography-double mass spectrometry. Major differences in the Fusarium species complex among the five cereals as well as great yearly variation were seen. Fusarium graminearum, F. culmorum, and F. avenaceum were dominant in wheat, with DON as the dominant mycotoxin. F. langsethiae, F. culmorum, and F. avenaceum were dominant in barley and oat, leading to relatively high levels of the mycotoxins T-2 and HT-2. F. graminearum, F. culmorum, and F. avenaceum dominated in triticale and rye. The nontoxigenic M. nivale/majus were present in significant amounts in all cereal species. Wheat and barley samples from 1957 to 1996 exhibited no or very low amounts of F. graminearum, indicating a recent increase of this pathogen. Biomass and mycotoxin data exhibited good correlations between Fusarium spp. and their corresponding mycotoxins under field conditions.