Shotgun Analysis of the Secretome of Fusarium graminearum

Fusarium head blight, caused predominately by Fusarium graminearum, is one of the most destructive diseases of wheat (Triticum aestivum L.) worldwide. To characterize the profile of proteins secreted by F. graminearum, the extracellular proteins were collectively obtained from F. graminearum culture supernatants and evaluated using one-dimensional SDS-PAGE and liquid chromatography-tandem mass spectrometry. A total of 87 proteins have been identified, of which 63 were predicted as secretory proteins including those with known functions. Meanwhile, 20 proteins that are not homologous to genomic sequences with known functions have also been detected. Some of the identified proteins are possible virulence factors and may play extracellular roles during F. graminearum infection. This study provides a valuable dataset of F. graminearum extracellular proteins, and a better understanding of the virulence mechanisms of the pathogen.     

Recent advances in genes involved in secondary metabolite synthesis,hyphal development,energy metabolism and pathogenicity in Fusarium graminearum (teleomorph Gibberella zeae)

The ascomycete fungus, Fusarium graminearum (teleomorph Gibberella zeae), is the most common causal agent of Fusarium head blight (FHB), a devastating disease for cereal crops worldwide. F. graminearum produces ascospores (sexual spores) and conidia (asexual spores), which can serve as disease inocula of FHB. Meanwhile, Fusarium-infected grains are often contaminated with mycotoxins such as trichothecenes (TRIs), fumonisins, and zearalenones, among which TRIs are related to the pathogenicity of F. graminearum, and these toxins are hazardous to humans and livestock. In recent years, with the complete genome sequencing of F. graminearum, an increasing number of functional genes involved in the production of secondary metabolites, hyphal differentiation, sexual and asexual reproduction, virulence and pathogenicity have been identified from F. graminearum. In this review, the secondary metabolite synthesis, hyphal development and pathogenicity related genes in F. graminearum were thoroughly summarized, and the genes associated with secondary metabolites, sexual reproduction, energy metabolism, and pathogenicity were highlighted.     

The Sch9 Kinase Regulates Conidium Size,Stress Responses,and Pathogenesis in Fusarium graminearum

Fusarium head blight caused by Fusarium graminearum is an important disease of wheat and barley worldwide. In a previous study on functional characterization of the F. graminearum kinome, one protein kinase gene important for virulence is orthologous to SCH9 that is functionally related to the cAMP-PKA and TOR pathways in the budding yeast. In this study, we further characterized the functions of FgSCH9 in F. graminearum and its ortholog in Magnaporthe oryzae. The ΔFgsch9 mutant was slightly reduced in growth rate but significantly reduced in conidiation, DON production, and virulence on wheat heads and corn silks. It had increased tolerance to elevated temperatures but became hypersensitive to oxidative, hyperosmotic, cell wall, and membrane stresses. The ΔFgsch9 deletion also had conidium morphology defects and produced smaller conidia. These results suggest that FgSCH9 is important for stress responses, DON production, conidiogenesis, and pathogenesis in F. graminearum. In the rice blast fungus Magnaporthe oryzae, the ΔMosch9 mutant also was defective in conidiogenesis and pathogenesis. Interestingly, it also produced smaller conidia and appressoria. Taken together, our data indicate that the SCH9 kinase gene may have a conserved role in regulating conidium size and plant infection in phytopathogenic ascomycetes.     

The FgHOG1 Pathway Regulates Hyphal Growth,Stress Responses,and Plant Infection in Fusarium graminearum

Fusarium head blight (FHB) caused by Fusarium graminearum is a destructive disease of wheat and barley worldwide. In a previous study of systematic characterization of protein kinase genes in F. graminearum, mutants of three putative components of the osmoregulation MAP kinase pathway were found to have distinct colony morphology and hyphal growth defects on PDA plates. Because the osmoregulation pathway is not known to regulate aerial hyphal growth and branching, in this study we further characterized the functions of the FgHog1 pathway in growth, pathogenesis, and development. The Fghog1, Fgpbs2, and Fgssk2 mutants were all reduced in growth rate, aerial hyphal growth, and hyphal branching angle. These mutants were not only hypersensitive to osmotic stress but also had increased sensitivity to oxidative, cytoplasm membrane, and cell wall stresses. The activation of FgHog1 was blocked in the Fgpbs2 and Fgssk2 mutants, indicating the sequential activation of FgSsk2-FgPbs2-FgHog1 cascade. Interestingly, the FgHog1 MAPK pathway mutants appeared to be sensitive to certain compounds present in PDA. They were female sterile but retained male fertility. We also used the metabolomics profiling approach to identify compatible solutes that were accumulated in the wild type but not in the Fghog1 deletion mutant. Overall, our results indicate that the FgSsk2-FgPbs2-FgHog1 MAPK cascade is important for regulating hyphal growth, branching, plant infection, and hyperosmotic and general stress responses in F. graminearum.     

The feruloyl esterase gene family of Fusarium graminearum is differentially regulated by aromatic compounds and hosts

Feruloyl esterases can liberate ferulic acid (FA) from plant cell wall polymers. They are expressed by plant pathogenic fungi and could play a role in pathogenicity, although this question has not been addressed yet. The fungus Fusarium graminearum is the principal causal agent of fusarium head blight (FHB) and gibberella ear rot (GER), major diseases of wheat, barley, and maize in all temperate regions of the world. The F. graminearum genome contains seven genes with strong homology to feruloyl esterase (FAE) sequences. Phylogenetic analysis showed that these included three type B, three type C, and one type D FAE genes. Expression profiling of the seven FAE genes showed complex regulation patterns unique to each gene. In F. graminearum-infected plant tissues, the FAE genes exhibited host-specific gene expression. On wheat, FAEB1 and FAED1 were strongly expressed while FAEB2, FAEB3, and FAEC1 were expressed at more modest levels. On maize, only FAEB3, FAEC1, and FAED1 were expressed and at low levels. When growing F. graminearum in liquid culture, only FAEB1 and FAEC1 were expressed. Both genes were induced by a small group of related aromatic compounds including FA, caffeic acid, and p-coumaric acid. FAEB1 was induced by xylose, while repressed by glucose and galactose. FAEC1 was constitutively expressed at low levels in the presence of those sugars. Expression of the other five FAE genes was not detected in the culture conditions used. To determine if FAE genes were important for pathogenicity of F. graminearum, mutant strains inactivated for faeB1?, faeD1? or both genes were constructed and tested on wheat plants. No statistically significant change in pathogenicity and no compensatory expression of the other FAE genes were observed in the fae gene mutants. Our results show that FAEB1 and FAED1 are not required for pathogenicity of F. graminearum on wheat.     

Glycinebetaine and choline in wheat: Distribution and relation to infection by Fusarium graminearum

Glycinebetaine and choline, which stimulate the growth of Fusarium graminearum both in vitro and in vivo, are present in higher concentrations in wheat anthers relative to any other organ as measured both by the periodide assay and a bioassay. This finding provides an explanation for the onset of greatly increased susceptibility of wheat to F. graminearum at anthesis.     

Cladistic and phenetic analyses of relationships among Fusarium spp. in Dongtan wetland by morphology and isozymes

Variations of 12 morphological characters and 78 isozymic bands among 78 isolates of five Fusarium spp. from Dongtan wetland were described and analysed with cladistic parsimony and phenetic UPGMA methods. Hierarchical cluster analysis of 12 morphological characters grouped 78 strains into five defined species with a high overlap between isolates. Hierarchical cluster analysis of isozyme patterns showed a higher degree of relationship among five Fusarium spp., in which Fusarium nivale, Fusarium semitectum and Fusarium oxysporum clustered as one group, and F. semitectum was closer to F. nivale than to F. oxysporum; Fusarium graminearum and Fusarium moniliforme formed one group and showed clearly distinct from the first group. Groups of individual isolates indicated by a plot of principal component analysis were consistent with these findings. The comparison of two different data sets revealed that isozyme patterns showed higher variations between species and among individual isolates than morphological characters. Parsimony analysis of morphological characters yielded unresolved cladograms. Parsimony analysis of isozymes as presence/absence characters revealed the same five species in general as the results indicated by phenetic analysis, differing in the relative position of species in subclusters.     

Functional evaluation of a homologue of plant rapid alkalinisation factor (RALF) peptides in Fusarium graminearum

The cereal infecting fungus Fusarium graminearum is predicted to possess a single homologue of plant RALF (rapid alkalinisation factor) peptides. Fusarium mutant strains lacking FgRALF were generated and found to exhibit wildtype virulence on wheat and Arabidopsis floral tissue. Arabidopsis lines constitutively overexpressing FgRALF exhibited no obvious change in susceptibility to F. graminearum leaf infection. In contrast transient virus-mediated over-expression (VOX) of FgRALF in wheat prior to F. graminearum infection, slightly increased the rate of fungal colonisation of floral tissue. Ten putative Feronia (FER) receptors of RALF peptide were identified bioinformatically in hexaploid wheat (Triticum aestivum). Transient silencing of two wheat FER homoeologous genes prior to F. graminearum inoculation did not alter the subsequent interaction outcome. Collectively, our VOX results show that the fungal RALF peptide may be a minor contributor in F. graminearum virulence but results from fungal gene deletion experiments indicate potential functional redundancy within the F. graminearum genome. We demonstrate that virus-mediated over-expression is a useful tool to provide novel information about gene/protein function when results from gene deletion/disruption experimentation were uninformative.     

Regulation of a novel Fusarium cytokinin in Fusarium pseudograminearum

Fusarium pseudograminearum is an agronomically important fungus, which infects many crop plants, including wheat, where it causes Fusarium crown rot. Like many other fungi, the Fusarium genus produces a wide range of secondary metabolites of which only few have been characterized. Recently a novel gene cluster was discovered in F. pseudograminearum, which encodes production of cytokinin-like metabolites collectively named Fusarium cytokinins. They are structurally similar to plant cytokinins and can activate cytokinin signalling in vitro and in planta. Here, the regulation of Fusarium cytokinin production was analysed in vitro. This revealed that, similar to deoxynivalenol (DON) production in Fusarium graminearum, cytokinin production can be induced in vitro by specific nitrogen sources in a pH-dependent manner. DON production was also induced in both F. graminearum and F. pseudograminearum in cytokinin-inducing conditions. In addition, microscopic analyses of wheat seedlings infected with a F. pseudograminearum cytokinin reporter strain showed that the fungus specifically induces its cytokinin production in hyphae, which are in close association with the plant, suggestive of a function of Fusarium cytokinins during infection.     

Deodorization of pig slurry and characterization of bacterial diversity using 16S rDNA sequence analysis

Fusarium graminearum is a filamentous fungal plant pathogen that infects major cereal crops. The fungus produces both sexual and asexual spores in order to endure unfavorable environmental conditions and increase their numbers and distribution across plants. In a model filamentous fungus, Aspergillus nidulans, early induction of conidiogenesis is orchestrated by the fluffy genes. The objectives of this study were to characterize fluffy gene homologs involved in conidiogenesis and their mechanism of action in F. graminearum. We characterized five fluffy gene homologs in F. graminearum and found that FlbD is the only conserved regulator for conidiogenesis in A. nidulans and F. graminearum. Deletion of fgflbD prevented hyphal differentiation and the formation of perithecia. Successful interspecies complementation using A. nidulans flbD demonstrated that the molecular mechanisms responsible for FlbD functions are conserved in F. graminearum. Moreover, abaA-wetA pathway is positively regulated by FgFlbD during conidiogenesis in F. graminearum. Deleting fgflbD abolished morphological effects of abaA overexpression, which suggests that additional factors for FgFlbD or an AbaA-independent pathway for conidiogenesis are required for F. graminearum conidiation. Importantly, this study led to the construction of a genetic pathway of F. graminearum conidiogenesis and provides new insights into the genetics of conidiogenesis in fungi.     

Relocation of genes generates non-conserved chromosomal segments in Fusarium graminearum that show distinct and co-regulated gene expression patterns

Background

Genome comparisons between closely related species often show non-conserved regions across chromosomes. Some of them are located in specific regions of chromosomes and some are even confined to one or more entire chromosomes. The origin and biological relevance of these non-conserved regions are still largely unknown. Here we used the genome of Fusarium graminearum to elucidate the significance of non-conserved regions.

Results

The genome of F. graminearum harbours thirteen non-conserved regions dispersed over all of the four chromosomes. Using RNA-Seq data from the mycelium of F. graminearum, we found weakly expressed regions on all of the four chromosomes that exactly matched with non-conserved regions. Comparison of gene expression between two different developmental stages (conidia and mycelium) showed that the expression of genes in conserved regions is stable, while gene expression in non-conserved regions is much more influenced by developmental stage. In addition, genes involved in the production of secondary metabolites and secreted proteins are enriched in non-conserved regions, suggesting that these regions could also be important for adaptations to new environments, including adaptation to new hosts. Finally, we found evidence that non-conserved regions are generated by sequestration of genes from multiple locations. Gene relocations may lead to clustering of genes with similar expression patterns or similar biological functions, which was clearly exemplified by the PKS2 gene cluster.

Conclusions

Our results showed that chromosomes can be functionally divided into conserved and non-conserved regions, and both could have specific and distinct roles in genome evolution and regulation of gene expression.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-191) contains supplementary material, which is available to authorized users.     

Sharing a Host Plant (Wheat [Triticum aestivum]) Increases the Fitness of Fusarium graminearum and the Severity of Fusarium Head Blight but Reduces the Fitness of Grain Aphids (Sitobion avenae)

We hypothesized that interactions between fusarium head blight-causing pathogens and herbivores are likely to occur because they share wheat as a host plant. Our aim was to investigate the interactions between the grain aphid, Sitobion avenae, and Fusarium graminearum on wheat ears and the role that host volatile chemicals play in mediating interactions. Wheat ears were treated with aphids and F. graminearum inoculum, together or separately, and disease progress was monitored by visual assessment and by quantification of pathogen DNA and mycotoxins. Plants exposed to both aphids and F. graminearum inoculum showed accelerated disease progression, with a 2-fold increase in disease severity and 5-fold increase in mycotoxin accumulation over those of plants treated only with F. graminearum. Furthermore, the longer the period of aphid colonization of the host prior to inoculation with F. graminearum, the greater the amount of pathogen DNA that accumulated. Headspace samples of plant volatiles were collected for use in aphid olfactometer assays and were analyzed by gas chromatography-mass spectrometry (GC-MS) and GC-coupled electroantennography. Disease-induced plant volatiles were repellent to aphids, and 2-pentadecanone was the key semiochemical underpinning the repellent effect. We measured aphid survival and fecundity on infected wheat ears and found that both were markedly reduced on infected ears. Thus, interactions between F. graminearum and grain aphids on wheat ears benefit the pathogen at the expense of the pest. Our findings have important consequences for disease epidemiology, because we show increased spread and development of host disease, together with greater disease severity and greater accumulation of pathogen DNA and mycotoxin, when aphids are present.     

Evaluation of Fusarium head blight in barley infected by Fusarium graminearum

Fusarium head blight, which is primarily caused by Fusarium graminearum, is a devastating disease in the barley field. A real-time PCR protocol was developed to evaluate the growth of this pathogen in the host plant tissues. All four strains harbored the gene encoding ATP-BINDING CASSETTE TRANSPORTER (FgABC; FGSG_00541) as a single copy within their genomes. Our Southern blot result was identical with the genomic data for F. graminearum strain PH-1. Based on the crossing point (CP) values obtained in our TaqMan real-time PCR analysis, two standard curves describing the relationship among the CP value, FgABC copy number, and amount of fungal DNA were constructed. Chronological enumeration of fungal growth was coincided with the symptom development.     

Analysis of simple sequence repeats (SSRs)dynamics in fungus Fusarium graminearum

The abundance and inherent potential for variations in simple sequence repeats (SSRs) or microsatellites resulted in valuable source for genetic markers in eukaryotes. We describe the organization and abundance of SSRs in fungus Fusarium graminearum (causative agent for Fusarium head blight or head scab of wheat). We identified 1705 SSRs of various nucleotide repeat motifs in the sequence database of F. graminearum. It is observed that mononucleotide repeats (62%) were most abundant followed by di- (20%) and trinucleotide repeats (14%). It is noted that tetra-, penta- and hexanucleotide repeats accounted for only 4% of SSRs. The estimated frequency of Class I SSRs (perfect repeats ≥20 nucleotides) was one SSR per 124.5 kb, whereas the frequency of Class II (perfect repeats >10 nucleotides and ≫20 nucleotides) was one SSR per 25.6 kb. The dynamics of SSRs will be a powerful tool for taxonomic, phylogenetic, genome mapping and population genetic studies as SSR based markers show high levels of allelic variation, codominant inheritance and ease of analysis.     

Production of trichothecene mycotoxins byFusarium graminearum andFusarium culmorum on barley and wheat

Wheat cultivars (Stoa, MN87150, SuMai-3, YMI-6, Wheaton) and barley cultivars (Robust, Excel, Chevron, M69) were inoculated in the field with isolates ofFusarium graminearum andF. culmorum. The diseased (Fusarium head blight) kernels were analyzed for deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON) and nivalenol (NIV).F. culmorum produced all three trichothecenes on all cultivars tested whereasF. graminearum only produced DON and 15-ADON. There was no well defined correlation between DON production in the host and resistance although the data tended to favor SuMai-3 as having definitive resistance to bothF. graminearum andF. culmorum.Minnesota Agricultural Experiment Station, Paper No. 20 279.     

Identification of a biocontrol agent Bacillus vallismortis BV23 and assessment of effects of its metabolites on Fusarium graminearum causing corn stalk rot

Corn stalk rot, caused by Fusarium graminearum, is one of the most destructive diseases of maize in many regions of the world. A bacterial strain BV23 was isolated from corn rhizosphere that reduced corn stalk rot significantly in greenhouse studies in 2016 and 2017. BV23 was identified as Bacillus vallismortis, which showed antagonistic effects against a number of fungal pathogens, including F. graminearum, Rhizoctonia solani, Athelia rolfsii, and Thanatephorus cucumeris. BV23 had the greatest fungistatic effect on F. graminearum, inhibiting mycelial growth by 66.2%, conidial germination by 90.1%, and conidial production by 86.7%. The probable antifungal mechanism was assessed by examining the morphology and ultrastructure of F. graminearum hyphae. Treatment by BV23 culture supernatant resulted in coarser hyphae, induced cytoplasmic granulation, and increased cell membrane permeability of F. graminearum, causing cytoplasm leakage. These effects became increasingly obvious with increasing concentration (1%, 5% and 10%). Furthermore, the antifungal active substances were sensitive to heat.