The transcription factor FgMed1 is involved in early conidiogenesis and DON biosynthesis in the plant pathogenic fungus Fusarium graminearum.

Fusarium graminearum is a prominent fungal pathogen that causes economically important losses by infesting a wide variety of cereal crops. F. graminearum produces both asexual and sexual spores which disseminate and inoculate hosts. Therefore, to better understand the disease cycle and to develop strategies to improve disease management, it is important to further clarify molecular mechanisms of F. graminearum conidiogenesis. In this study, we functionally characterized the FgMed1, a gene encoding an ortholog of a conserved MedA transcription factor known to be a key conidiogenesis regulator in Aspergillus nidulans. The gene deletion mutants ΔFgMed1 produced significantly less conidia, and these were generated from abnormal conidiophores devoid of phialides. Additionally, we observed defective sexual development along with reduced virulence and deoxynivalenol (DON) production in ΔFgMed1. The GFP-tagged FgMed1 protein localized to the nuclei of conidiophores and phialides during early conidiogenesis. Significantly, RNA-Seq analyses showed that a number of the conidiation- and toxin-related genes are differentially expressed in the ΔFgMed1 mutant in early conidiogenesis. These data strongly suggest that FgMed1 involved in regulation of genes associated with early conidiogenesis, DON production, and virulence in F. graminearum.

A phosphorylated transcription factor regulates sterol biosynthesis in Fusarium graminearum.

Sterol biosynthesis is controlled by transcription factor SREBP in many eukaryotes. Here, we show that SREBP orthologs are not involved in the regulation of sterol biosynthesis in Fusarium graminearum, a fungal pathogen of cereal crops worldwide. Instead, sterol production is controlled in this organism by a different transcription factor, FgSR, that forms a homodimer and binds to a 16-bp cis-element of its target gene promoters containing two conserved CGAA repeat sequences. FgSR is phosphorylated by the MAP kinase FgHog1, and the phosphorylated FgSR interacts with the chromatin remodeling complex SWI/SNF at the target genes, leading to enhanced transcription. Interestingly, FgSR orthologs exist only in Sordariomycetes and Leotiomycetes fungi. Additionally, FgSR controls virulence mainly via modulating deoxynivalenol biosynthesis and responses to phytoalexin.

Effects of validamycin in controlling Fusarium head blight caused by Fusarium graminearum: Inhibition of DON biosynthesis and induction of host resistance.

Validamycin, known to interfere with fungal energy metabolism by inhibiting trehalase, has been extensively used to control plant diseases caused by Rhizoctonia spp. However, the effect of validamycin on controlling Fusarium graminearum has not been previously reported. In this study, when applied to F. graminearum in vitro, validamycin inhibited the synthesis of deoxynivalenol (DON), which is a mycotoxin and virulence factor, by decreasing trehalase activity and the production of glucose and pyruvate, which are precursors of DON biosynthesis. Because FgNTH encodes the main trehalase in F. graminearum, these effects were nullified in the FgNTH deletion mutant ΔFgNTH but restored in the complemented strain ΔFgNTHC. In addition, validamycin also increased the expression of pathogenesis-related genes (PRs) PR1, PR2, and PR5 in wheat, inducing resistance responses of wheat against F. graminearum. Therefore, validamycin exhibits dual efficacies on controlling Fusarium head blight (FHB) caused by F. graminearum: inhibition of DON biosynthesis and induction of host resistance. In addition, field trials further confirmed that validamycin increased FHB control and reduced DON contamination in grain. Control of FHB and DON contamination by validamycin increased when the antibiotic was applied with the triazole fungicide metconazole. Overall, this study is a successful case from foundational research to applied research, providing useful information for wheat protection programs against toxigenic fungi responsible for FHB and the consequent mycotoxin accumulation in grains.

The molecular link between tyrosol binding to tri6 transcriptional regulator and downregulation of trichothecene biosynthesis.

Tri cluster is responsible for the biosynthesis of trichothecenes in Trichoderma spp. Tri6 gene present within the cluster encodes for a transcriptional regulator and is vital for the expression of all other tri genes of the cluster. Tri6 encodes for a 218-amino-acid residues long protein which contains three zinc finger motifs. Tri6 is able to regulate and bind within the GTGA/TCAC promoter region of tri genes. Here we highlight the binding of tri6 with the regulatory DNA element present at the upstream of tri3 gene and effect of two quorum-sensing molecules tyrosol and farnesol on its binding. Analysis showed that tyrosol binds at sequence GTGA/TCAC specific for tri6 binding and thus do not allow tri6 to bind on promoter region. Interactions of tyrosol with zinc finger motif of tri6 protein also resulted in structural changes making tri6 unsuitable for binding with the regulatory DNA element of tri3 gene promoter resulting in downregulation of the gene. Structural changes also resulted in loss of zinc from zinc finger 2 motif. In contrast to the tyrosol, tri6-DNA complex could easily accommodate farnesol molecule without showing any interference with the functional conformation of the tri6-DNA complex. Therefore, tri gene expression seems not to be negatively regulated by the farnesol.

Inhibition of Fusarium trichothecene biosynthesis by yeast extract components extractable with ethyl acetate.

While Fusarium graminearum readily produces trichothecenes in complex media containing sucrose as the carbon source (YS_60), the amount of the mycotoxin is quite limited when other sugars, such as glucose and fructose, are used. We found that autoclaving of media containing fructose and yeast extract (YF_60) results in the formation of inhibitors of trichothecene biosynthesis by F. graminearum JCM 9873, a strain that produces 15-acetyldeoxynivalenol (15-ADON) in liquid culture. Removal of the solvent fraction from the autoclaved media after ethyl acetate extraction attenuated the inhibitory activity against trichothecene production. In addition, extraction of the non-autoclaved complex media with ethyl acetate, followed by removal of the solvent fraction, similarly resulted in increased accumulation of the mycotoxin. Although the increase in trichothecene production differed considerably among fungal strains and yeast extract products, F. graminearum species complex generally responded to the medium treatments in the same way. These results suggest that some hydrophobic substances that arise during the drying and heating of yeast extract negatively affected trichothecene production in liquid culture. Modes of actions of inhibitory substances were partially characterized using strain JCM 9873, with focus on the transcriptional and functional analyses of Tri6, a key regulator gene in trichothecene biosynthesis. The presence of the ethyl acetate-extractable substances in autoclaved YF_60 media decreased the relative transcription level of Tri6, as well as that of a trichodiene synthase gene Tri5. Thus, the substances exerted their inhibitory action through suppression of Tri6 expression. By using a yeast extract lot that completely prevented trichothecene production by the wild-type strain in autoclaved YS_60 medium, we prepared YF_60 media and cultured a constitutive Tri6 overexpressor strain described by Maeda et al. (2018). Despite the high transcription level of Tri6, the presence of the ethyl acetate extractable-substances suppressed 15-ADON production. These results suggested that both Tri6p-independent initial activation of Tri6 expression and subsequent Tri6p-dependent activation of Tri expression were affected by the hydrophobic substances in the yeast extract products.

Identification of amino acids negatively affecting Fusarium trichothecene biosynthesis.

Nitrogen sources in media have a significant impact on the onset of secondary metabolism in fungi. For transcriptional activation of many nitrogen catabolic genes, an AreA transcription factor is indispensable. This also holds true for Fusarium graminearum that produces trichothecenes, an important group of mycotoxin, in axenic culture. Despite the presence of numerous consensus AreA-binding sites in the promoters of Tri genes in the trichothecene cluster core region, the effect of medium amino acids on trichothecene biosynthesis is poorly understood. In this study, we examined the effect of certain amino acids, which were predicted to activate AreA function and increase Tri gene transcription, on trichothecene production in liquid culture. By frequent monitoring and adjustments in the pH of the culture medium, including replacement of the spent medium with fresh medium, we demonstrate the suppressive effects of the amino acids, used as the sole nitrogen source, on trichothecene biosynthesis. When the medium pH was maintained at 4.0, Gly, L-Ser, and L-Thr suppressed trichothecene production by F. graminearum. Enhanced trichothecene-inducing effects were observed when the medium pH was 3.5, with only L-Thr suppressing trichothecene synthesis.

The transcription factor FgCrz1A is essential for fungal development, virulence, deoxynivalenol biosynthesis and stress responses in Fusarium graminearum.

The zinc finger transcription factor Crz1 is an important downstream regulator of calcium-dependent signal transduction pathways in many organisms. The function of Crz1 in the wheat-head blight pathogen Fusarium graminearum remains unclear. In this study, we identified and functionally characterised FgCrz1A, a potential ortholog of yeast Crz1. The deletion mutant ΔFgCrz1A exhibited slower hyphal growth on basic medium, and conidia formation and sexual reproduction were completely blocked. ΔFgCrz1A also displayed increased sensitivity to metal cations Ca2+, Mg2+, Mn2+ and Li+, but decreased sensitivity to Zn2+. Unexpectedly, the deletion mutant was more resistant to osmotic stress and cell wall-damaging agents than the wild-type fungus. Pathogenicity assays showed that virulence of the mutant was dramatically decreased on flowering wheat heads and corn silks, consistent with the observed reduction in deoxynivalenol production. Moreover, GFP-fused FgCrz1A was mainly localised in the nucleus, and was required for transcriptional induction of abaA and wetA that are involved in conidiogenesis, as well as genes of the MAT locus during sexual reproduction, and TRI genes responsible for deoxynivalenol biosynthesis. Taken together, the results indicate that FgCrz1A plays critical roles not only in regulating fungal development, secondary metabolism and virulence in F. graminearum, but also in multiple stress responses.

Requirement of Two Acyltransferases for 4- O-Acylation during Biosynthesis of Harzianum A, an Antifungal Trichothecene Produced by Trichoderma arundinaceum.

Trichothecenes are sesquiterpenoid toxins produced by multiple fungi, including plant pathogens, entomopathogens, and saprotrophs. Most of these fungi have the acyltransferase-encoding gene tri18. Even though its function has not been determined, tri18 is predicted to be involved in trichothecene biosynthesis because of its pattern of expression and its location near other trichothecene biosynthetic genes. Here, molecular genetic, precursor feeding, and analytical chemistry experiments indicate that in the saprotroph Trichoderma arundinaceum the tri18-encoded acyltransferase (TRI18) and a previously characterized acyltransferase (TRI3) are required for conversion of the trichothecene biosynthetic intermediate trichodermol to harzianum A, an antifungal trichothecene analog with an octa-2,4,6-trienedioyl acyl group. On the basis of the results, we propose that TRI3 catalyzes trichothecene 4- O-acetylation, and subsequently, TRI18 catalyzes replacement of the resulting acetyl group with octa-2,4,6-trienedioyl to form harzianum A. Thus, the findings provide evidence for a previously unrecognized two-step acylation process during trichothecene biosynthesis in T. arundinaceum and possibly other fungi.

Resistance-Related l-Pyroglutamic Acid Affects the Biosynthesis of Trichothecenes and Phenylpropanoids by F. graminearum Sensu Stricto.

Fungicide application remains amongst the most widely used methods of fungal control in agroecosystems. However, the extensive use of fungicides poses hazards to human health and the natural environment and does not always ensure the effective decrease of mycotoxins in food and feed. Nowadays, the rising threat from mycotoxin contamination of staple foods has stimulated efforts in developing alternative strategies to control plant pathogenic fungi. A substantial effort is focused on the identification of plant-derived compounds inhibiting mycotoxin production by plant pathogenic fungi. l-Pyroglutamic acid has recently been suggested as playing a role in the response of barley to toxigenic Fusaria. Considering the above, we studied the response of various strains of F. graminearum sensu stricto to different levels of l-pyroglutamic acid on solid YES (yeast extract sucrose) media. l-Pyroglutamic acid decreased the accumulation of trichothecenes in all examined strains. Gene expression studies addressing Tri genes (Tri4, Tri5, and Tri10), which induce the biosynthesis of trichothecenes, revealed the production of mycotoxins by l-pyroglutamic acid to be inhibited at the transcriptional level. Besides inhibitory effects on mycotoxin production, l-pyroglutamic acid exhibited variable and concentration-related effects on phenylpropanoid production by fungi. Accumulation of most of the fungal-derived phenolic acids decreased in the presence of 100 and 400 µg/g of l-pyroglutamic acid. However, a higher dose (800 µg/g) of l-pyroglutamic acid increased the accumulation of trans-cinnamic acid in the media. The accumulation of fungal-derived naringenin increased in the presence of l-pyroglutamic acid. Contrasting results were obtained for quercetin, apigenin, luteolin, and kaempferol, the accumulation of which decreased in the samples treated with 100 and 400 µg/g of l-pyroglutamic acid, whereas the highest l-pyroglutamic acid concentration (800 µg/g) seemed to induce their biosynthesis. The results obtained in this study provide new insights for breeders involved in studies on resistance against Fusaria.

Effect of Zingiber officinale Roscoe essential oil in fungus control and deoxynivalenol production of Fusarium graminearum Schwabe in vitro.

Members of the Fusarium genus are capable of contaminating agricultural commodities, compromising the quality of maize and other grains, which leads to severe quality and yield losses. Contamination with mycotoxins is also a concern. Essential oils are possible alternatives to the use of synthetic pesticides for control of fungal contamination, as many have antifungal and anti-mycotoxigenic properties and are innocuous to human health. They also do not cause any sort of microbial resistance and do not promote environmental pollution. The aim of this study was to evaluate the antifungal and anti-mycotoxigenic effects of Zingiber officinale Roscoe essential oil (GEO) upon Fusarium graminearum Schwabe in vitro. The essential oil was extracted by hydrodistillation and analysed by GC/MS. Antifungal and anti-mycotoxigenic activities were assessed by HPLC/UV by quantifying ergosterol and deoxynivalenol (DON), respectively. Results indicated that GEO inhibited ergosterol production at a concentration of 1000 µg/mL and DON production at a concentration of 500 µg/mL, evidencing that the anti-mycotoxigenic effect is independent of the antifungal effect due to its probable direct action upon toxin biosynthesis.

Involvement of a dihydrodipicolinate synthase gene (FaDHDPS1) in fungal development, pathogenesis and stress responses in Fusarium asiaticum.

BACKGROUND: Dihydrodipicolinate synthase (DHDPS) is an allosteric enzyme, which catalyzes the first unique step of lysine biosynthesis in prokaryotes, higher plants and some fungi. To date, the biological roles of DHDPS in filamentous fungi are poorly understood. RESULTS: In this study, on the basis of comparative genome resequencing, a DHDPS gene was found to be specific in Fusarium asiaticum, named FaDHDPS1, which showed high amino acid identity to that of entomopathogenic fungus. Subcellular localization of the FaDHDPS1-GFP fusion protein was mainly concentrated in the cytoplasm of conidia and dispersed in the cytoplasm during conidial germination. To reveal the biological functions, both deletion and complementation mutants of FaDHDPS1 were generated. The results showed that the FaDHDPS1 deletion mutant was defective in conidiation, virulence and DON biosynthesis. In addition, deletion of FaDHDPS1 resulted in tolerance to sodium pyruvate, lysine, low temperature and Congo red. CONCLUSION: Results of this study indicate that FaDHDPS1 plays an important role in the regulation of vegetative differentiation, pathogenesis and adaption to multiple stresses in F. asiaticum.

Effect of temperature on growth, wheat head infection, and nivalenol production by Fusarium poae.

Fusarium poae is one of the Fusarium species commonly detected in wheat kernels affected by Fusarium Head Blight. Fusarium poae produces a wide range of mycotoxins including nivalenol (NIV). The effect of temperature on colony growth and NIV production was investigated in vitro at 5-40 °C with 5 °C intervals. When the data were fit to a Beta equation (R2 ≥ 0.97), the optimal temperature was estimated to be 24.7 °C for colony growth and 27.5 °C for NIV production. The effects of temperature on infection incidence, fungal biomass, and NIV contamination were investigated by inoculating potted durum wheat plants at full anthesis; inoculated heads were kept at 10-40 °C with 5 °C intervals for 3 days and then at ambient temperature until ripening. Temperature significantly affected the incidence of floret infection and fungal biomass (as indicated by DNA amount) in the affected heads but did not affect NIV content in the head tissue. Inoculation of potted plants with F. poae did not reduce yield.

FgSec2A, a guanine nucleotide exchange factor of FgRab8, is important for polarized growth, pathogenicity and deoxynivalenol production in Fusarium graminearum.

Sec4/Rab8 is one of the well-studied members of the Rab GTPase family, previous studies have shown that Sec4/Rab8 crucially promotes the pathogenesis of phytopathogens, but the upstream regulators of Rab8 are still unknown. Here, we have identified two Sec2 homologues FgSec2A and FgSec2B in devastating fungal pathogen Fusarium graminearum and investigated their functions and interactions with FgRab8 by live-cell imaging, genetic and functional analyses. Yeast two-hybrid assay shows that FgSec2A specifically interacts with FgRab8DN(N123I) and itself. Importantly, FgSec2A is required for growth, conidiation, DON production and virulence of F. graminearum. Live-cell imaging shows that FgSec2A and FgSec2B are both localized to the tip region of hyphae and conidia. Both N-terminal region and Sec2 domain of FgSec2A are essential for its function, but not for localization, whereas the C-terminal region is important for its polarized localization. Furthermore, constitutively active FgRab8CA(Q69L) partially rescues the defects of ΔFgsec2A. Consistently, FgSec2A is required for the polarized localization of FgRab8. Finally, FgSec2A and FgSec2B show partial functions, but FgSec2A does not interact and co-localize with FgSec2B. Taken together, these results indicate that FgSec2A acts as a FgRab8 guanine nucleotide exchange factor and is necessary for polarized growth, DON production and pathogenicity in F. graminearum.

Effect of water activity and temperature on growth and trichothecene production by Fusarium meridionale.

Fusarium meridionale has been frequently isolated from soybean in Argentina and showed similar pathogenicity as F. graminearum sensu stricto. However, no data on their growth and mycotoxin production under different environmental conditions are yet available. The aims of this study were: to determine the effect of temperature, water activity (aW) and strain on growth of F. meridionale and to evaluate deoxynivalenol (DON) and nivalenol (NIV) production in a soybean based medium. The results showed that optimal conditions for F. meridionale growth were at 25 °C and 0.98-0.99 aW. Deoxynivalenol production was favored at 25 °C and 0.96 aW while NIV production was strain-dependent, being 30 °C and 0.98 aW optimal conditions for F. meridionale B2300 strain and 20 °C and 0.98 aW for F. meridionale F5043 and F. meridionale 5048 strains. These conditions are similar to those observed at pre-harvest stage in soybean crop, thus control strategies need to be considered to reduce the risk of the occurrence of DON and NIV in harvested grains.

The Fungicidal Activity of Tebuconazole Enantiomers against Fusarium graminearum and Its Selective Effect on DON Production under Different Conditions.

Tebuconazole, which consists of a pair of enantiomers with different fungicidal activities, is one of the most common fungicides used in the control of Fusarium graminearum. In this study, the fungicidal activity of rac-tebuconazole and its enantiomers against F. graminearum was determined at 0.997, 0.975, and 0.950 aw and at 20, 25, and 30 °C on wheat-based media. Then, F. graminearum was treated with rac-tebuconazole and its enantiomers at the EC10, EC50, and EC90 levels under different culture conditions, and DON production was measured. Finally, expression of the DON biosynthetic genes ( TRI5 and TRI6) was quantified by real-time RT-PCR after incubation with EC50 doses of rac-tebuconazole and its enantiomers for 4, 8, and 14 days at 30 °C and aw 0.997. The results showed that the fungicidal activity of tebuconazole was strongly influenced by temperature, aw, and the combined factors. (-)-Tebuconazole is higher in fungicidal activity than (+)-tebuconazole and rac-tebuconazole with 24-99-fold and 1.8-6.7-fold, respectively. However, (-)-tebuconazole was generally more favorable for DON production than (+)-tebuconazole under the same conditions. Additionally, (-)-tebuconazole and rac-tebuconazole induced significantly increased expression of the DON biosynthetic genes ( TRI5 and TRI6) compared to the control by the 14th day of treatment. In this research, the combination condition of 30 °C and 0.997 aw is the most suitable for DON production by F. graminearum. The test strains of F. graminearum treated with the EC10 dose of (-)-tebuconazole produced the greatest amounts of DON.

Identification and Characterization of Small Molecule Compounds That Modulate Trichothecene Production by Fusarium graminearum.

From the RIKEN Natural Products Depository (NPDepo) chemical library, we identified small molecules that alter trichothecene 15-acetyldeoxynivalenol (15-ADON) production by Fusarium graminearum. Among trichothecene production activators, a furanocoumarin NPD12671 showed the strongest stimulatory activity on 15-ADON production by the fungus cultured in a 24-well plate. NPD12671 significantly increased the transcription of Tri6, a transcription factor gene necessary for trichothecene biosynthesis, in both trichothecene-inducing and noninducing culture conditions. Dihydroartemisinin (DHA) was identified as the most effective inhibitor of trichothecene production in 24-well plate culture; DHA inhibited trichothecene production (>50% inhibition at 1 µM) without affecting fungal mass by suppressing Tri6 expression. To determine the effect of DHA on trichothecene pathway Tri gene expression, we generated a constitutively Tri6-overexpressing strain that produced 15-ADON in YG_60 medium in Erlenmeyer flasks, conditions under which no trichothecenes are produced by the wild-type. While 5 µM DHA failed to inhibit trichothecene biosynthesis by the overexpressor in trichothecene-inducing YS_60 culture, trichothecene production was suppressed in the YG_60 culture. Regardless of a high Tri6 transcript level in the constitutive overexpressor, the YG_60 culture showed reduced accumulation of Tri5 and Tri4 mRNA upon treatment with 5 µM DHA. Deletion mutants of FgOs2 were also generated and examined; both NPD12671 and DHA modulated trichothecene production as they did in the wild-type strain. These results are discussed in light of the mode of actions of these chemicals on trichothecene biosynthesis.

Population genomics of Fusarium graminearum reveals signatures of divergent evolution within a major cereal pathogen.

The cereal pathogen Fusarium graminearum is the primary cause of Fusarium head blight (FHB) and a significant threat to food safety and crop production. To elucidate population structure and identify genomic targets of selection within major FHB pathogen populations in North America we sequenced the genomes of 60 diverse F. graminearum isolates. We also assembled the first pan-genome for F. graminearum to clarify population-level differences in gene content potentially contributing to pathogen diversity. Bayesian and phylogenomic analyses revealed genetic structure associated with isolates that produce the novel NX-2 mycotoxin, suggesting a North American population that has remained genetically distinct from other endemic and introduced cereal-infecting populations. Genome scans uncovered distinct signatures of selection within populations, focused in high diversity, frequently recombining regions. These patterns suggested selection for genomic divergence at the trichothecene toxin gene cluster and thirteen additional regions containing genes potentially involved in pathogen specialization. Gene content differences further distinguished populations, in that 121 genes showed population-specific patterns of conservation. Genes that differentiated populations had predicted functions related to pathogenesis, secondary metabolism and antagonistic interactions, though a subset had unique roles in temperature and light sensitivity. Our results indicated that F. graminearum populations are distinguished by dozens of genes with signatures of selection and an array of dispensable accessory genes, suggesting that FHB pathogen populations may be equipped with different traits to exploit the agroecosystem. These findings provide insights into the evolutionary processes and genomic features contributing to population divergence in plant pathogens, and highlight candidate genes for future functional studies of pathogen specialization across evolutionarily and ecologically diverse fungi.

Tolerance of triazole-based fungicides by biocontrol agents used to control Fusarium head blight in wheat in Argentina.

Fusarium head blight (FHB) caused by Fusarium graminearum species complex is a devastating disease that causes extensive yield and quality losses to wheat around the world. Fungicide application and breeding for resistance are among the most important tools to counteract FHB. Biological control is an additional tool that can be used as part of an integrated management of FHB. Bacillus velezensisRC 218, Brevibacillus sp. RC 263 and Streptomyces sp. RC 87B were selected by their potential to control FHB and deoxynivalenol production. The aim of this work was to test the tolerance of these biocontrol agents to triazole-based fungicides such as prothioconazole, tebuconazole and metconazole. Bacterial growth was evaluated in Petri dishes using the spread plating technique containing the different fungicides. Bacillus velezensisRC 218 and Streptomyces sp. RC 87B showed better tolerance to fungicides than Brevibacillus sp. RC 263. Complete growth inhibition was observed at concentrations of 20 µg ml-1 for metconazole, 40 µg ml-1 for tebuconazole and 80 µg ml-1 for prothioconazole. The results obtained indicate the possibility of using these biocontrol agents in combination with fungicides as part of an integrated management to control FHB of wheat. SIGNIFICANCE AND IMPACT OF THE STUDY: This study evaluates the possibility to use biocontrol agents (Bacillus velezensisRC 218, Brevibacillus sp. RC 263 and Streptomyces sp. RC 87B) in combination with triazole-based fungicides to control Fusarium head blight in wheat. The evaluation of biocontrol agents' growth under in vitro conditions was carried out in Petri dishes containing either prothioconazole, tebuconazole or metconazole. Viability studies demonstrated that B. velezensisRC 218 and Streptomyces sp. RC 87B were more tolerant to the fungicides evaluated. Results obtained reflect the possibility to use fungicides at low doses combined with biocontrol agents.

A subunit of the HOPS endocytic tethering complex, FgVps41, is important for fungal development and plant infection in Fusarium graminearum.

The signals by which eukaryotic cells communicate with the environment are usually mediated by vesicle trafficking to be attenuated or terminated. However, vesicle trafficking-mediated signal transmission during interactions between pathogens and host plants is poorly understood. Here, we identified and characterized the vacuole sorting protein FgVps41, which is the yeast HOPS tethering complex subunit Vps41 homolog in Fusarium graminearum. Targeted gene deletion demonstrated that FgVps41 is important for vegetative growth, asexual/sexual development, conidial morphology, plant infection and deoxynivalenol production. Cellular localization and cytological examinations revealed that FgVps41 localizes to early/late endosomes and vacuole membrane, and is recruited to prevacuolar compartments and vacuole membrane by interacting with FgRab7 in F. graminearum. Furthermore, we found FgVps41 mediates vacuole membrane fusion and sorting of FgApeI, a cargo protein involving in the cytosol-to-vacuole targeting pathway. In addition, we found that FgVps41 interacts with FgYck3, a vacuolar type I casein kinase, which regulates vesicle fusion in the AP-3 pathway. Deletion of FgYck3 showed similar phenotypes to the ΔFgvps41 mutant, and both FgRab7 and FgYck3 regulate the normal localization of FgVps41. Collectively, our results demonstrate that FgVps41 acts as a HOPS tethering complex subunit and is important for the development of infection-related morphogenesis in F. graminearum.

The 5-oxoprolinase is required for conidiation, sexual reproduction, virulence and deoxynivalenol production of Fusarium graminearum.

In eukaryotic organisms, the 5-oxoprolinase is one of the six key enzymes in the γ-glutamyl cycle that is involved in the biosynthetic pathway of glutathione (GSH, an antioxidative tripeptide counteracting the oxidative stress). To date, little is known about the biological functions of the 5-oxoprolinase in filamentous phytopathogenic fungi. In this study, we investigated the 5-oxoprolinase in Fusarium graminearum for the first time. In F. graminearum, two paralogous genes (FgOXP1 and FgOXP2) were identified to encode the 5-oxoprolinase while only one homologous gene encoding the 5-oxoprolinase could be found in other filamentous phytopathogenic fungi or Saccharomyces cerevisiae. Deletion of FgOXP1 or FgOXP2 in F. graminearum led to significant defects in its virulence on wheat. This is likely caused by an observed decreased deoxynivalenol (DON, a mycotoxin) production in the gene deletion mutant strains as DON is one of the best characterized virulence factors of F. graminearum. The FgOXP2 deletion mutant strains were also defective in conidiation and sexual reproduction while the FgOXP1 deletion mutant strains were normal for those phenotypes. Double deletion of FgOXP1 and FgOXP2 led to more severe defects in conidiation, DON production and virulence on plants, suggesting that both FgOXP1 and FgOXP2 play a role in fungal development and plant colonization. Although transformation of MoOXP1into ΔFgoxp1 was able to complement ΔFgoxp1, transformation of MoOXP1 into ΔFgoxp2 failed to restore its defects in sexual development, DON production and pathogenicity. Taken together, these results suggest that FgOXP1 and FgOXP2 are likely to have been functionally diversified and play significant roles in fungal development and full virulence in F. graminearum.