Fumonisin Biosynthesis Research Articles

Characterisation of sorghum cultivated in Hungary: potential health risk resulting from the occurrence of toxigenic fungi and effect of endogenous lipophilic antioxidants on mycotoxin production

Abstract Fusarium grain mould, or FGM, is caused by Fusarium species that most frequently infect sorghum during the early stages of grain development. Several toxigenic Fusarium species have been shown to be involved in FGM, the most frequently isolated being Fusarium verticillioides, Fusarium andiyazi, Fusarium proliferatum, Fusarium thapsinum, and Fusarium sacchari. The aim of the present study was to provide new insights on the occurrence of toxigenic fungi infecting sorghum cultivated in Hungary and the risk of contamination with mycotoxins. To reach this objective, the presence of toxigenic Fusarium isolates in sorghum samples was investigated; fungal species and capacity to produce mycotoxins were characterised for each isolated Fusarium strain. In a second step, the profile in tocochromanols and carotenoids of the two most widely cultivated sorghum varieties in Hungary was detailed and the effect of the predominant compounds on the biosynthesis of fumonisins and type B trichothecenes was investigated.

FvOshC Is a Key Global Regulatory Target in Fusarium verticillioides for Fumonisin Biosynthesis and Disease Control.

Fusarium verticillioides has a substantial impact on maize production, commonly leading to maize ear rot and the production of fumonisin, a mycotoxin that poses health risks to both humans and animals. Currently, there is a lack of molecular targets for preventing the disease and controlling the toxin. The biological functions of oxysterol-binding proteins (OSBP) in filamentous fungi remain unclear. In this research, 7 oxysterol-binding protein-related proteins were identified in F. verticillioides, and these proteins were obtained through prokaryotic expression and purification. FvOshC was identified as the specific protein that binds to ergosterol through fluorescence titration. Gene knockout complementation techniques confirmed that FvOSHC plays a positive role, establishing it as a novel global regulatory protein involved in the pathogenicity and FB1 biosynthesis in F. verticillioides. Additionally, the interaction between FvOshC and FvSec14 was identified using yeast two-hybrid techniques. Moreover, computer-aided drug design technology was utilized to identify the receptor molecule Xanthatin based on FvOshC. The inhibitory effect of Xanthatin on the growth of F. verticillioides and the synthesis of FB1 was significantly demonstrated. These findings provide valuable insights that can aid in the management of mycotoxin pollution.

Development of an Efficient CRISPR/Cas9 System in Fusarium verticillioides and Its Application in Reducing Mycotoxin Contamination.

Fusarium verticillioides (F. verticillioides) is a globally recognized and highly impactful fungal pathogen of maize, causing yield losses and producing harmful mycotoxins that pose a threat to human and animal health. However, the genetic tools available for studying this crucial fungus are currently limited in comparison to other important fungal pathogens. To address this, an efficient CRISPR/Cas9 genome editing system based on an autonomously replicating plasmid with an AMA1 sequence was established in this study. First, gene disruption of pyrG and pyrE via nonhomologous end-joining (NHEJ) pathway was successfully achieved, with efficiency ranging from 66 to 100%. Second, precise gene deletions were achieved with remarkable efficiency using a dual sgRNA expression strategy. Third, the developed genome editing system can be applied to generate designer chromosomes in F. verticillioides, as evidenced by the deletion of a crucial 38 kb fragment required for fumonisin biosynthesis. Fourth, the pyrG recycling system has been established and successfully applied in F. verticillioides. Lastly, the developed ΔFUM1 and ΔFUM mutants can serve as biocontrol agents to reduce the fumonisin B1 (FB1) contamination produced by the toxigenic strain. Taken together, these significant advancements in genetic manipulation and biocontrol strategies provide valuable tools for studying and mitigating the impact of F. verticillioides on maize crops.

Incidence of fungal contamination in fresh ginseng samples and mycotoxigenic potential of representative fungal isolates.

Fresh ginseng is typically accompanied by soil after harvest, leading to contamination with harmful fungi during storage and distribution. In this study, we investigated the incidence of fungal contamination in fresh ginseng (5-6 years old) purchased from 22 different stores in Geumsan, Korea. The incidence of fungal contamination in the samples was 67.4-111.5%. Fusarium solani was the most abundant species in the head (38.5%) and fine root (19.3%) parts of the ginseng samples, whereas F. oxysporum was the most abundant in the main root (22.0%) part. We isolated Aspergillus, Fusarium and Penicillium spp. (total number of isolates: 395) from the ginseng samples, and 138 isolates were identified using phylogenetic analysis. Polymerase chain reaction-based screening of 65 mycotoxin-producing species revealed that two P. expansum isolates were positive for citrinin and/or patulin, and five F. oxysporum isolates were positive for fumonisin biosynthesis gene. One P. expansum isolate produced 738.0 mg kg-1 patulin, and the other produced 10.4 mg kg-1 citrinin and 12.0 mg kg-1 patulin on potato dextrose agar (PDA) medium. Among the 47 representative F. oxysporum isolates, 43 (91.5%) produced beauvericin (0.1-15.4 mg kg-1) and four of them (8.5%) produced enniatin B and enniatin B1 (0.1-1.8 mg kg-1) as well. However, none of these toxins was detected in fresh ginseng samples. Fusarium solani and F. oxysporum were the most abundant species in fresh ginseng samples. Most F. oxysporum (43) and P. expansum (2) strains isolated from fresh ginseng produced beauvericin and enniatins (B and B1), and patulin or citrinin, respectively, on PDA medium. This is the first report of the mycotoxigenic potential of P. expansum and F. oxysporum strains isolated from fresh ginseng. © 2024 Society of Chemical Industry.

FvVam6 is associated with fungal development and fumonisin biosynthesis via vacuole morphology regulation in Fusarium verticillioides1

As the largest multifunctional and dynamic organelle in fungi, vacuoles are associated with different organelles through membrane contact sites and participate in various cellular processes. Vacuole and mitochondria patch (vCLAMP), the membrane contact site that tethers vacuoles and mitochondria, is indispensable for reciprocal interplay between these two organelles. The impairment of vacuoles and mitochondria significantly suppressed FB1 production in Fusarium verticillioides. However, the understanding of how vCLAMP complex regulates fumonisin biosynthesis remained unknown. Herein, the biological functions of vCLAMP component Vam6 were investigated in F. verticillioides. Our results showed that FvVam6 deletion mutant ΔFvVam6 exhibited palpable defects in fungal development, stresses responses and pathogenicity. In addition, abnormal vacuolar morphology and significantly reduced FB1 production were observed in ΔFvVam6. Furthermore, we demonstrated that two vacuolar sorting protein 39 (Vps39) domains and clathrin domain were critical for the biological functions of FvVam6, while clathrin and Vps39-2 domains played dominant roles in the regulation of virulence and FB1 production. Taken together, our results advanced our understanding of vCLAMP in fumonisin biosynthesis in plant pathogenic fungus F. verticillioides.

Mechanism Insights into the Enantioselective Bioactivity and Fumonisin Biosynthesis of Mefentrifluconazole to Fusarium verticillioides.

The occurrence of maize ear rot caused by Fusarium verticillioides (F. verticillioides) poses a threat to the yield and quality of maize. Mefentrifluconazole enantiomers appear to have strong stereoselective activity against F. verticillioides and cause differences in fumonisin production. We evaluated the stereoselective activity of mefentrifluconazole enantiomers by determining inhibition of the strain, hyphae, and conidia. Strain inhibition by R-(-)-mefentrifluconazole was 241 times higher than S-(+)-mefentrifluconazole and 376 times higher in conidia inhibition. For the mechanism of the enantioselective bioactivity, R-mefentrifluconazole had stronger binding to proteins than S-(+)-mefentrifluconazole. Under several concentration conditions, the fumonisin concentration was 1.3-24.9-fold higher in the R-(-)-mefentrifluconazole treatment than in the S-(+)-mefentrifluconazole treatment. The R-enantiomer stimulated fumonisin despite a higher bioactivity. As the incubation time increased, the stimulation of the enantiomers on fumonisin production decreased. R-(-)-Mefentrifluconazole stimulated higher fumonisin production in F. verticillioides at 25 °C compared to 30 °C. This study established a foundation for the development of high-efficiency and low-risk pesticides.

Development of an RPA-based CRISPR/Cas12a assay in combination with a lateral flow strip for rapid detection of toxigenic Fusarium verticillioides in maize

Fusarium verticillioides is an important phytopathogenic fungus that poses a threat to maize yield and quality in global maize-growing regions by causing Fusarium ear and stalk rot. The fungus is known to produce fumonisins, which are toxic secondary metabolites and have been associated with high incidences of esophageal cancer. The FUM1 gene is responsible for producing a crucial polyketide synthase required for fumonisin biosynthesis and is present in all pathogenic strains of F. verticillioides. This study aims to develop a rapid and accurate detection assay for F. verticillioides based on the FUM1 gene, by utilizing Chelex-100 resin for DNA extraction, recombinase polymerase amplification coupled with CRISPR/Cas12a cleavage and lateral flow detection (RPA-Cas12a-LFD) assay. The developed RPA-Cas12a-LFD assay exhibited remarkable specificity for F. verticillioides, and the lowest limit of detection was 2 ag DNA of F. verticillioides. The entire diagnostic process was completed in just 73 min, including sample DNA extraction, RPA reaction, Cas12a cleavage, and result readout. Furthermore, RPA-Cas12a-LFD assay was found to be equivalent to single-spore isolation and partial translation elongation factor 1α gene (TEF-1α) sequencing in identifying diseased samples in the field. In summary, this accurate and portable detection equipment has great potential for detecting and recognizing F. verticillioides, especially in areas where sophisticated lab equipment is not available.

Detection of fumonisin among different strains of Fusarium spp. associated with bakanae disease of rice (Oryza sativa L.) using molecular markers

Bakanae disease caused by Fusarium fujikuroi of basmati rice causes huge economic losses varying with varieties produced, with a frequency of 3.0-95.4%. The Fusarium spp. associated with bakanae disease produce fumonisins, a group of structurally similar sphingosine analogue mycotoxins, among which Fumonisin B1 is the most prevalent and active (FB1). The worst harm to both people and animal wellbeing is created by fumonisins, which infect feed and food sources. IARC, a global organization dedicated to cancer research, classified FB1 as a potential causing human cancer (Group 2B). Altogether 26 strains of Fusarium spp. from bakanae infected samples of various popular basmati rice varieties collected from Hisar, Jind, Fatehabad, Bhiwani, Sirsa, Panipat, Sonipat, Karnal, Yamunanagar, Kaithal and Kurukshetra (eleven) districts of Haryana state. Two specific primers namely VERTF and polyketide synthase (PKS) (involved in fumonisin biosynthesis) FUM (rp 32 and rp 33) were utilized in this investigation to differentiation between fumonisin-producing and non-producing strains employing PCR technique. Twenty-two strains were significant for the VERTF primer and showed the capacity to generate fumonisin, while four isolates evaluated negative for both primers. The FUM specific primer displayed positive respose only in nine strains and rest were negative. The present study provides a rapid and specific method that helped in accurate differentiation between fumonisin-producing and non-producing strains.

Vacuole Proteins with Optimized Microtubule Assembly Is Required for Fum1 Protein Localization and Fumonisin Biosynthesis in Mycotoxigenic Fungus Fusarium verticillioides.

Fumonisin contamination of corn caused by Fusarium verticillioides is a major concern worldwide. While key genes involved in fumonisin biosynthesis are known, the location within the fungal cell where this process occurs has yet to be fully characterized. In this study, three key enzymes, i.e., Fum1, Fum8, and Fum6, associated with early steps of fumonisin biosynthesis pathway, were tagged with GFP, and we examined their cellular localization. Results showed that these three proteins co-localized with the vacuole. To further understand the role of the vacuole in fumonisin B1 (FB1) biosynthesis, we disrupted two predicted vacuole associated proteins, FvRab7 and FvVam7, resulting in a significant reduction of FB1 biosynthesis and a lack of Fum1-GFP fluorescence signal. Furthermore, we used the microtubule-targeting drug carbendazim to show that proper microtubule assembly is critical for proper Fum1 protein localization and FB1 biosynthesis. Additionally, we found that α1 tubulin is a negative regulator in FB1 biosynthesis. We concluded that vacuole proteins with optimized microtubule assembly play a crucial role in proper Fum1 protein localization and fumonisin production in F. verticillioides.

Transcriptome analysis of maize pathogen Fusarium verticillioides revealed FvLcp1, a secreted protein with type-D fungal LysM and chitin-binding domains, that plays important roles in pathogenesis and mycotoxin production

Fusarium verticillioides is a key maize pathogen and produces fumonisins, a group of mycotoxins detrimental to humans and animals. Unfortunately, our understanding on how this fungus interacts with maize to trigger mycotoxin biosynthesis is limited. We performed a systematic computational network-based analysis of large-scale F. verticillioides RNA-seq datasets to identify gene subnetwork modules associated with virulence and fumonisin regulation. F. verticillioides was inoculated on two different maize lines, moderately resistant line hybrid 33K44 and highly susceptible line maize inbred line B73, to generate time-course RNA-Seq data. Among the highly discriminative subnetwork modules, we identified a putative hub gene FvLCP1, which encodes a putative a type-D fungal LysM protein with a signal peptide, three LysM domains, and two chitin binding domains. FvLcp1 is a unique protein that harbors these domains amongst five representative Fusarium species. FvLcp1 is a secreted protein important for fumonisin production with the LysM domain playing a critical role. The chitin-binding domain was essential for in vitro chitin binding. Using Magnaporthe oryzae, we learned that FvLcp1 accumulates in appressoria, suggesting that FvLcp1 is involved in host recognition and infection. Full length FvLcp1 suppressed BAX-triggered plant cell death in Nicotiana benthamiana. This unique type-D LysM secreted protein with a chitin-binding domain in F. verticillioides was shown to be potentially involved in suppressing host cell death and promoting fumonisin biosynthesis while the pathogen colonizes maize kernels.

Variation in Tocochromanols Level and Mycotoxins Content in Sweet Maize Cultivars after Inoculation with Fusarium verticillioides and F. proliferatum.

A major problem in maize production is the contamination of the grain with Fusarium spp., mainly F. proliferatum and F. verticillioides and their secondary metabolites—mycotoxins. Under biotic stress conditions, caused by a fungal pathogen, plants initiate a series of defense mechanisms that may cause quantitative and qualitative changes in the composition of phenolic compounds. We analyzed the resistance of four sweet maize cultivars (Syngenta Group: Overland, Sweetstar, GSS 8529, Shinerock) to the infection with Fusarium verticillioides and F. proliferatum isolates, along with fumonisins B1, B2, and B3 grain contamination and the levels of tocopherols and tocotrienols accumulated. Differences in ear rot levels were found between the cultivars and isolates used. The phenotypic evaluation positively correlated with the concentrations of fumonisins. The results obtained also indicate a significant dependence on tocochromanols content in sweet maize cultivars tested on the infection of plants with Fusarium isolates and fumonisin biosynthesis. Further studies are needed to investigate the mechanisms of the plant reaction and the effect of different levels of tocopherols and tocotrienols on Fusarium resistance and grain contamination with mycotoxins.

FfCOX17 is Involved in Fumonisins Production, Growth, Asexual Reproduction, and Fungicide Sensitivity in Fusarium fujikuroi.

Fusarium fujikuroi, a causal agent of Rice Bakanae Disease, produces secondary metabolites such as gibberellin, pigments bikaverin, and mycotoxins fumonisins. Fumonisins produced by F. fujikuroi pose a severe threat to human and animal health. The copper chaperone protein plays a critical role in different growth stages of plants, fungi, and yeasts, but their functions and regulation in fumonisin biosynthesis are still unclear. Here, a copper chaperone protein, FfCOX17, was identified in F. fujikuroi. The FfCOX17 deletion mutant (∆FfCOX17) exhibited decreased vegetative growth and asexual reproduction. The transcriptional level of the FfFUM2 gene was significantly induced in ∆FfCOX17, and the fumonisin production in ∆FfCOX17 mutants was significantly increased compared to wild-type F. fujikuroi, but the pathogenicity of ∆FfCOX17 mutants was unaffected, which may be caused by the no significantly changed gibberellin content. ∆FfCOX17 showed decreased sensitivity to oxidative stress, osmotic stress, and increased sensitivity to cell wall stress, heat shock stress, and high concentration glucose. In addition, ∆FfCOX17 also showed increased sensitivity to fungicide fluazinam and fludioxonil, and decreased sensitivity to phenamacril and prochloraz. Taken together, this study suggested that FfCOX17 is critical for fumonisin production, vegetative growth, asexual reproduction, and fungicide sensitivity, but is not required for the virulence function of F. fujikuroi on rice.

Cross-genus inhibitory activity of polyoxins against aflatoxin production by Aspergillus parasiticus and fumonisin production by Fusarium fujikuroi.

Co-exposure to aflatoxin and fumonisin is a health concern where corn is a staple food, and a method to prevent co-contamination of these mycotoxins in foods is urgently needed. Polyoxins are chitin synthase inhibitors produced by Streptomyces cacaoi var. asoensis. The aflatoxin production inhibitory activity of a commercially available polyoxin D and four polyoxins purified from polyoxin AL water-soluble powder, an agricultural chemical containing polyoxins, was tested. The five polyoxins dose-dependently inhibited aflatoxin production by Aspergillus parasiticus and the IC50 values of polyoxin A, B, D, K and L were 16, 74, 110, 9 and 280µmol L-1, respectively. Polyoxins also inhibited fumonisin production by Fusarium fujikuroi, and the IC50 values of polyoxin B, D, K and L were 270, 42, 65 and 62µmol L-1, respectively. Polyoxins repressed the transcription of genes encoding proteins required for aflatoxin biosynthesis in A. parasiticus and fumonisin biosynthesis in F. fujikuroi. Polyoxin K and D also inhibited conidiation in A. parasiticus and F. fujikuroi, respectively. These results suggest that a mixture of polyoxins may effectively prevent co-contamination of aflatoxin and fumonisin in foods.

The autophagy-related proteins FvAtg4 and FvAtg8 are involved in virulence and fumonisin biosynthesis in Fusarium verticillioides

ABSTRACT Autophagy is the main intracellular degradation system by which cytoplasmic materials are transported to and degraded in the vacuole/lysosome of eukaryotic cells, and it also controls cellular differentiation and virulence in a variety of filamentous fungi. However, the contribution of the autophagic pathway to fungal development and pathogenicity in the important maize pathogen and mycotoxigenic fungus Fusarium verticillioides is still unknown. In this study, we characterized two autophagy-related proteins, FvAtg4 and FvAtg8. The F. verticillioides deletion mutants ΔFvAtg4 and ΔFvAtg8 were impaired in autophagosome formation, aerial hyphal formation, sexual growth, lipid turnover, pigmentation and fungal virulence. Interestingly, ΔFvAtg4 and ΔFvAtg8 were defective in fumonisin B1 (FB1) synthesis, which may have resulted from decreased intracellular levels of alanine in the mutants. Our results indicate that FvAtg4 and FvAtg8 contribute to F. verticillioides pathogenicity by regulating the autophagic pathway to control lipid turnover, fumonisin biosynthesis, and pigmentation during its infectious cycle.

Ensiling process and pomegranate peel extract as a natural additive in potential prevention of fungal and mycotoxin contamination in silage

A study was conducted on six animal feed centers in Israel where fungal and mycotoxin presence was examined in maize and wheat silages. Fumonisin mycotoxins FB1 and FB2 were present in every maize silage sample analyzed. Interestingly, no correlation was found between the occurrence of specific mycotoxins and the presence of the fungal species that might produce them in maize and wheat silages. We further investigated the effect of pomegranate peel extract (PPE) on Fusarium infection and fumonisin biosynthesis in laboratory-prepared maize silage. PPE had an inhibitory effect on FB1 and FB2 biosynthesis by Fusarium proliferatum, which resulted in up to 90 % reduction of fumonisin production in silage samples compared to untreated controls. This finding was supported by qRT-PCR analysis, showing downregulation of key genes involved in the fumonisin-biosynthesis pathway under PPE treatment. Our results present promising new options for the use of natural compounds that may help reduce fungal and mycotoxin contamination in agricultural foodstuff, and potentially replace traditionally used synthetic chemicals.

Detection of a Gene Involved in Fumonisin Production in F usarium Strains Isolated from Moringa ( Moringa oliefera ) Sold in the Markets of Abidjan (Côte d'Ivoire)

Fumonisins produced by Fusarium constitute a concern group of mycotoxins contaminating food products. In Cote d'Ivoire, data on the sanitary quality of marketed Moringa powder remains unknown. This study was carried out to characterize molds of the genus Fusarium isolated from Moringa powder by detecting the presence of a gene encoding the biosynthesis of fumonisins. A total of 192 Moringa leaf powder samples of approximately 250g each were taken from various sales sites in Abidjan markets including Abobo, Adjame, Koumassi and Yopougon. The isolation and purification of the Fusarium strains were carried out on Sabouraud’s medium supplemented with 10 μg / mL of chloramphenicol. The Polymerase Chain Reaction (PCR) method was used for molecular identification of Fusarium strains and the detection of the presence of the Fum13 gene involved in the fumonisins biosynthesis. The contamination rate of Moringa powder by Fusarium-type molds was 15.43%. The predominantly isolated species was Fusarium solani (11.43%) followed by Fusarium sp (4%). The presence of the Fum13 gene was detected in 62.96% (17/27) of the Fusarium strains isolated. The presence of molds of the genus Fusarium and the detection of a gene coding for the fumonisins biosynthesis, requires the implementation of health safety measures during the manufacturing process of Moringa powder.

Carbon sources to enhance the biosynthesis of useful secondary metabolites in Fusarium verticillioides submerged cultures

Fusarium verticillioides is a prolific producer of useful secondary metabolites such as naphthoquinone pigments, monoterpenes, and sesquiterpenes, as well as the harmful mycotoxins fumonisins. A strategy to increase their production includes creating a proper nutritional environment that enables the fungus to produce the compounds of interest. The aim of the present work was to study the effect of different carbon sources (glucose, fructose, xylose, sucrose, and lactose) on secondary metabolites biosynthesis in F. verticillioides submerged cultures. The production of volatile terpenes was evaluated through gas chromatography coupled to mass spectrometry. The quantification and identification of pigments was conducted using a UV/VIS spectrophotometer and NMR spectrometer, respectively. The quantification of fumonisin B1 and fumonisin B2 was performed by high-performance liquid chromatography. Our results showed that the biosynthesis of naphthoquinone pigments, monoterpenes, and sesquiterpenes was highest in cultures with fructose (13.00 ± 0.71mmol/g), lactose [564.52 × 10-11 ± 11.50 × 10-11μg/g dry weight (DW)], and xylose (54.41 × 10-11 ± 1.55 × 10-11μg/g DW), respectively, with fumonisin being absent or present in trace amounts in the presence of these carbon sources. The highest biosynthesis of fumonisins occurred in sucrose-containing medium (fumonisin B1: 7.85 × 103 ± 0.25 × 103μg/g DW and fumonisin B2: 0.38 × 103 ± 0.03 × 103μg/g DW). These results are encouraging since we were able to enhance the production of useful fungal metabolites without co-production with harmful mycotoxins by controlling the carbon source provided in the culture medium.

Sphinganine-Analog Mycotoxins (SAMs): Chemical Structures, Bioactivities, and Genetic Controls.

Sphinganine-analog mycotoxins (SAMs) including fumonisins and A. alternata f. sp. Lycopersici (AAL) toxins are a group of related mycotoxins produced by plant pathogenic fungi in the Fusarium genus and in Alternaria alternata f. sp. Lycopersici, respectively. SAMs have shown diverse cytotoxicity and phytotoxicity, causing adverse impacts on plants, animals, and humans, and are a destructive force to crop production worldwide. This review summarizes the structural diversity of SAMs and encapsulates the relationships between their structures and biological activities. The toxicity of SAMs on plants and animals is mainly attributed to their inhibitory activity against the ceramide biosynthesis enzyme, influencing the sphingolipid metabolism and causing programmed cell death. We also reviewed the detoxification methods against SAMs and how plants develop resistance to SAMs. Genetic and evolutionary analyses revealed that the FUM (fumonisins biosynthetic) gene cluster was responsible for fumonisin biosynthesis in Fusarium spp. Sequence comparisons among species within the genus Fusarium suggested that mutations and multiple horizontal gene transfers involving the FUM gene cluster were responsible for the interspecific difference in fumonisin synthesis. We finish by describing methods for monitoring and quantifying SAMs in food and agricultural products.

The in vitro Production Potentialities of Secondary Toxic Metabolites by the Fungal Factory Fusarium verticillioides Is, Fortunately, Largely Underestimated in Fields: Pioneering Study on Fumonisins.

This study presents fungi infrequently viewed as fungal factories for secondary metabolite production resources such as mycotoxins in Ascomycota. Additionally, we demonstrated that biochemical warfare of Fusarium verticillioides factory against animal cells is not only due to mycotoxins such as fumonisins, but acute cytotoxic firing is based on different excreted secondary metabolite series, potentially leading to animal and human diseases. In this study, fumonisins, which can be followed by in situ localization, quantification, or expression of the key gene implicated in their synthesis, are used to understand secondary metabolite production by this fungus. It is known that F. verticillioides produces mycotoxins such as fumonisins on cereals, but until now, there is no evidence demonstrating a method to totally block fumonisin production on feed and food. In this paper, we explained, what was never clearly established before, that fumonisin production depends on two bottlenecks. The fumonisin synthesis and secretion in fungal articles of the mycelium are medium-independent and follow the fungal cell cycle developmental program (ontogenesis). Conversely, the fumonisin excretion into the medium depends on its composition, which also impacts fumonisin biosynthesis level. Using a high-pressure freezing method, we showed that, in non-permissive fumonisin excretion (NPFE) medium, FB1 is sequestered inside extra-vesicles and in the first third of the cell wall next to the plasmalemma, leading to the hypothesis that the fungus develops mechanisms to protect its cytosolic homeostasis against this cytotoxic. In permissive fumonisin excretion (PFE) medium, leading to very high quantities of excreted fumonisins, FB1 localized inside extra-vesicles, crosses the entire cell wall thickness, and then releases into the medium. Our results demonstrated a delayed and lower expression of Fvpks gene in mycelium developed on NPFE medium as compared to PFE medium. Conversely, higher amounts of fumonisins were accumulated in NPFE-grown mycelium than in PFE-grown mycelium. Thus, our results demonstrated for the first time that we have to take into account that the synthesis and secretion inside the article of secondary metabolites depend on the occurrence of cryptic biochemical specialized articles, differentiated in the mycelium. However, those are not morphologically different from other colonial hyphae.

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

BackgroundSphingolipids 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.ResultsUsing 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.ConclusionOur 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.