Benzoxazinoids secreted by wheat root weaken the pathogenicity of Fusarium oxysporum f. sp. fabae by inhibiting linoleic acid and nucleotide metabolisms.

KEY MESSAGE: The regulatory action of BXs secreted by wheat on the pathogenicity of FOF causing Fusarium wilt in faba bean were analyzed. DIMBOA and MBOA weakened the pathogenicity of FOF. A large number of pathogenic bacteria in continuous cropping soil infect faba bean plants, leading to the occurrence of wilt disease, which restricts their production. Faba bean-wheat intercropping is often used to alleviate this disease. This study investigates the effect of benzoxazinoids (BXs) secreted by wheat root on the pathogenicity of Fusarium oxysporum f. sp. Fabae (FOF) and underlying molecular mechanisms. The effects of DIMBOA(2,4-dihydroxy-7-methoxy-1,4-benzoxazine-4-one) and MBOA(6-methoxybenzoxazolin-2-one) on the activity of cell-wall-degrading enzymes in FOF(cellulase, pectinase, amylase, and protease), FOF Toxin (fusaric acid, FA) content were investigated through indoor culture experiments. The effect of BXs on the metabolic level of FOF was analyzed by metabonomics to explore the ecological function of benzoxazines intercropping control of Fusarium wilt in faba bean. The results show that the Exogenous addition of DIMBOA and MBOA decreased the activity of plant-cell-wall-degrading enzymes and fusaric acid content and significantly weakened the pathogenicity of FOF. DIMBOA and MBOA significantly inhibited the pathogenicity of FOF, and metabolome analysis showed that DIMBOA and MBOA reduced the pathogenicity of FOF by down-regulating related pathways such as nucleotide metabolism and linoleic acid metabolism, thus effectively controlling the occurrence of Fusarium wilt in faba bean.

Exogenous application of mycotoxin fusaric acid improve the morphological, cytogenetic, biochemical and anatomical parameters in salt (NaCl) stressed Allium cepa L.

Salinity is one of the most important abiotic stress factors that negatively affect plant growth and development. In contrast, fusaric acid (FA), a mycotoxin produced by Fusarium and Giberella fungal genera, has biological and metabolic effects in various plants. In this study, it was aimed to investigate the protective effect of externally applied FA (0.1 nM) against the damage caused by salt (0.15 M NaCl) stress in onion (Allium cepa L.) plant. Salt stress resulted in an increase in the chromosomal aberrations (CAs) and micronucleus (MN) frequency, a decrease in the mitotic index (MI), fresh weight, root number, germination percentage, and root length. It promoted CAs such as irregular mitosis, bilobulated nuclei, chromosome loss, bridge, unequal seperation of chromosome, vagrant chromosome and polar slip in root meristem cells. In addition, salt stress caused a enhancement in free proline (PR), catalase (CAT), superoxide dismutase (SOD) and malondialdehyde (MDA) contents in the roots of onion plant. Moreover, it revealed damage and changes that include the accumulation of some chemical substances such as proline and sugars in epidermis and cortex layer cells, epidermal cell injury, flattening of the cell nucleus, wall thickening in cortex cells, necrotic areas and indistinct transmission tissue in the anatomical structure of onion roots. On the other hand, FA application promoted bulb germination and mitotic activity, strengthened the antioxidant defense system, and reduced chromosome and anatomical structure damages. In conclusion; it has been revealed that exogenous FA application may have a positive effect on increasing the resistance of onion plants to salt stress.

Computational methods meet in vitro techniques: A case study on fusaric acid and its possible detoxification through cytochrome P450 enzymes.

Mycotoxins are known environmental pollutants that may contaminate food and feed chains. Some mycotoxins are regulated in many countries to limit the trading of contaminated and harmful commodities. However, the so-called emerging mycotoxins are poorly understood and need to be investigated further. Fusaric acid is an emerging mycotoxin, noxious to plants and animals, but is known to be less toxic to plants when hydroxylated. The detoxification routes effective in animals have not been elucidated yet. In this context, this study integrated in silico and in vitro techniques to discover potential bioremediation routes to turn fusaric acid to its less toxic metabolites. The toxicodynamics of these forms in humans have also been addressed. An in silico screening process, followed by molecular docking and dynamics studies, identified CYP199A4 from the bacterium Rhodopseudomonas palustris HaA2 as a potential fusaric acid biotransforming enzyme. Its activity was confirmed in vitro. However, the effect of hydroxylation seemed to have a limited impact on the modelled toxicodynamics against human targets. This study represents a starting point to develop a hybrid in silico/in vitro pipeline to find bioremediation agents for other food, feed and environmental contaminants.

Genetic dissection of the degradation pathways for the mycotoxin fusaric acid in Burkholderia ambifaria T16.

IMPORTANCE: Fusaric acid (FA) is an important virulence factor produced by several Fusarium species. These fungi are responsible for wilt and rot diseases in a diverse range of crops. FA is toxic for animals, humans and soil-borne microorganisms. This mycotoxin reduces the survival and competition abilities of bacterial species able to antagonize Fusarium spp., due to its negative effects on viability and the production of antibiotics effective against these fungi. FA biodegradation is not a common characteristic among bacteria, and the determinants of FA catabolism have not been identified so far in any microorganism. In this study, we identified genes, enzymes, and metabolic pathways involved in the degradation of FA in the soil bacterium Burkholderia ambifaria T16. Our results provide insights into the catabolism of a pyridine-derivative involved in plant pathogenesis by a rhizosphere bacterium.

Fusaric acid-evoked oxidative stress affects plant defence system by inducing biochemical changes at subcellular level.

Fusaric acid (FA) is one of the most harmful phytotoxins produced in various plant-pathogen interactions. Fusarium species produce FA as a secondary metabolite, which can infect many agronomic crops at all stages of development from seed to fruit, and FA production can further compromise plant survival because of its phytotoxic effects. FA exposure in plant species adversely affects plant growth, development and crop yield. FA exposure in plants leads to the generation of reactive oxygen species (ROS), which cause cellular damage and ultimately cell death. Therefore, FA-induced ROS accumulation in plants has been a topic of interest for many researchers to understand the plant-pathogen interactions and plant defence responses. In this study, we reviewed the FA-mediated oxidative stress and ROS-induced defence responses of antioxidants, as well as hormonal signalling in plants. The effects of FA phytotoxicity on lipid peroxidation, physiological changes and ultrastructural changes at cellular and subcellular levels were reported. Additionally, DNA damage, cell death and adverse effects on photosynthesis have been explained. Some possible approaches to overcome the harmful effects of FA in plants were also discussed. It is concluded that FA-induced ROS affect the enzymatic and non-enzymatic antioxidant system regulated by phytohormones. The effects of FA are also associated with other photosynthetic, ultrastructural and genotoxic modifications in plants.

Hepialiamides A-C: Aminated Fusaric Acid Derivatives and Related Metabolites with Anti-Inflammatory Activity from the Deep-Sea-Derived Fungus Samsoniella hepiali W7.

A systematic investigation combined with a Global Natural Products Social (GNPS) molecular networking approach, was conducted on the metabolites of the deep-sea-derived fungus Samsoniella hepiali W7, leading to the isolation of three new fusaric acid derivatives, hepialiamides A-C (1-3) and one novel hybrid polyketide hepialide (4), together with 18 known miscellaneous compounds (5-22). The structures of the new compounds were elucidated through detailed spectroscopic analysis. as well as TD-DFT-based ECD calculation. All isolates were tested for anti-inflammatory activity in vitro. Under a concentration of 1 µM, compounds 8, 11, 13, 21, and 22 showed potent inhibitory activity against nitric oxide production in lipopolysaccharide (LPS)-activated BV-2 microglia cells, with inhibition rates of 34.2%, 30.7%, 32.9%, 38.6%, and 58.2%, respectively. Of particularly note is compound 22, which exhibited the most remarkable inhibitory activity, with an IC50 value of 426.2 nM.

Fusaric acid inhibits cell proliferation and downregulates expressions of toll-like receptors pathway genes in Ishikawa endometrial cancer cells.

OBJECTIVE: Fusaric acid is a derivative of picolinic acid produced by some Fusarium species. In this study, we aimed to determine the mRNA expression and antiproliferative effects of fusaric acid in Ishikawa endometrium cancer cells in signal pathway genes associated with Toll-like receptors (TLRs). The effect of fusaric acid on the viability of Ishikawa cells was evaluated using XTT. PATIENTS AND METHODS: After total RNA was isolated from control and dose group cells, cDNA synthesis was performed, and mRNA expression changes of genes involved in the Toll-like signaling pathway were evaluated by real-time reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: The decrease in viability of Ishikawa cells was observed in a time- and dose-dependent manner. The inhibitory concentration (IC50) dose of fusaric acid at the 72nd hour in the Ishikawa cell line was 142.81 µM. When the dose group treated with 125 µM fusaric acid at the 72nd hour was compared with the control group, significantly decreased toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, and Myeloid differentiation primary response protein 88 (MYD88) gene expressions were observed. CONCLUSIONS: Fusaric acid inhibits cell proliferation and downregulates Toll-like receptors pathway gene expression in Ishikawa endometrial cancer cells.

A MAPK signaling cascade regulates the fusaric acid-induced cell death in Arabidopsis.

Mycotoxin contamination of foods and feeds is a global problem. Fusaric acid (FA) is a mycotoxin produced by Fusarium species that are phytopathogens of many economically important plant species. FA can cause programmed cell death (PCD) in several plant species. However, the signaling mechanisms of FA-induced cell death in plants are largely unknown. Here we showed that FA induced cell death in the model plant Arabidopsis thaliana, and MPK3/6 phosphorylation was triggered by FA in Arabidopsis. Both the acid nature and the radical of FA are required for its activity in inducing MPK3/6 activation and cell death. Expression of the constitutively active MKK5DD resulted in the activation of MPK3/6 and promoted the FA-induced cell death. Our work demonstrates that the MKK5-MPK3/6 cascade positively regulates FA-induced cell death in Arabidopsis and also provides insight into the mechanisms of how cell death is induced by FA in plants.

Fusaric acid inhibits proliferation and induces apoptosis through triggering endoplasmic reticulum stress in MCF-7 human breast cancer cells.

Breast cancer has replaced lung cancer to be the leading cancer in the world. Currently, chemotherapy is still the major method for breast cancer therapy, but its overall effect remains unsatisfactory. Fusaric acid (FSA), a mycotoxin derived from fusarium species, has shown potency against the proliferation of several types of cancer cells, but its effect on breast cancer cells has not been examined. Therefore, we explored the possible effect of FSA on the proliferation of MCF-7 human breast cancer cells and uncovered the underlying mechanism in the present study. Our results showed that FSA has a strong anti-proliferative effect on MCF-7 cells through inducing ROS production, apoptosis and arresting cell cycle at G2/M transition phase. Additionally, FSA triggers endoplasmic reticulum (ER) stress in the cells. Notably, the cell cycle arrest and apoptosis inducing effect of FSA can be attenuated by ER stress inhibitor, tauroursodeoxycholic acid. Our study provide evidence that FSA is a potent proliferation inhibition and apoptosis inducing agent against human breast cancer cells, and the possible mechanism involves the activation of ER stress signaling pathways. Our study may highlight that FSA is promising for the future in vivo study and development of potential agent for breast cancer therapy.

Screening Biocontrol Agents for Cash Crop Fusarium Wilt Based on Fusaric Acid Tolerance and Antagonistic Activity against Fusarium oxysporum.

Fusarium wilt, caused by Fusarium oxysporum, is one of the most notorious diseases of cash crops. The use of microbial fungicides is an effective measure for controlling Fusarium wilt, and the genus Bacillus is an important resource for the development of microbial fungicides. Fusaric acid (FA) produced by F. oxysporum can inhibit the growth of Bacillus, thus affecting the control efficacy of microbial fungicides. Therefore, screening FA-tolerant biocontrol Bacillus may help to improve the biocontrol effect on Fusarium wilt. In this study, a method for screening biocontrol agents against Fusarium wilt was established based on tolerance to FA and antagonism against F. oxysporum. Three promising biocontrol bacteria, named B31, F68, and 30833, were obtained to successfully control tomato, watermelon, and cucumber Fusarium wilt. Strains B31, F68, and 30833 were identified as B. velezensis by phylogenetic analysis of the 16S rDNA, gyrB, rpoB, and rpoC gene sequences. Coculture assays revealed that strains B31, F68, and 30833 showed increased tolerance to F. oxysporum and its metabolites compared with B. velezensis strain FZB42. Further experiments confirmed that 10 µg/mL FA completely inhibited the growth of strain FZB42, while strains B31, F68, and 30833 maintained normal growth at 20 µg/mL FA and partial growth at 40 µg/mL FA. Compared with strain FZB42, strains B31, F68, and 30833 exhibited significantly greater tolerance to FA.

Potential toxicity assessment of mycotoxin fusaric acid with the spectral shift profile on DNA.

In this study, the multiple toxicities induced by three different doses (1, 5, and 10 µM) of fusaric acid (FA), a mycotoxin, was investigated with Allium test. Physiological (percent germination, root number, root length, and weight gain), cytogenetic (micronucleus = MN, chromosomal abnormalities = CAs, and mitotic index = MI), biochemical (proline level, malondialdehyde = MDA level, catalase = CAT activity, and superoxide dismutase = SOD activity), and anatomical parameters were used as indicators of toxicity. Allium cepa L. bulbs were divided into four groups as one control and three applications. The bulbs in the control group were germinated with tap water for 7 days, and the bulbs in the treatment groups were germinated with three different doses of FA for 7 days. As a result, FA exposure caused a decrease in all physiological parameters examined at all three doses. Besides, all FA doses caused a decrease in MI and an increase in the frequency of MN and the number of CAs. FA promoted CAs such as nucleus with vacuoles, nucleus buds, irregular mitosis, bridge, and misdirection in root meristem cells. DNA and FA interactions, which are the possible causes of genotoxic effects, were examined by spectral analysis, and FA could interact with DNA through intercalation, causing bathochromic and hypochromic shifts in the spectrum. FA also causes toxicity by inducing oxidative stress in cells, confirming this; dose-related increases in root MDA and proline levels were measured as a result of FA exposure. In the root SOD and CAT enzyme activities, increases up to 5 µM doses and decreases at 10 µM doses were measured. FA exposure induced anatomical damage such as necrosis, epidermis cell damage, flattened cell nucleus, thickening of the cortex cell wall, and unclear vascular tissue in root tip meristem cells. As a result, FA caused a comprehensive toxicity by showing an inhibitory effect in A. cepa test material, and the Allium test was a very useful test in determining this toxicity.

Ethylene-dependent regulation of oxidative stress in the leaves of fusaric acid-treated tomato plants.

The mycotoxin fusaric acid (FA) induces rapid oxidative burst leading to cell death in plants. At the same time, plant defence reactions are mediated by several phytohormones for instance ethylene (ET). However, previously conducted studies leave research gaps on how ET plays a regulatory role under mycotoxin exposure. Therefore, this study aims to the time-dependent effects of two FA concentrations (0.1 mM and 1 mM) were explored on the regulation of reactive oxygen species (ROS) in leaves of wild-type (WT) and ET receptor mutant Never ripe (Nr) tomatoes. FA induced superoxide and H2O2 accumulation in both genotypes in a mycotoxin dose- and exposure time-dependent pattern. 1 mM FA activated NADPH oxidase (+34% compared to the control) and RBOH1 transcript levels in WT leaves. However, superoxide production was significantly higher in Nr with 62% which could contribute to higher lipid peroxidation in this genotype. In parallel, the antioxidative defence mechanisms were also activated. Both peroxidase and superoxide dismutase activities were lower in Nr but ascorbate peroxidase showed one-fold higher activity under 1 mM FA stress than in WT leaves. Interestingly, catalase (CAT) activity decreased upon FA in a time- and concentration-dependent manner and the encoding CAT genes were also downregulated, especially in Nr leaves at 20%. Ascorbate level was decreased and glutathione remained lower in Nr than WT plants under FA exposure. Conclusively, Nr genotype showed more sensitivity to FA-induced ROS suggesting that ET serves defence reactions of plants by activating several enzymatic and non-enzymatic antioxidants to detoxify excess ROS accumulation.

Potential of Burkholderia sp. IMCC1007 as a biodetoxification agent in mycotoxin biotransformation evaluated by mass spectrometry and phytotoxicity analysis.

Microbial degradation is considered as an attractive method to eliminate exposure to mycotoxin that cause a serious threat in agriculture global industry and severe human health problems. Compared with other more prominent mycotoxin compounds, fusaric acid (FA) biodegradation has not been widely investigated. In this study, a fusaric acid-degrading bacterium Burkholderia sp. IMCC1007 was identified by 16 S rRNA gene sequencing and its detoxification characteristics were evaluated. This strain able to utilize FA as sole energy and carbon source with growth rate (µ) of 0.18 h- 1. Approximately 93% from the initial substrate FA concentration was almost degraded to the residual about 4.87 mg L- 1 after 12 h of incubation. The optimal degradation conditions for pH and temperature were recorded at 6.0 with 30 °C respectively. An efficient FA degradation of strain IMCC1007 suggested its potential significance to detoxification development. Accroding to LC-MS/Q-TOF analysis, FA was bio-transformed to 4-hydroxybenzoic acid (C7H6O3) and other possible metabolites. Plant treated with detoxified FA products exhibited reduction of wilting index, mitigating against FA phytoxicity effect on plant growth and photosynthesis activity. Phytotoxicity bioassay suggested that degradation product of IMCC1007 was not a potent harmful compound towards plants as compared to the parent compound, FA. As a conslusion, our study provides a new insight into the practical application of biodetoxifcation agent in controlling mycotoxin contamination.

Alkaline pH, Low Iron Availability, Poor Nitrogen Sources and CWI MAPK Signaling Are Associated with Increased Fusaric Acid Production in Fusarium oxysporum.

Fusaric acid (FA) is one of the first secondary metabolites isolated from phytopathogenic fungi belonging to the genus Fusarium. This molecule exerts a toxic effect on plants, rhizobacteria, fungi and animals, and it plays a crucial role in both plant and animal pathogenesis. In plants, metal chelation by FA is considered one of the possible mechanisms of action. Here, we evaluated the effect of different nitrogen sources, iron content, extracellular pH and cellular signalling pathways on the production of FA siderophores by the pathogen Fusarium oxysporum (Fol). Our results show that the nitrogen source affects iron chelating activity and FA production. Moreover, alkaline pH and iron limitation boost FA production, while acidic pH and iron sufficiency repress it independent of the nitrogen source. FA production is also positively regulated by the cell wall integrity (CWI) mitogen-activated protein kinase (MAPK) pathway and inhibited by the iron homeostasis transcriptional regulator HapX. Collectively, this study demonstrates that factors promoting virulence (i.e., alkaline pH, low iron availability, poor nitrogen sources and CWI MAPK signalling) are also associated with increased FA production in Fol. The obtained new insights on FA biosynthesis regulation can be used to prevent both Fol infection potential and toxin contamination.

Exploring the chemical diversity of phytopathogenic fungi infecting edible fruits.

Two fungi, Fusarium guttiforme and Colletotrichum horii, were cultured under different conditions to obtain fourteen compounds. The axenic cultures of F. guttiforme and C. horii in potato dextrose broth (PDB) medium yielded fusaric acid (1), 9,10-dehydrofusaric acid (2), and tyrosol, whereas their co-cultivation produced fusarinol (5), a fusaric acid complex with magnesium (3), 9,10-dehydrofusaric acid complex with magnesium (4), and 5-butyl-5-(hydroxymethyl) dihydrofuranone (9). Upon changing the medium from PDB to Czapek, different compounds (uracil, p-hydroxy acetophenone, and cyclo(L-Leu-L-Pro) were obtained. Fusaric acid (1) was biotransformed into fusarinol (5) by C. horii, suggesting a detoxification process, and three other compounds were obtained: 7-hydroxyfusarinol (7), 9,10-dehydrofusarinol (6), and fusarinyl acetate (8). Epigenetic modulation of suberohydroxamic acid against F. guttiforme afforded gibepyrone B (10). These compounds were subjected to a papain inhibition enzymatic assay; the highest inhibitory activity was displayed by the two magnesium complexes, at 56 and 54% inhibition, respectively.

FoRSR1 Is Important for Conidiation, Fusaric Acid Production, and Pathogenicity in Fusarium oxysporum f. sp. ginseng.

The root rot disease caused by Fusarium oxysporum f. sp. ginseng is one of the most destructive diseases of ginseng, an economically important herb. However, little is known about the pathogen's toxin biosynthesis or the molecular mechanisms regulating infection of ginseng. In this study we identified and functionally characterized the FoRSR1 gene that encodes a Ras-related (RSR) small GTPase homologous to yeast Rsr1 in F. oxysporum f. sp. ginseng. Disruption of FoRSR1 resulted in a significant reduction in mycelial dry weight in liquid cultures, although vegetative growth rate was not affected on culture plates. Notably, the Forsr1 mutant exhibited blunted and swollen hyphae with multi-nucleated compartments. It produced fewer and morphologically abnormal conidia and was defective in chlamydospore formation. In infection assays with ginseng roots, the Forsr1 mutant was significantly less virulent and caused only limited necrosis at the wounding sites. Deletion of FoRSR1 also affected pigmentation, autophagy, and production of fusaric acid. Furthermore, the expression of many candidate genes involved in secondary metabolism was significantly downregulated in the mutant, suggesting that FoRSR1 is also important for secondary metabolism. Overall, our results indicated that FoRSR1 plays important roles in conidiation, vacuolar morphology, secondary metabolism, and pathogenesis in F. oxysporum f. sp. ginseng.

Biosensor approach for electrochemical quantitative assessment and qualitative characterization of the effect of fusaric acid on a culture-receptor.

Fusaric acid (FA) is a secondary fungal metabolite, which is widespread on corn and corn-based feed and food; FA has non-specific toxicity. Biosensor method is an express and easy-to-use method for quantitative and qualitative assessment of FA effect. Search for cultures has been performed for the formation of laboratory models of FA biosensor with the Clark-type oxygen electrode as transducer: respiration intensity of chosen cultures changed in the presence of FA. Resting cells of Fusarium oxysporum f. sp. vasinfectum and Bacillus subtilis were used as receptors of the amperometric biosensor for FA determination in aqueous solution. To enhance the sensitivity of detection, induction by substrate was performed for Bacillus subtilis. Response-concentration linear dependencies were obtained in a range of 0.5-500 FA mg/L. Biosensor models were applied to characterize influence of FA on microbial cells and investigate some features of FA transport. The dependences of the cells' response to FA on FA concentration were obtained; the kinetic parameters S0.5 and Vmax were determined for each culture. Inhibition-threshold FA (Sit) concentrations were similar for both studied cultures. At concentrations lower than Sit, the process of simple diffusion governed FA transport into cells and caused the cells' response to FA for non-induced culture.

Genome mining of Burkholderia ambifaria strain T16, a rhizobacterium able to produce antimicrobial compounds and degrade the mycotoxin fusaric acid.

Burkholderia ambifaria T16 is a bacterium isolated from the rhizosphere of barley plants that showed a remarkable antifungal activity. This strain was also able to degrade fusaric acid (5-Butylpyridine-2-carboxylic acid) and detoxify this mycotoxin in inoculated barley seedlings. Genes and enzymes responsible for fusaric acid degradation have an important biotechnological potential in the control of fungal diseases caused by fusaric acid producers, or in the biodegradation/bio catalysis processes of pyridine derivatives. In this study, the complete genome of B. ambifaria T16 was sequenced and analyzed to identify genes involved in survival and competition in the rhizosphere, plant growth promotion, fungal growth inhibition, and degradation of aromatic compounds. The genomic analysis revealed the presence of several operons for the biosynthesis of antimicrobial compounds, such as pyrrolnitrin, ornibactin, occidiofungin and the membrane-associated AFC-BC11. These compounds were also detected in bacterial culture supernatants by mass spectrometry analysis. In addition, this strain has multiple genes contributing to its plant growth-promoting profile, including those for acetoin, 2,3-butanediol and indole-3-acetic acid production, siderophores biosynthesis, and solubilisation of organic and inorganic phosphate. A pan-genomic analysis demonstrated that the genome of strain T16 possesses large gene clusters that are absent in the genomes of B. ambifaria reference strains. According to predictions, most of these clusters would be involved in aromatic compounds degradation. One genomic region, encoding flavin-dependent monooxygenases of unknown function, is proposed as a candidate responsible for fusaric acid degradation.

Fusaric acid detoxification: a strategy of Gliocladium roseum involved in its antagonism against Fusarium verticillioides.

Fungal co-culture has several biotechnological applications including the discovery or the enhanced production of secondary metabolites. It is also a powerful tool aiding to elucidate the involvement of secondary metabolism in fungus-fungus interactions. The aim of this work was to investigate secondary metabolites produced when Fusarium verticillioides is co-cultured with Gliocladium roseum. Secreted metabolites were analyzed by HPLC-MS, and fusaric acid (FA) was quantified by HPLC-DAD. Four FA derivatives were identified only in the F. verticillioides-G. roseum co-culture. Mass spectrometry and one- and two-dimensional NMR spectra indicated that they were 5-butylpyridine-2-carboxylic acid methyl ester (5B2CAM), 4-(5-butylpicolinamido) butanoic acid (45BBA), methyl 4-(5-butylpicolinamido) butanoate (M45BBA), and bis(5-butyl-2-pyridinecarboxylate-N1,O2)-copper (B52P). 45BBA and M45BBA are reported for the first time and were FA biotransformation products generated by G. roseum. The antifungal activity of 5B2CAM, 45BBA, and M45BBA was evaluated in vitro against Botrytis cinerea and Aspergillus niger. They were less fungitoxic than FA, with 45BBA as the least toxic. Our results suggest that the effective antagonism exerted by G. roseum against F. verticillioides is due, at least in part, to its detoxifying ability against FA.

A target fishing study to spot possible biological targets of fusaric acid: Inhibition of protein kinase-A and insights on the underpinning mechanisms.

Fusaric acid is a secondary metabolite produced by various Fusarium fungi, present with relatively high incidence in Fusarium-contaminated foods. It was already described as phytotoxic and cytotoxic. However, the understanding of its molecular mechanisms is still fragmentary and further data are needed to ensure an informed assessment of the risk related to its presence in food. This work applied an integrated in silico/in vitro approach to reveal novel potential biological activities of fusaric acid and to investigate the underpinning mechanisms. An in silico reverse screening was used to identify novel biological targets for fusaric acid. Computational results indicated as target protein kinase-A, which was confirmed with biochemical cell-free assays providing evidence of its actual inhibitory potential. Cell-based experiments on intestinal cells (HCEC-1CT cells) identified the mitochondrial network and cell membranes as potentially affected organelles, possibly resulting from PKA inhibition. The integration of 3D molecular modeling supported the plausibility of fusaric acid-dependent inhibition. From the hazard identification perspective, considering the Low Observed Adverse Effect Level described here (0.1 mM) and the possible level of contamination in food, fusaric acid might raise concern from a food safety standpoint and the gastrointestinal tract was described as a meaningful system to investigate with priority.