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.

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.

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.

Evaluation of Hydrangea Cultivars for Tolerance Against Root Rot Caused by Fusarium oxysporum.

Root rot caused by Fusarium oxysporum Schltdl. is a newly identified disease in oakleaf hydrangea. Some cultivars such as Pee Wee and Queen of Hearts grown in pot-in-pot container systems showed root rot symptoms after late spring frost in May 2018 with 40 and 60% incidence in the infected nursery, respectively. This experiment was carried out to evaluate the tolerance among different hydrangea cultivars against root rot caused by F. oxysporum. Fifteen hydrangea cultivars from four different species were selected, and rooted cuttings were prepared from new spring flushes. Twelve plants from each cultivar were transplanted in a 1-gallon pot. Half of transplanted plants (six single plants) were inoculated by drenching 150 ml of F. oxysporum conidial suspension to maintain the concentration of 1 × 106 conidia/ml. Half of the plants remain noninoculated (control) and were drenched with sterile water. After 4 months, root rot was assessed using a scale of 0 to 100% root area affected, and recovery of F. oxysporum was recorded by plating 1-cm root sections in Fusarium selective medium. Fusaric acid (FA) and mannitol were extracted from the roots of inoculated and noninoculated plants to see the effect and role on pathogenesis. Further, mannitol concentration was analyzed using absorption wavelength in a spectrophotometer, and FA was analyzed using high-performance liquid chromatography (HPLC). Results indicated that no cultivars were resistant to F. oxysporum. Cultivars from Hydrangea arborescens, H. macrophylla, and H. paniculata were more tolerant to F. oxysporum compared to cultivars from H. quercifolia. Among H. quercifolia, cultivars Snowflake, John Wayne, and Alice were more tolerant to F. oxysporum.

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.

Development of fusarium wilt resistant mutants of Musa spp. cv.Rasthali (AAB, Silk subgroup) and comparative proteomic analysis along with its wild type.

MAIN CONCLUSION: Induced mutagenesis using embryogenic cell suspension (ECS) explants with toxin based screening is an effective tool to create non-chimeral Fusarium wilt resistant mutants in banana. Global proteomics unravel the molecular mechanism behind resistance. Race 1 of Fusarium wilt is a serious threat to Musa spp. cv.Rasthali (AAB, Silk subgroup) which is a choice variety traditionally grown in most of the south East Asian countries. Resistant gene introgression into susceptible varieties through conventional breeding has several limitations and the predominant ones being sterility and long generation time. Under such circumstances, induced mutagenesis combined with toxin based in vitro screening remains as the viable alternative for the development of fusarium wilt resistant Rasthali. Therefore, induced mutagenesis was attempted by using ethylmethane sulfonate (EMS) in embryogenic cell suspension (ECS) of Rasthali followed by in vitro screening for fusarium wilt resistance using new generation toxins and pot screening through challenge inoculation with Foc race 1. This ultimately resulted in the identification of 15 resistant lines. Global proteomic analysis in one of the resistant mutant lines namely NRCBRM15 and its wild type revealed 37 proteins, of which 20 showed differential expression. Out of 20 proteins, nineteen were significantly abundant in NRCBRM15 and only one was abundant in wild Rasthali. A total of nine genes based on protein expression were further validated using quantitative real time polymerase chain reaction (qRT-PCR). Annotation results revealed that some of the genes namely Enolase, ATP synthase-alpha subunit, Actin 2, Actin 3,-glucanase, UTP-glucose-1-phosphate uridylyltransferase, Respiratory burst oxidase homolog, V type proton ATPase catalytic subunit A and DUF292 domain containing protein are involved in diverse functions such as carbohydrate metabolism, energy production, electron carrier, response to wounding, binding proteins, cytoskeleton organization, extracellular region, structural molecule and defense.

Fusaric acid instigates the invasion of banana by Fusarium oxysporum f. sp. cubense TR4.

Fusaric acid (FSA) is a phytotoxin produced by several Fusarium species and has been associated with plant disease development, although its role is still not well understood. Mutation of key genes in the FSA biosynthetic gene (FUB) cluster in Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) reduced the FSA production, and resulted in decreased disease symptoms and reduced fungal biomass in the host banana plants. When pretreated with FSA, both banana leaves and pseudostems exhibited increased sensitivity to Foc TR4 invasion. Banana embryogenic cell suspensions (ECSs) treated with FSA exhibited a lower rate of O2 uptake, loss of mitochondrial membrane potential, increased reactive oxygen species (ROS) accumulation, and greater nuclear condensation and cell death. Consistently, transcriptomic analysis of FSA-treated ECSs showed that FSA may induce plant cell death through regulating the expression of genes involved in mitochondrial functions. The results herein demonstrated that the FSA from Foc TR4 functions as a positive virulence factor and acts at the early stage of the disease development before the appearance of the fungal hyphae in the infected tissues.

Hypothermia induced by central injection of sucralose potentially occurs via monoaminergic pathways in the hypothalamus of chicks.

Oral administration of sucralose has been reported to stimulate food intake through inducing hypothalamic neuropeptide Y (NPY) in mice and fruit flies. However, the underlying mechanisms of action of sucralose in hypothermia and NPY and monoamine regulation remain unknown. The aim of the present study was to investigate central effects of sucralose on body temperature, NPY, and monoamine regulation, as well as its peripheral effects, in chicks. In Experiment 1, 5-day-old chicks were centrally injected with 1 µmol of sucralose, other sweeteners (erythritol and glucose), or saline. In Experiment 2, chicks were centrally injected with 0.2, 0.4, and 1.6 µmol of sucralose or saline. In Experiment 3, chicks were centrally injected with 0.8 µmol of sucralose or saline, with a co-injection of 100 µg fusaric acid (FA), an inhibitor of dopamine-ß-hydroxylase, to examine the role dopamine in sucralose induced hypothermia. In Experiment 4, 7-16-day-old chicks were orally administered with 75, 150, and 300 mg/2 ml distilled water or sucralose, daily. We observed that the central injection of sucralose, but not other sweeteners, decreased body temperature (P < .05) in chicks; however, the oral injection did not influence body temperature, food intake, and body weight gain. Central sucralose administration decreased dopamine and serotonin and stimulated dopamine turnover rate in the hypothalamus significantly (P < .05). Notably, sucralose co-injection with FA impeded sucralose-induced hypothermia. Sucralose decreases body temperature potentially via central monoaminergic pathways in the hypothalamus.

Evaluation of the cytotoxic and genotoxic effects of mycotoxin fusaric acid.

Fusaric acid (FA) is produced by several Fusarium species and is commonly found in grains. This investigation was performed to evaluate the cytotoxic and genotoxic effects of FA either in human cervix carcinoma (HeLa) cell line using 3-(4,5-dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide (MTT) assay and in human lymphocytes using chromosome aberrations (CAs), sister chromatid exchanges (SCEs), micronuclei (MN) as well as comet assay in vitro. The cells were treated with 0.78, 1.56, 3.125, 6.25, 12.50, 25, 50, 100, 200, and 400 µg/mL concentrations of FA. It has potent cytotoxic effect on HeLa cell line measured by MTT assay especially at higher concentrations (200, 400 µg/mL). The half of inhibitory concentration (IC50) evidenced by FA in the HeLa cells was 200 µg/mL at 24 h and between 200 and 400 µg/mL at 48 h. It was also observed that FA produced a significant decrease in mitotic index (MI) at 12.50 µg/mL compared to solvent control. Furthermore, it indicated a cytotoxic effect at the concentrations ranging from 25 to 400 µg/mL in human lymphocytes. The results of this research point out that being exposed to FA at high concentrations show cytotoxicity. Besides FA induced comet tail intensity at 3.125, 6.25, and 12.50 µg/mL concentrations in isolated human lymphocytes. On the other hand, no genotoxic effects were seen in human lymphocytes in vitro using CA, SCE and MN assays.

Zinc and Copper Enhance Cucumber Tolerance to Fusaric Acid by Mediating Its Distribution and Toxicity and Modifying the Antioxidant System.

Fusaric acid (FA), the fungal toxin produced by Fusarium oxysporum, plays a predominant role in the virulence and symptom development of Fusarium wilt disease. As mineral nutrients can be protective agents against Fusarium wilt, hydroponic experiments employing zinc (Zn) and copper (Cu) followed by FA treatment were conducted in a glasshouse. FA exhibited strong phytotoxicity on cucumber plants, which was reversed by the addition of Zn or Cu. Thus, Zn or Cu dramatically reduced the wilt index, alleviated the leaf or root cell membrane injury and mitigated against the FA inhibition of plant growth and photosynthesis. Cucumber plants grown with Zn exhibited decreased FA transportation to shoots and a 17% increase in toxicity mitigation and showed minimal hydrogen peroxide, lipid peroxidation level with the increased of antioxidant enzymes activity in both roots and leaves. Cucumber grown with additional Cu absorbed less FA but showed more toxicity mitigation at 20% compared to with additional Zn and exhibited decreased hydrogen peroxide level and increased antioxidant enzymes activity. Thus, adding Zn or Cu can decrease the toxicity of the FA by affecting the absorption or transportation of the FA in plants and mitigate toxicity possibly through chelation. Zn and Cu modify the antioxidant system to scavenge hydrogen peroxide for suppressing FA induction of oxidative damage. Our experiments could provide a theoretical basis for the direct application of micro-fertilizer as protective agents in farming.

Fusarium oxysporum f. sp. lycopersici C2H2 transcription factor FolCzf1 is required for conidiation, fusaric acid production, and early host infection.

The soil-borne, asexual fungus Fusarium oxysporum f.sp. lycopersici (Fol) is a causal agent of tomato wilt disease. The infection process of Fol comprises root recognition, adhesion, penetration, colonization of the root cortex and hyphal proliferation within the xylem vessels, which are under the regulation of virulence-involved transcription factors (TFs). In this study, we identified a gene, designated FolCZF1, which encodes a C2H2 TF in Fol. The homologs of FolCzf1 are also known to affect pathogenicity in F. graminearum and Magnaporthe oryzae on wheat and rice, respectively. We learned that FolCZF1 transcript level is upregulated in conidia and early host infection stage, which led us to hypothesize that FolCzf1 is associated with early host infection in Fol. The FolCZF1 deletion mutant (ΔFolCZF1) exhibited defects in growth rate, conidiation, conidia morphology and a complete loss of virulence on tomato root. Further microscopic observation showed that ΔFolCZF1 can penetrate the root but the primary infection hypha cannot extend its colonization inside the host tissue, suggesting that FolCzf1 TF plays an important role in early infection. Fusaric acid, a secondary metabolite produced by Fusarium species, is suggested as a virulence factor in many crop diseases. We found that FolCzf1 plays a critical role in fusaric acid production by regulating the expression of fusaric acid biosynthesis genes. In summary, FolCzf1 is required for conidiation, secondary metabolism, and early host infection in Fol, and we propose that homologs of FolCzf1 are required for early parasitic growth in other plant pathogenic filamentous fungi.

Histological and Gene Expression Analyses in Banana Reveals the Pathogenic Differences between Races 1 and 4 of Banana Fusarium Wilt Pathogen.

Fusarium oxysporum f. sp. cubense, the causative agent of Panama disease, is classified into three races: Foc1, Foc2, and Foc4. However, the histological characteristics, the accumulation of fusaric acid (FA), and resistant gene expression in banana infected with different races remain unclear. In this study, we compared the infection processes, FA contents, and gene expression levels in a Cavendish banana cultivar (Musa AAA Brazilian) inoculated with Foc1 and Foc4. Results showed that Foc4 can rapidly extend from the roots to the leaves, whereas Foc1 expands slowly from the roots to the rhizomes but cannot expand further upward. In addition, the colonization of plants by Foc4 was significantly higher compared with Foc1, as was the content of FA in those infected plant tissues. We observed that a large amount of starch granules was produced in the rhizomes and the number of starch granules was significantly higher after infection with Foc1 than after infection with Foc4. We further found that starch has an important inhibitory effect on the phytotoxicity induced by FA, thus leading to more resistance to the pathogens in the plants with high amounts of starch accumulation than in those with a low amount of starch accumulation. Moreover, the expression levels of 10 defense-related genes were analyzed and the results showed that the induction levels of those genes were higher after infection with Foc1 than after infection with Foc4. These results suggest that the observed differences in the invasion of host tissues and FA accumulation, and the number of starch granules and expression of defense-related genes, may contribute to a difference in virulence between the two races and the resulting difference in host resistance response, respectively.

Role of Phenolic Acids from the Rhizosphere Soils of Panax notoginseng as a Double-Edge Sword in the Occurrence of Root-Rot Disease.

Chemical agents in the rhizosphere soils of plants might have an influence on root-rot disease, which therefore might reveal the mechanism of root rot in Panax notoginseng (P. notoginseng). With this hypothesis the alterations of phenolic acids (PAs) in the rhizosphere soils of P. notoginseng after pathogen infection were determined. The effects of PAs on the growth of Fusarium oxysporum (F. oxysporum), a fungal pathogenic factor for P. notoginseng, as well as production of fusaric acid, a wilting agent for the plants, were also examined. The results indicate the presence of five PAs (ferulic acid, syringic acid, p-hydroxybenzoic acid, p-coumaric acid, and vanillic acid) in the rhizosphere soils of P. notoginseng, whose contents in the rhizosphere soils of healthy plants are higher than those of the diseased ones. Further we found that individual PA could inhibit the mycelium growth and spore production of F. oxysporum, but stimulate fusaric acid production as well, disclosing the double-edge sword role of PAs in the occurrence of root rot of P. notoginseng and paving the way for the intervention of P. notoginseng root rot via balancing PAs.

Fusaric Acid Induces DNA Damage and Post-Translational Modifications of p53 in Human Hepatocellular Carcinoma (HepG2 ) Cells.

Fusaric acid (FA), a common fungal contaminant of maize, is known to mediate toxicity in plants and animals; however, its mechanism of action is unclear. p53 is a tumor suppressor protein that is activated in response to cellular stress. The function of p53 is regulated by post-translational modifications-ubiquitination, phosphorylation, and acetylation. This study investigated a possible mechanism of FA induced toxicity in the human hepatocellular carcinoma (HepG2 ) cell line. The effect of FA on DNA integrity and post-translational modifications of p53 were investigated. Methods included: (a) culture and treatment of HepG2 cells with FA (IC50 : 580.32 µM, 24 h); (b) comet assay (DNA damage); (c) Western blots (protein expression of p53, MDM2, p-Ser-15-p53, a-K382-p53, a-CBP (K1535)/p300 (K1499), HDAC1 and p-Ser-47-Sirt1); and (d) Hoechst 33342 assay (apoptosis analysis). FA caused DNA damage in HepG2 cells relative to the control (P < 0.0001). FA decreased the protein expression of p53 (0.24-fold, P = 0.0004) and increased the expression of p-Ser-15-p53 (12.74-fold, P = 0.0126) and a-K382-p53 (2.24-fold, P = 0.0096). This occurred despite the significant decrease in the histone acetyltransferase, a-CBP (K1535)/p300 (K1499) (0.42-fold, P = 0.0023) and increase in the histone deacetylase, p-Ser-47-Sirt1 (1.22-fold, P = 0.0020). The expression of MDM2, a negative regulator of p53, was elevated in the FA treatment compared to the control (1.83-fold, P < 0.0001). FA also inhibited cell proliferation and induced apoptosis in HepG2 cells as evidenced by the Hoechst assay. Together, these results indicate that FA is genotoxic and post-translationally modified p53 leading to HepG2 cell death. J. Cell. Biochem. 118: 3866-3874, 2017. © 2017 Wiley Periodicals, Inc.

Microbial Resistance Mechanisms to the Antibiotic and Phytotoxin Fusaric Acid.

Fusaric acid (FA) produced by Fusarium oxysporum plays an important role in disease development in plants, including cotton. This non-specific toxin also has antibiotic effects on microorganisms. Thus, one expects a potential pool of diverse detoxification mechanisms of FA in nature. Bacteria and fungi from soils infested with Fusarium and from laboratory sources were evaluated for their ability to grow in the presence of FA and to alter the structure of FA into less toxic compounds. None of the bacterial strains were able to chemically modify FA. Highly FA-resistant strains were found only in Gram-negative bacteria, mainly in the genus of Pseudomonas. The FA resistance of the Gram-negative bacteria was positively correlated with the number of predicted genes for FA efflux pumps present in the genome. Phylogenetic analysis of predicted FA resistance proteins (FUSC, an inner membrane transporter component of the efflux pump) revealed that FUSC proteins having high sequence identities with the functionally characterized FA resistance protein FusC or Fdt might be the major contributors of FA resistance. In contrast, most fungi converted FA to less toxic compounds regardless of the level of FA resistance they exhibited. Five derivatives were detected, and the detoxification of FA involved either oxidative reactions on the butyl side chain or reductive reactions on the carboxylic acid group. The production of these metabolites from widely different phyla indicates that resistance to FA by altering its structure is highly conserved. A few FA resistant saprophytic or biocontrol strains of fungi were incapable of altering FA, indicating a possible involvement of efflux transporters. Deployment of both efflux and derivatization mechanisms may be a common feature of fungal FA resistance.

A Rapid Method with UPLC for the Determination of Fusaric Acid in Fusarium Strains and Commercial Food and Feed Products.

A rapid, sensitive and validated method for the determination of fusaric acid (FA) in several Fusarium strains and different commercial food and feed products is reported based on ultra-performance liquid chromatography. This method requires only crude sample by a simple extraction with methanol, and requires a very short time of 8 min for completion. Separation of FA was performed at injection volume of 1 µl with a 20:80 (v/v) water/acetonitrile mobile phase containing 0.1 % formic acid at a flow rate of 0.05 ml/min and detected with UV at 220 nm. Nice linearity and good correlation coefficient (R2 > 0.99) were obtained in the concentration range of 1-200 µg/ml. Validation was demonstrated using blank samples spiked at three different concentrations with standard solution, and the method yielded more than 98.2 % recovery efficiencies and below 2.56 % R.S.D. when applied in the analysis of FA produced by Fusarium verticillioides and a set of transgenic strains of this fungus. Satisfactory recoveries in the range of 79.1-105.8 % and R.S.D lower than 10 % were also obtained for the tested commercial food and feed products. The concentration FA detection in the transgenic strains ranged from 9.65 to 135 µg/kg (0.29-4.05 µg per gram of biomass). However, FA was not detected in most of the commercial products with the exception of niblet, oatmeal, red kidney bean and soybean, for which the concentrations of FA ranged from 2.5 to 18 µg/kg (below the permitted maximum). These results show that the proposed method has a great potential application to analyze FA from different sources rapidly.

Nitrate Protects Cucumber Plants Against Fusarium oxysporum by Regulating Citrate Exudation.

Fusarium wilt causes severe yield losses in cash crops. Nitrogen plays a critical role in the management of plant disease; however, the regulating mechanism is poorly understood. Using biochemical, physiological, bioinformatic and transcriptome approaches, we analyzed how nitrogen forms regulate the interactions between cucumber plants and Fusarium oxysporum f. sp. cucumerinum (FOC). Nitrate significantly suppressed Fusarium wilt compared with ammonium in both pot and hydroponic experiments. Fewer FOC colonized the roots and stems under nitrate compared with ammonium supply. Cucumber grown with nitrate accumulated less fusaric acid (FA) after FOC infection and exhibited increased tolerance to chemical FA by decreasing FA absorption and transportation in shoots. A lower citrate concentration was observed in nitrate-grown cucumbers, which was associated with lower MATE (multidrug and toxin compound extrusion) family gene and citrate synthase (CS) gene expression, as well as lower CS activity. Citrate enhanced FOC spore germination and infection, and increased disease incidence and the FOC population in ammonium-treated plants. Our study provides evidence that nitrate protects cucumber plants against F. oxysporum by decreasing root citrate exudation and FOC infection. Citrate exudation is essential for regulating disease development of Fusarium wilt in cucumber plants.

Production of Fusaric Acid by Fusarium spp. in Pure Culture and in Solid Medium Co-Cultures.

The ability of fungi isolated from nails of patients suffering from onychomycosis to induce de novo production of bioactive compounds in co-culture was examined. Comparison between the metabolite profiles produced by Sarocladium strictum, by Fusarium oxysporum, and by these two species in co-culture revealed de novo induction of fusaric acid based on HRMS. Structure confirmation of this toxin, using sensitive microflow NMR, required only three 9-cm Petri dishes of fungal culture. A targeted metabolomics study based on UHPLC-HRMS confirmed that the production of fusaric acid was strain-dependent. Furthermore, the detected toxin levels suggested that onychomycosis-associated fungal strains of the F. oxysporum and F. fujikuroi species complexes are much more frequently producing fusaric acid, and in higher amount, than strains of the F. solani species complex. Fusarium strains producing no significant amounts of this compound in pure culture, were shown to de novo produce that compound when grown in co-culture. The role of fusaric acid in fungal virulence and defense is discussed.