Bikaverin and fusaric acid from Fusarium oxysporum show antioomycete activity against Phytophthora infestans

Aims: To isolate and identify antioomycete substances from Fusarium oxysporum EF119 against Phytophthora infestans and to investigate their antimicrobial activities against various plant pathogenic bacteria, oomycetes and true fungi. Methods and Results: Two antioomycete substances were isolated from liquid cultures of F. oxysporum EF119, which shows a potent disease control efficacy against tomato late blight caused by P. infestans. They were identified as bikaverin and fusaric acid by mass and nuclear magnetic resonance spectral analyses. They inhibited the mycelial growth of plant pathogenic oomycetes and fungi. Fusaric acid also effectively suppressed the cell growth of various plant pathogenic bacteria, but bikaverin was virtually inactive. Treatment with bikaverin at 300 μg ml^?1 suppressed the development of tomato late blight by 71%. Fusaric acid provided effective control against tomato late blight and wheat leaf rust over 67% at concentrations more than 100 μg ml^?1. Conclusions: Both bikaverin and fusaric acid showed in vitro and in vivo antioomycete activity against P. infestans. Significance and Impact of the Study: Fusarium oxysporum EF119 producing both bikaverin and fusaric acid may be used as a biocontrol agent against tomato late blight caused by P. infestans.     

Stimulation of bikaverin production by sucrose and by salt starvation in Fusarium fujikuroi

The fungus Fusarium fujikuroi (Gibberella fujikuroi mating group C) exhibits a rich secondary metabolism that includes the synthesis of compounds of biotechnological interest, such as gibberellins, bikaverin, and carotenoids. The effect of the carbon source on their production was checked using a two-phase incubation protocol, in which nine different sugars were added upon transfer of the fungus from repressed to appropriate inducing conditions, i.e., nitrogen starvation for gibberellins and bikaverin and illumination for carotenoids production. Most of the carbon sources allowed the synthesis of these metabolites in significant amounts. However, bikaverin production was strongly increased by the presence of sucrose in comparison to other carbon sources, an effect not exhibited for the production of gibberellins and carotenoids. The bikaverin inducing effect was enhanced in the absence of phosphate and/or sulfate. Similar results were also observed in carotenoid-overproducing strains known to be altered in bikaverin production. The induction by salt starvation, but not by sucrose, correlated with an increase in messenger RNA levels of gene bik1, encoding a polyketide synthase of the bikaverin pathway.     

Selective production of bikaverin in a fluidized bioreactor with immobilized Gibberella fujikuroi

The best culture medium composition for the production of bikaverin by Gibberella fujikuroi in shake-flasks, i.e. 100 g glucose l^–1; 1 g NH₄Cl l^–1; 2 g rice flour l^–1; 5 g KH₂PO₄ l^–1 and 2.5 g MgSO₄ l^–1, was obtained through a fractional factorial design and then scaled-up to a fluidized bioreactor. The effects of carbon and nitrogen concentrations, inoculum size, aeration, flow rate and bead sizes on batch bikaverin production using immobilized G. fujikuroi in a fluidized bioreactor were determined by an orthogonal experimental design. Concentrations of up to 6.83 g bikaverin l^–1 were obtained when the medium contained 100 g glucose l^–1 and 1 g NH₄Cl l^–1 with an inoculum ratio of 10% v/v, an aeration rate of 3 volumes of air per volume of medium min^–1, and a bead size of 3 mm. Based on dry weight, the bikaverin production was 30–100 times larger than found in submerged culture and approximately three times larger than reported for solid substrate fermentation.     

Bikaverin production and applications

Bikaverin is a reddish pigment produced by different fungal species, most of them from the genus Fusarium, with antibiotic properties against certain protozoa and fungi. Chemically, bikaverin is a polyketide with a tetracyclic benzoxanthone structure, resulting from the activity of a specific class I multifunctional polyketide synthase and subsequent group modifications introduced by a monooxygenase and an O-methyltransferase. In some fungi, bikaverin is found with smaller amounts of a precursor molecule, called norbikaverin. Production of these metabolites by different fungal species depends on culture conditions, but it is mainly affected by nitrogen availability and pH. Regulation of the pathway has been investigated in special detail in the gibberellin-producing fungus Fusarium fujikuroi, whose genes and enzymes responsible for bikaverin production have been recently characterized. In this fungus, the synthesis is induced by nitrogen starvation and acidic pH, and it is favored by other factors, such as aeration, sulfate and phosphate starvation, or sucrose availability. Some of these inducing agents increase mRNA levels of the enzymatic genes, organized in a coregulated cluster. The biological properties of bikaverin include antitumoral activity against different cancer cell lines. The diverse biological activities and the increasing information on the biochemical and genetic basis of its production make bikaverin a metabolite of increasing biotechnological interest.     

Mathematical description of bikaverin production in a fluidized bed bioreactor

Summary  Growth of Gibberella fujikuroi in submerged cultures occurs as micelles or filamentous hyphae dispersed in fluid and pellets or stable, spherical agglomerations. Gibberella fujikuroi growth, substrate consumption and bikaverin production kinetics obtained from submerged batch fermentation were fitted to three different sigmoid models: two and three-parameter Gompertz models and one Logistic model. Growth fitting was used to compare between models and select the best one by means of an F test. The best model for describing growth was the two-parameter Gompertz model and was used for glucose consumption and bikaverin production fitting. Data from eight different schemes of fermentations were analysed and parameter estimation was carried out by means of minimization of residual sum of squares. Some characteristic values obtained with the two-parameter Gompertz model fit are: μ=0.028 h⁻¹, Y_x/s=0.1089 g substrate/g biomass, α =0.1384 g product/g biomass.     

Growth substrates control the ability of Fusarium oxysporum to solubilize low-rank coal

The ability of the fungus Fusarium oxysporum to solubilize lignite was found to depend on the presence of a specific carbon source. When grown on glucose or another carbohydrate, the fungus is unable to solubilize coal but it produces the red dye bikaverin. In the coal-solubilizing state, which can be induced by cultivation in the presence of glutamate or gluconate, the fungus does not produce bikaverin. The presence or absence of the pigment can therefore be taken as an indicator of the ability of the fungus to solubilize coal. Addition of extracted and purified bikaverin to F. oxysporum growing on glutamate or gluconate inhibits coal solubilization. Hence, F. oxysporum offers a suitable system for investigating the mechanism of microbial coal degradation by comparing the two growth-substrate-controlled physiological states.     

Neuroprotective Effects of Bikaverin on H2O2-Induced Oxidative Stress Mediated Neuronal Damage in SH-SY5Y Cell Line

The generation of free radicals and oxidative stress has been linked to several neurodegenerative diseases including Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and Amyotrophic lateral sclerosis. The use of free radical scavenging molecules for the reduction of intracellular reactive oxygen species is one of the strategies used in the clinical management of neurodegeneration. Fungal secondary metabolism is a rich source of novel molecules with potential bioactivity. In the current study, bikaverin was extracted from Fusarium oxysporum f. sp. lycopersici and its structural characterization was carried out. Further, we explored the protective effects of bikaverin on oxidative stress and its anti-apoptotic mechanism to attenuate H₂O₂-induced neurotoxicity using human neuroblastoma SH-SY5Y cells. Our results elucidate that pretreatment of neurons with bikaverin attenuates the mitochondrial and plasma membrane damage induced by 100 µM H₂O₂ to 82 and 26 % as evidenced by MTT and LDH assays. H₂O₂ induced depletion of antioxidant enzyme status was also replenished by bikaverin which was confirmed by Realtime Quantitative PCR analysis of SOD and CAT genes. Bikaverin pretreatment efficiently potentiated the H₂O₂-induced neuronal markers, such as BDNF, TH, and AADC expression, which orchestrate the neuronal damage of the cell. The H₂O₂-induced damage to cells, nuclear, and mitochondrial integrity was also restored by bikaverin. Bikaverin could be developed as a preventive agent against neurodegeneration and as an alternative to some of the toxic synthetic antioxidants.     

Changes in protein secretion of Aspergillus niger caused by the reduction of the water activity by potassium chloride

The effect of the rapid reduction of the water activity (a_w) on the extracellular protein and amylolytic activity of Aspergillus niger was studied. An a_w value gradient from 0.90 to 0.99 in KCl solutions was applied for the mycelium treatment. It was found that the a_w reduction considerably influenced the protein secretion. This phenomenon was dependent on the age of the treated mycelium and the range of the a_w gradient. The highest protein and enzyme secretion yields were obtained at a_w = 0.98 using a 72-h old mycelium. In comparison with the non-treated mycelium, the increase in the secretion amounted to about 60% for the amylolytic activity and 37% for the soluble protein, respectively. It was shown that the mycelium incubated in KCl solutions of an a_w value from 0.90 to 0.99 had the ability for regeneration in fresh CZAPEK-DOX medium. The effect of the osmotic shock on the protein secretion was limited only for the treated cell population and declined in the mycelium which was regenerated after the transfer into the culture medium.     

Aflatoxin is degraded by heated and unheated mycelia,filtrates of homogenized mycelia and filtrates of broth cultures of Aspergillus parasiticus

Steaming one-half of a lot of 9-day-old mycelia of Aspergillus parasiticus NRRL 2999 for 6 min resulted in little or no subsequent degradation of aflatoxin B₁ or G₁ by these mycelia. The other half of these mycelia was not heat-treated and degraded aflatoxins B₁ and G₁ Filtrates of the growth substrate which remained after the mycelium was removed from 8- to 15-day old cultures of A. parasiticus NRRL 2999 did not degrade substantial amounts of aflatoxin B₁ or G₁, whereas mycelia originally produced on these filtrates degraded substantial amounts of both aflatoxins. The supernatant fluid from homogenates of 9-day-old mycelia of A. parasiticus NRRL 2999 degraded aflatoxins B₁ and G₁ when 0.1 M or 1.0 M phosphate buffer, pH 6.5, was used to suspend the homogenate. These data support the hypothesis that the aflatoxin degrading factor(s) present in the mycelium of A. purasiticus is/are enzyme(s) or at least influenced by enzyme(s).     

Lipid-to-gibberellin metabolic switching in Fusarium moniliforme via the action of sesamol

Sesamol (3,4-methylenedioxyphenol) at 2.5 mM inhibited growth of Fusarium moniliforme by about 40% and lipid accumulation by 35%. Gibberellin (GA₃) accumulation was increased by 20-fold, to 63 mg g^–1 biomass, in the presence of sesamol indicating that the acetyl-CoA destined for fatty acid biosynthesis was now being switched into secondary metabolite (GA₃) accumulation. Synthesis of other metabolites from acetyl-CoA, such as bikaverin and carotenoids, though were not increased in the presence of sesamol. Metabolic switching is therefore feasible by judicious use of selected inhibitors that can thus block primary metabolic routes but which do not affect secondary metabolites.     

A Functional Bikaverin Biosynthesis Gene Cluster in Rare Strains of Botrytis cinerea Is Positively Controlled by VELVET

The gene cluster responsible for the biosynthesis of the red polyketidic pigment bikaverin has only been characterized in Fusarium ssp. so far. Recently, a highly homologous but incomplete and nonfunctional bikaverin cluster has been found in the genome of the unrelated phytopathogenic fungus Botrytis cinerea. In this study, we provided evidence that rare B. cinerea strains such as 1750 have a complete and functional cluster comprising the six genes orthologous to Fusarium fujikuroi ffbik1-ffbik6 and do produce bikaverin. Phylogenetic analysis confirmed that the whole cluster was acquired from Fusarium through a horizontal gene transfer (HGT). In the bikaverin-nonproducing strain B05.10, the genes encoding bikaverin biosynthesis enzymes are nonfunctional due to deleterious mutations (bcbik2-3) or missing (bcbik1) but interestingly, the genes encoding the regulatory proteins BcBIK4 and BcBIK5 do not harbor deleterious mutations which suggests that they may still be functional. Heterologous complementation of the F. fujikuroi Δffbik4 mutant confirmed that bcbik4 of strain B05.10 is indeed fully functional. Deletion of bcvel1 in the pink strain 1750 resulted in loss of bikaverin and overproduction of melanin indicating that the VELVET protein BcVEL1 regulates the biosynthesis of the two pigments in an opposite manner. Although strain 1750 itself expresses a truncated BcVEL1 protein (100 instead of 575 aa) that is nonfunctional with regard to sclerotia formation, virulence and oxalic acid formation, it is sufficient to regulate pigment biosynthesis (bikaverin and melanin) and fenhexamid HydR2 type of resistance. Finally, a genetic cross between strain 1750 and a bikaverin-nonproducing strain sensitive to fenhexamid revealed that the functional bikaverin cluster is genetically linked to the HydR2 locus.     

Fungal biomass production in response to elevated atmospheric CO2 in a Glomus mosseae–Prunus cerasifera model system

Biomass and length of intraradical and extraradical mycorrhizal mycelium under ambient (aCO₂) and elevated (eCO₂ ) atmospheric CO₂ was investigated using a non-destructive in vivo experimental model system. Time-course experiments allowed measurements of intact extraradical mycelium spreading from mycorrhizal roots of Prunus cerasifera micropropagated plants inoculated with the arbuscular mycorrhizal fungus Glomus mosseae, in controlled environmental chambers. The length of extraradical mycelium was significantly increased at the highest CO₂ concentration, ranging from 10.7 to 20.3 m at aCO₂ and eCO₂, respectively. The biochemical determination of mycelial glucosamine content allowed the evaluation of intraradical and extraradical fungal biomass, which were 2 and 3 times larger at eCO₂ than at aCO₂. Present data show that Glomus mosseae responds to increases of CO₂ concentrations producing larger mycorrhizal networks which may potentially represent carbon sink agents in soil ecosystems.     

Simple method for separating the chlamydoconidia of Candida albicans from its mycelium

Different physical and chemical methods were used to detach the chlamydoconidia of Candida albicans from its mycelium. The action of concentrated H₂SO₄ acid for a 4-min period on cultures lysed both the mycelium and the outer but not the inner wall layer of the chlamydoconidia.The sulfuric acid procedure is recommended as the best method to obtain mycelium free chlamydoconidia because of its simplicity, rapidity and low cost.     

Viability of ectomycorrhizal fungus mycelium entrapped in calcium alginate gel

 We studied the viability of fragmented mycelium of Pisolithus tinctorius and Paxillus involutus entrapped in calcium alginate gel to determine the efficacy of this method of producing ectomycorrhizal fungus inoculum. Fungi were grown in MMN solution at 28  °C before being fragmented in a blender and subsequently entrapped in calcium alginate. We tested different ratios of alginate and mycelium suspension to 0.7 M CaCl₂. The ratio 8 : 10 resulted in well-formed beads of the highest viability for Paxillus involutus (99%) and for Pisolithus tinctorius (75%). Paxillus involutus mycelium was more than 90% viable when entrapped mycelium was 10 to 50 days old, and Pisolithus tinctorius attained its highest viability (55%) for 20- to 40-day-old mycelium. Gel entrapped Paxillus involutus mycelium grew well at all temperatures after 30 days of storage, but viability significantly decreased after 60 days storage at 6  °C on dry filter paper. For gel-entrapped Pisolithus tinctorius mycelium, viability was highest when stored at 25  °C in 0.7 M CaCl₂. Entrapment of Paxillus involutus fragmented mycelium in calcium alginate beads under the conditions that we propose can be used successfully to produce inoculum. Accepted: 11 October 1998     

Optimization of protoplast formation,regeneration, and viability in Microsporum gypseum

Factors affecting high yields, regeneration frequencies, and viability of protoplasts from clonal cultures of Microsporum gypseum were investigated. Maximum yields of protoplasts were obtained after 6 hrs digestion of 2–4 days old mycelium with Novozyme 234 using CaCl₂ (0.4 M) as an osmotic stabilizer and glycine + HCl (pH 4.5) as the buffer system. Mercaptoethanol + dithiothreitol (0.01 M) proved to be the best pretreatment of mycelium prior to digestion with enzyme. A regeneration frequency of 94.4% was obtained using the top agar method with complete medium (pH 6.5) containing 0.5% agar and 0.4 M CaCl₂ as an osmoticum. Colonies from regenerated protoplasts on medium containing CaCl₂ were pigmented and completely powdery with high sporulation. Protoplast viability was studied in osmotic stabilizer supplemented with glucose or glutamine. After 24 hrs, glucose (2%) and glutamine (2%) enhanced protoplast viability by 22% and 23%, respectively. Protein synthesis, as measured by ³H-lysine uptake, matched the viability profile determined by fluorescence microscopy.     

Effect of phosphate on the toxicity of phosphite in Phytophthora palmivora

The concentration of P_i in the growth medium had a marked effect on the amount of the fungicide phosphite accumulated by Phytophthora palmivora. The mass of mycelium, produced after 7 days growth in a medium containing excess P_i, was not markedly inhibited until phosphite concentrations of 10 mM or greater were supplied. In contrast, in a P_i-deficient medium, growth was inhibited by 0.1 mM phosphite. In the latter medium, the fungus took up significantly more phosphite once P_i had been depleted from the medium (from day 4 to 6). When 7 day old mycelium was presented with either 1 mM P_i or phosphite, the P_i-deficient mycelium transported either anion at almost eight times the rate found in the P_i-excess mycelium. The level of alkaline phosphatase present in the P_i-deficient mycelium was also significantly elevated.     

Degradation of lignin and lignin related compounds by protoplasts isolated from Fomes annosus

Protoplasts from a lignolytic fungus Fomes annosus were prepared through enzymatic hydrolysis of mycelium utilizing Novozym, a wall lytic enzyme preparation. Isolated protoplasts and living mycelium were compared in their ability to degrade ¹⁴C-labelled lignin related phenols and dehydropolymers of labelled coniferyl alcohol (synthetic lignin). The amounts of ¹⁴CO₂ released from O¹⁴CH₃-groups, ¹⁴C-2-side chains and ¹⁴C-rings by protoplasts was in the same range as those for intact mycelium. The methoxyl groups of synthetic lignin were more rapidly metabolized by protoplasts than by mycelium. When calculated in dpm of released ¹⁴CO₂ per mg protein the decomposition of ¹⁴C-labelled synthetic lignin and lignin-related monomers in a hyphae-free system of protoplasts was considerable higher than that obtained by the intact mycelium. The presence of intact hyphae is thus not necessary for lignin degradation to occur.Non-common-abbreviations used DHP Dehydropolymer of coniferyl alcohol - LS lignosulfonates prepared from DHP     

The Effects of Inorganic Elements on the Reduction of Phytophthora Stem Rot Disease of Soybean, the Growth Rate and Zoospore Release of Phytophthora sojae

The effects of several inorganic elements contained in B5 medium on Phytophthora stem rot disease reduction of Glycine max (L.) Merr. cv. Chusei‐Hikarikuro, fungal growth of Phytophthora sojae isolate and zoospore release were investigated. Application of B5 solution and macro inorganic nutrients in the B5 medium prior to inoculation significantly inhibited infection, compared with controls. Various concentrations of KNO₃, (NH₄)₂SO₄, MgSO₄, CaCl₂ and NaH₂PO₄ in the presence of macro inorganic nutrients were investigated in an effort to determine the elements most effective in suppressing the incidence of disease. A concentration of 2.47–24.7 mm KNO₃ and 0.1–10.2 mm CaCl₂ greatly inhibited infection. Although mycelium growth of the isolate was affected by the potassium and calcium concentration, no significant relationship was observed between inhibition of the growth rate and disease reduction at 2.47 mm KNO₃ and 0.1–5.1 mm CaCl₂ application. Disease suppression recorded in laboratory experiments using pathogen mycelium was due to the response of plant tissues rather than a direct inhibition of pathogen fungal growth by the application of potassium or calcium. The extent of disease reduction was related to an increased potassium and calcium uptake by plants, suggesting that the effective elements in reducing Phytophthora stem rot were potassium and calcium. The presence of 2.47–247 mm KNO₃ and 5.1–10.2 mm CaCl₂ decreased the release of zoospores, although 0.1–2.5 mm CaCl₂ significantly induced zoospore release. These results suggest that applying a solution containing more than 2.47 mm of potassium and 5.1 mm of calcium can decrease the incidence of disease in agricultural fields by the inhibition of zoospore release.     

Cyathuscavins A, B, and C, new free radical scavengers with DNA protection activity from the Basidiomycete Cyathus stercoreus

Three new polyketides, cyathuscavins A (1), B (2), and C (3) were isolated from the mycelium culture of Cyathus stercoreus. The structures of the compounds were elucidated on the basis of NMR and mass spectroscopic data. Antioxidant activities of the compounds were evaluated by the scavenging ability against ABTS⁺, DPPH, and superoxide anion radicals. Cyathuscavins A–C showed significant antioxidant activity comparable to those of reference antioxidants, BHA and Trolox. Cyathuscavins A–C protected supercoiled plasmid DNA from Fe²⁺/H₂O₂-induced breakage.