Design, synthesis and biological evaluation of novel flavone Mannich base derivatives as potential antibacterial agents.

A series of novel Mannich base derivatives of flavone containing benzylamine moiety was synthesized using the Mannich reaction. The results of antifungal activity are not ideal, but its antifungal effect has a certain increase compared to flavonoids. After that, four bacteria were used to test antibacterial experiments of these compounds; compound 5g (MIC = 0.5, 0.125 mg/L) showed significant inhibitory activity against Staphylococcus aureus and Salmonella gallinarum compared with novobiocin (MIC = 2, 0.25 mg/L). Compound 5s exhibited broad spectrum antibacterial activity (MIC = 1, 0.5, 2, 0.05 mg/L) against four bacteria. The selected compounds 5g and 5s exhibit potent inhibition against Topo II and Topo IV with IC50 values (0.25-16 mg/L). Molecular docking model showed that the compounds 5g and 5s can bind well to the target by interacting with amino acid residues. It will provide some valuable information for the commercial antibacterial agents.

Screening of lactic acid bacteria and yeast strains to select adapted anti-fungal co-cultures for cocoa bean fermentation.

Contamination with filamentous fungi during cocoa bean fermentation and drying reduces the quality of cocoa beans and poses a health risk for consumers due to the potential accumulation of mycotoxins. The aim of this study was to develop anti-fungal lactic acid bacteria (LAB)-yeast co-cultures by selecting anti-fungal strains best adapted to the cocoa bean fermentation process from 362 LAB and 384 yeast strains isolated from cocoa bean post-harvest processes. The applied multiphasic screening approach included anti-fungal activity tests in vitro and in vivo and assessment of the carbon metabolism and stress tolerance of the anti-fungal strains in a cocoa pulp simulation medium. The anti-fungal strains, Lactobacillus fermentum M017, Lb. fermentum 223, Hanseniaspora opuntiae H17, and Saccharomyces cerevisiae H290, were selected based on their high fungal growth inhibition capacity and their well-adapted metabolism. Up to seven filamentous fungal strains of the genera Aspergillus, Penicillium, and Gibberella were inhibited on average by 63 and 75% of the maximal inhibition zone by M017 and 223, respectively, and by 25 and 31% by the strains H17 and H290, respectively. Both Lb. fermentum strains converted the medium's glucose, fructose, and citric acid into 20.4-23.0 g/l of mannitol, 3.9-6.2 g/l acetic acid, and 8.6-10.3 g/l lactic acid, whereas the two yeast strains metabolized glucose and fructose to produce 7.4-18.4 g/l of ethanol. The Lb. fermentum strains were further characterized as particularly tolerant towards ethanol, acetic acid, and heat stress and both yeast strains tolerated high amounts of ethanol and lactic acid in the medium. Finally, the anti-fungal in vivo assays revealed that the two Lb. fermentum strains completely inhibited growth of the citrinin-producing strain, P. citrinum S005, and the potentially fumonisin-producing strain, G. moniliformis S003, on the surface of cocoa beans. Furthermore, growth of the aflatoxin-producer A. flavus S075 was inhibited after 10-14 days by all four selected anti-fungal strains, i.e. Lb. fermentum M017, Lb. fermentum 223, H. opuntiae H17, and Sacc. cerevisiae H290, at 51-95% when applied as single cultures and at 100% when the strains were combined into four co-cultures, each composed of a Lb. fermentum and one of the two yeast strains. As a conclusion, these four LAB-yeast co-cultures are recommended for future applications to limit the growth of filamentous fungi and the concomitant mycotoxin production during the fermentation of cocoa beans.

Characterization and mechanism of copper biosorption by a highly copper-resistant fungal strain isolated from copper-polluted acidic orchard soil.

In this paper, a highly copper-resistant fungal strain NT-1 was characterized by morphological, physiological, biochemical, and molecular biological techniques. Physiological response to Cu(II) stress, effects of environmental factors on Cu(II) biosorption, as well as mechanisms of Cu(II) biosorption by strain NT-1 were also investigated in this study. The results showed that NT-1 belonged to the genus Gibberella, which exhibited high tolerance to both acidic conditions and Cu(II) contamination in the environment. High concentrations of copper stress inhibited the growth of NT-1 to various degrees, leading to the decreases in mycelial biomass and colony diameter, as well as changes in morphology. Under optimal conditions (initial copper concentration: 200 mg L-1, temperature 28 °C, pH 5.0, and inoculum dose 10%), the maximum copper removal percentage from solution through culture of strain NT-1 within 5 days reached up to 45.5%. The biosorption of Cu(II) by NT-1 conformed to quasi-second-order kinetics and Langmuir isothermal adsorption model and was confirmed to be a monolayer adsorption process dominated by surface adsorption. The binding of NT-1 to Cu(II) was mainly achieved by forming polydentate complexes with carboxylate and amide group through covalent interactions and forming Cu-nitrogen-containing heterocyclic complexes via Cu(II)-π interaction. The results of this study provide a new fungal resource and key parameters influencing growth and copper removal capacity of the strain for developing an effective bioremediation strategy for copper-contaminated acidic orchard soils.

C-coordinated O-carboxymethyl chitosan metal complexes: Synthesis, characterization and antifungal efficacy.

A novel type of O-carboxymethyl chitosan Schiff bases (O-CSPX) was synthesized via a condensation reaction. After the coordination reaction of cupric ions, zinc ions and nickel ions, metal complexes (O-CSPX-M) were achieved. The theoretical structure of O-CSPX-M calculated by Gaussian 09 reveals that the copper ions and nickel ions underwent dsp2 hybridization, the zinc ions underwent sp3 hybridization, and they all coordinated by the carbon atom in the p-π conjugate group. Then, the structures were confirmed by FT-IR, 1H NMR, CP-MAS 13C NMR, elemental analysis, DSC and XRD. The antifungal properties of O-CSPX-M against Phytophthora capsici (P. capsici), Gibberella zeae (G. zeae), Fusarium oxysporum (F. oxysporum) and Botrytis cinerea (B. cinerea) were evaluated at concentrations ranging from 0.05mg/mL to 0.40mg/mL. The experiments indicated that the derivatives have significantly enhanced antifungal activity after metal ions complexation compared with the original chitosan. Moreover, it was shown that 0.20mg/mL of O-CSPX-Cu can 100% inhibit the growth of P. capsici and 0.20mg/mL of O-CSPX-Ni can 87.5% inhibit the growth of B. cinerea. In addition, the phytotoxicity assay and cell viability assay were also evaluated. The experimental results may provide a novel direction for the development of metal fungicides.

Gibberellic Acid Production by Different Fermentation Systems Using Citric Pulp as Substrate/Support.

Gibberellic acid (GA3) is an important phytohormone, a member of gibberellins family, which acts as a promoter and regulator of plant growth. This study aimed to evaluate GA3 production by Fusarium moniliforme LPB03 and Gibberella fujikuroi LPB06 using different techniques of fermentation, solid state fermentation (SSF), submerged fermentation (SmF), and semisolid state fermentation (SSSF), and different types of bioreactors. In all techniques, citric pulp (CP), a subproduct obtained from the extraction of orange juice, was employed as the substrate/support. GA3 production by SSF reached 7.60 g kg-1 and 7.34 g kg-1 in Erlenmeyer flasks and column bioreactors, respectively. For SmF, the highest concentration of GA3 obtained was 236.00 mg L-1 in Erlenmeyer flasks, 273.00 mg L-1 in a 10 L stirred tank reactor (STR), and 203.00 mg L-1 in a 1.5 L bubble column reactor (BCR). SSSF was conducted with a CP suspension. In this case, GA3 concentration reached 331.00 mg L-1 in Erlenmeyer flasks and 208 mg L-1 in a BCR. The choice of the fermentation technique is undoubtedly linked to the characteristics and productivity of each process. The methods studied are inexpensive and were found to produce good proportions of GA3, making them suitable for several applications.

Larval western bean cutworm feeding damage encourages the development of Gibberella ear rot on field corn.

BACKGROUND: A 2 year study was conducted to determine whether western bean cutworm (Striacosta albicosta Smith) (WBC) larval feeding damage increases severity of the fungal disease Gibberella ear rot [Fusarium graminearum (Schwein.) Petch] in field corn (Zea mays L.). The effect of a quinone-outside inhibiting fungicide, pyraclostrobin, on Gibberella ear rot severity and mycotoxin production, both with and without WBC pressure, was also evaluated. The impact of each variable was assessed individually and in combination to determine the effect of each upon ear disease severity. RESULTS: There was a positive correlation between the presence of WBC larvae in field corn and Gibberella ear rot severity under inoculated conditions in the 2 years of the experiment. An application of pyraclostrobin did not impact Gibberella ear rot development when applied at corn growth stage R1 (silks first emerging). CONCLUSION: Feeding damage from WBC larvae significantly increases the development of F. graminearum in field corn. We conclude that an effective integrated management strategy for Gibberella ear rot should target the insect pest first, in an effort to limit disease severity and subsequent mycotoxin production by F. graminearum in kernels. © 2016 Society of Chemical Industry.

Kinetic analysis of the uptake of glucose and corn oil used as carbon sources in batch cultures of Gibberella fujikuroi.

This study determined the specific uptake rate of glucose and corn oil substrates used as carbon sources in batch cultures of Gibberella fujikuroi. We tested three biological models of growth rate: Monod, logistic and lag-exponential. With respect to the substrate consumption rate, we tested two models: constant cell yield (CCY) and law of mass action (LMA). The experimental data obtained from the culture with glucose as substrate correlated satisfactorily with the logistic/LMA model, indicating that the cell yield was variable. In the case of corn oil as carbon source, considering total residual lipids as substrate in the culture broth, the model with the best correlation was the lag-exp/CCY model. The quantification by GC of the three main fatty acids (linoleic, oleic and palmitic) in the culture medium showed a cumulative behavior, with a maximum concentration of each acid at 36 h. We established a more explicit mechanism of the consumption of corn oil, consisting of two stages: generation of fatty acids by hydrolysis and consumption by cellular uptake. The kinetic of hydrolysable lipids was of first order. We found that the hydrolysis rate of corn oil is not a limiting factor for the uptake of fatty acids by the microorganism. We also established, based on the analysis of the identical mathematical structure of consumption kinetics, that the uptake of fatty acids is faster than the uptake of glucose.

Influence of prebiotics, probiotics and protein ingredients on mycotoxin bioaccessibility.

The aim of this study was to investigate the influence of prebiotic compounds (cellulose and inulin), food ingredients (milk whey, ß-lactoglobulin and calcium caseinate) and several probiotic microorganisms on the bioaccessibility of beauvericin (BEA), enniatins (ENs A, A1, B, B1), deoxynivalenol (DON) and zearalenone (ZEA) present in wheat crispy bread produced with wheat flour previously fermented with F. tricinctum, F. culmorum and G. zeae. The bioaccessibility of mycotoxins was determined by a dynamic simulated gastrointestinal digestion system, imitating the human digestive physiological conditions of the gastrointestinal tract. Mycotoxins were determined in the simulated intestinal fluids by liquid chromatography-tandem mass spectrometry (LC-MS/MS). EN bioaccessibility ranged from 15.1 to 30.6%, whereas the values evidenced for BEA ranged from 12 to 19%. DON showed bioaccessibility data ranging from 0.8 to 5.6% whereas for ZEA the data evidenced ranged from 26 to 44%. The bioaccessibility reduction evidenced using probiotic microorganisms for the mycotoxins studied ranged from 21 to 27.1% for ENs, from 29 to 39.7% for DON, from 41 to 57% for ZEA and from 6.6 to 10.5% for BEA. The addition of prebiotic and bioactive microorganisms decreased the bioaccessibility of mycotoxins, with a concentration-dependent behavior, thus being a potential strategy for reducing human exposure to these minor mycotoxins.

Skin nodules in a patient with acute lymphoblastic leukaemia.

Opportunistic infections cause a significant morbidity and mortality in immunocompromised patients. We describe the case of a patient with skin fusariosis and a probable cerebral toxoplasmosis after UCB stem cell transplantation for B-cell acute lymphoblastic leukaemia. Fusarium species (spp) infections are difficult to treat. To date, there has been no consensus on the treatment of fusariosis and the management of its side effects. Given the negative pretransplant Toxoplasma serology in this case, identifying the origin of the Toxoplasma infection was challenging. All usual transmission routes were screened for and ruled out. The patient's positive outcome was not consistent with that of the literature reporting 60% mortality due to each infection.

Extractive fermentation of gibberellic acid with free and immobilized Gibberella fujikuroi.

Gibberelic acid fermentation using extractive methods was carried out in the presence of corn oil and Alamine 336. Gibberella fujikuroi fungus (NRRL 2278) was used to produce gibberellic acid. Oleyl alcohol was a diluting agent for Alamine 336. The effects of oleyl alcohol (100%, v/v), corn oil (5-25%, v/v), the concentration of Alamine 336 in oleyl alcohol, and feeding air were examined in this study. According to the results, oleyl alcohol was not effective on the production. On the other hand, oleyl alcohol solutions containing 15-30% (v/v) Alamine 336 showed effects as a toxic substance. In order to reduce solvent toxicity, corn oil was used. Addition of corn oil increased the concentration of gibberellic acid 1.3-fold compared to the control. Then the effects of immobilization and co-immobilization on extractive gibberelic acid fermentation were investigated. The highest total gibberellic acid concentration of 158.9 mg/L was produced with immobilized cells and feeding air by using extractive fermentation. The yield of gibberellic acid increased about 2.6-fold compared with the shake-flask fermentation (60.5 mg/L) without organic solutions.

[Screening and condition optimization of a strain for efficiently biotransformation of saponins in Dioscorea zingiberensis into diosgenin].

Diosgenin is an important raw material in steroid hormone and widely used in pharmaceutical industry. The traditional method for diosgenin production is acidolysis, which causes serious pollution. In order to obtain a cleaner and more efficient approach of diosgenin production, a strain of Gibberella intermedia WX12 (the sexual stage of Fusarium proliferatum) was screened from the strains deposited in our laboratory. This strain converted saponins in Dioscorea zingiberensis C.H. Wright (DZW) into diosgenin. The conversion medium was optimized by statistical experimental design. The optimized conversion medium was as follows (g/L): glucose 20.6, yeast extract 5.0, NaCl 1, K2PO4 3, ZnSO4 x 7H2O 1.5 and saponins 3. Under the optimal conditions, the yield of diosgenin achieved to (31 +/- 0.3) mg/g DZW, which was 3 times higher than that of the original medium.

[Breeding of high 3beta,7alpha,15alpha-trihydroxy-5-androsten-17-one transforming strains and their conversion process optimization].

In order to improve transformation efficiency of dehydroepiandrosterone (DHEA) into 3beta,7alpha,15alpha-trihydroxy-5-androsten-17-one (7alpha,15alpha-diOH-DHEA) by Gibberella intermedia CA3-1, we investigated the strains breeding and their conversion process optimization. G. intermedia CA3-1 strains were treated with 0.12 mg/mL 1-methyl-3-nitro-1-nitroso-guanidin (NTG) for 30 min and chosen by 350 micromol/L minimum inhibitory concentration ketoconazole resistance marker. The high production strain named M-10 with a good genetic stability was selected and the product molar yield achieved to 70.2%, which was 20% higher than that of original strain. Under the improved conversion process with the DHEA concentration of 5 g/L, the product molar yield of the mutant M-10 reached 75.6%, which was improved by 31.3% than that of original strain.

Mitochondrial carnitine-dependent acetyl coenzyme A transport is required for normal sexual and asexual development of the ascomycete Gibberella zeae.

Fungi have evolved efficient metabolic mechanisms for the exact temporal (developmental stages) and spatial (organelles) production of acetyl coenzyme A (acetyl-CoA). We previously demonstrated mechanistic roles of several acetyl-CoA synthetic enzymes, namely, ATP citrate lyase and acetyl-CoA synthetases (ACSs), in the plant-pathogenic fungus Gibberella zeae. In this study, we characterized two carnitine acetyltransferases (CATs; CAT1 and CAT2) to obtain a better understanding of the metabolic processes occurring in G. zeae. We found that CAT1 functioned as an alternative source of acetyl-CoA required for lipid accumulation in an ACS1 deletion mutant. Moreover, deletion of CAT1 and/or CAT2 resulted in various defects, including changes to vegetative growth, asexual/sexual development, trichothecene production, and virulence. Although CAT1 is associated primarily with peroxisomal CAT function, mislocalization experiments showed that the role of CAT1 in acetyl-CoA transport between the mitochondria and cytosol is important for sexual and asexual development in G. zeae. Taking these data together, we concluded that G. zeae CATs are responsible for facilitating the exchange of acetyl-CoA across intracellular membranes, particularly between the mitochondria and the cytosol, during various developmental stages.

Functional analyses of the nitrogen regulatory gene areA in Gibberella zeae.

Fusarium head blight caused by Gibberella zeae is a prominent disease of cereal crops that poses serious human health concerns due to the contamination of grains with mycotoxins. In this study, we deleted an orthologue of areA, which is a global nitrogen regulator in filamentous fungi, to characterize its functions in G. zeae. The areA deletion resulted in an inability to use nitrate as a sole nitrogen source, whereas urea utilization was partially available. The virulence of ΔareA strains on wheat heads was markedly reduced compared with the wild-type strain. The areA mutation triggered loss of trichothecene biosynthesis but did not affect zearalenone biosynthesis. The ΔareA strains showed immaturity of asci and did not produce mature ascospores. Chemical complementation by urea restored normal sexual development, whereas the virulence and trichothecene production were not affected by urea addition. GFP-AreA fusion protein was localized to nuclei, and its expression increased in response to nitrogen-limiting conditions. These results suggest that areA-dependent regulation of nitrogen metabolism is required for vegetative growth, sexual development, trichothecene biosynthesis, and virulence in G. zeae.

Synthesis and bioactivity of pyrazole acyl thiourea derivatives.

Sixteen novel pyrazole acyl thiourea derivatives 6 were synthesized from monomethylhydrazine (phenylhydrazine) and ethyl acetoacetate. The key 5-chloro-3-methyl-1-substituted-1H-pyrazole-4-carbonyl chloride intermediates 4 were first generated in four steps through cyclization, formylation, oxidation and acylation. Thess were then reacted with ammonium thiocyanate in the presence of PEG-400 to afford 5-chloro-3-methyl-1-substituted-1H-pyrazole-4-carbonyl isothiocyanates 5. Subsequent reaction with fluorinated aromatic amines resulted in the formation of the title compounds. The synthesized compound were unequivocally characterized by IR, ¹H-NMR, ¹³C-NMR and elemental analysis and some of the synthesized compounds displayed good antifungal activities against Gibberella zeae, Fusarium oxysporum, Cytospora mandshurica and anti-TMV activity in preliminary antifungal activity tests.

ß-Tubulins in Gibberella zeae: their characterization and contribution to carbendazim resistance.

BACKGROUND: Fusarium head blight caused by Gibberella zeae is an important disease of wheat and barley because it reduces grain yield and quality and results in the contamination of grain with mycotoxins. Recent studies have shown that carbendazim resistance in field strains of G. zeae is not caused by mutation of the ß-tubulin gene (ß1 tub), which is the case with other filamentous fungi, but that fungicide resistance is greatly increased by deletion of ß1 tub. The aim of the present study was to clarify the function of ß1 tub and its role in carbendazim resistance in G. zeae by artificial gene operation. RESULTS: Deletion of ß1 tub reduced vegetative growth and pathogenicity but increased asexual reproduction in G. zeae. All the mutants were more resistant to carbendazim than parent strains. A three-dimensional model of ß1 tub was constructed, and the possible carbendazim binding site was analysed. CONCLUSION: ß1 tub is not an essential gene in G. zeae, but it affects the sensitivity of the fungus to carbendazim.

Effect of simulated space gravity environment on Gibberella moniliformis EZG0807.

To determine the feasibility of inducing mutation for Gibberella moniliformis EZG0807 with a superconducting magnet, this paper investigated the effects of this instrument on the filamentous fungus G. moniliformis EZG0807. The superconducting magnet could simulate space gravity environment from hypo-gravity (0 g) to hyper-gravity (2 g). After G. moniliformis EZG0807 was exposed to the superconducting magnet for 72 h, the morphological observation, agar diffusion method, and amplified fragment length polymorphism were performed to detect the mutagenic effects in the aspect of morphology, the activity of metabolites, and genomic DNA, respectively. The mutant strain M7212 in 1 g (16 T) was different from the control in the morphology, showing no activity against the four tested bacteria Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Proteus vulgaris, and lost a size of 675 bp band on the genomic DNA. These results indicated that the superconducting magnet could be used to induce mutation for G. moniliformis EZG0807, which enabled improving the production of G. moniliformis EZG0807 and providing an effective approach for fungal breeding.

A putative transcription factor MYT1 is required for female fertility in the ascomycete Gibberella zeae.

Gibberella zeae is an important pathogen of major cereal crops. The fungus produces ascospores that forcibly discharge from mature fruiting bodies, which serve as the primary inocula for disease epidemics. In this study, we characterized an insertional mutant Z39P105 with a defect in sexual development and identified a gene encoding a putative transcription factor designated as MYT1. This gene contains a Myb DNA-binding domain and is conserved in the subphylum Pezizomycotina of Ascomycota. The MYT1 protein fused with green fluorescence protein localized in nuclei, which supports its role as a transcriptional regulator. The MYT1 deletion mutant showed similar phenotypes to the wild-type strain in vegetative growth, conidia production and germination, virulence, and mycotoxin production, but had defect in female fertility. A mutant overexpressing MYT1 showed earlier germination, faster mycelia growth, and reduced mycotoxin production compared to the wild-type strain, suggesting that improper MYT1 expression affects the expression of genes involved in the cell cycle and secondary metabolite production. This study is the first to characterize a transcription factor containing a Myb DNA-binding domain that is specific to sexual development in G. zeae.

Functional analyses of two acetyl coenzyme A synthetases in the ascomycete Gibberella zeae.

Acetyl coenzyme A (acetyl-CoA) is a crucial metabolite for energy metabolism and biosynthetic pathways and is produced in various cellular compartments with spatial and temporal precision. Our previous study on ATP citrate lyase (ACL) in Gibberella zeae revealed that ACL-dependent acetyl-CoA production is important for histone acetylation, especially in sexual development, but is not involved in lipid synthesis. In this study, we deleted additional acetyl-CoA synthetic genes, the acetyl-CoA synthetases (ACS genes ACS1 and ACS2), to identify alternative acetyl-CoA production mechanisms for ACL. The ACS1 deletion resulted in a defect in sexual development that was mainly due to a reduction in 1-palmitoyl-2-oleoyl-3-linoleoyl-rac-glycerol production, which is required for perithecium development and maturation. Another ACS coding gene, ACS2, has accessorial functions for ACS1 and has compensatory functions for ACL as a nuclear acetyl-CoA producer. This study showed that acetate is readily generated during the entire life cycle of G. zeae and has a pivotal role in fungal metabolism. Because ACSs are components of the pyruvate-acetaldehyde-acetate pathway, this fermentation process might have crucial roles in various physiological processes for filamentous fungi.

Mid1, a mechanosensitive calcium ion channel, affects growth, development, and ascospore discharge in the filamentous fungus Gibberella zeae.

The role of Mid1, a stretch-activated ion channel capable of being permeated by calcium, in ascospore development and forcible discharge from asci was examined in the pathogenic fungus Gibberella zeae (anamorph Fusarium graminearum). The Δmid1 mutants exhibited a >12-fold reduction in ascospore discharge activity and produced predominately abnormal two-celled ascospores with constricted and fragile septae. The vegetative growth rate of the mutants was ∼50% of the wild-type rate, and production of macroconidia was >10-fold lower than in the wild type. To better understand the role of calcium flux, Δmid1 Δcch1 double mutants were also examined, as Cch1, an L-type calcium ion channel, is associated with Mid1 in Saccharomyces cerevisiae. The phenotype of the Δmid1 Δcch1 double mutants was similar to but more severe than the phenotype of the Δmid1 mutants for all categories. Potential and current-voltage measurements were taken in the vegetative hyphae of the Δmid1 and Δcch1 mutants and the wild type, and the measurements for all three strains were remarkably similar, indicating that neither protein contributes significantly to the overall electrical properties of the plasma membrane. Pathogenicity of the Δmid1 and Δmid1Δcch1 mutants on the host (wheat) was not affected by the mutations. Exogenous calcium supplementation partially restored the ascospore discharge and vegetative growth defects for all mutants, but abnormal ascospores were still produced. These results extend the known roles of Mid1 to ascospore development and forcible discharge. However, Neurospora crassa Δmid1 mutants were also examined and did not exhibit defects in ascospore development or in ascospore discharge. In comparison to ion channels in other ascomycetes, Mid1 shows remarkable adaptability of roles, particularly with regard to niche-specific adaptation.