Production of kojic acid by Aspergillus flavus OL314748 using box-Behnken statistical design and its antibacterial and anticancer applications using molecular docking technique.

Kojic acid is a wonderful fungal secondary metabolite that has several applications in the food, medical, and agriculture sectors. Many human diseases become resistant to normal antibiotics and normal treatments. We need to search for alternative treatment sources and understand their mode of action. Aspergillus flavus ASU45 (OL314748) was isolated from the caraway rhizosphere as a non-aflatoxin producer and identified genetically using 18S rRNA gene sequencing. After applying the Box-Behnken statistical design to maximize KA production, the production raised from 39.96 to 81.59 g/l utilizing (g/l) glucose 150, yeast extract 5, KH2PO4 1, MgSO4.7H2O 2, and medium pH 3 with a coefficient (R2) of 98.45%. Extracted KA was characterized using FTIR, XRD, and a scanning electron microscope. Crystalized KA was an effective antibacterial agent against six human pathogenic bacteria (Bacillus cereus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, Serratia marcescens, and Serratia plymuthica). KA achieves high inhibition activity against Bacillus cereus, K. pneumonia, and S. plymuthica at 100 µg/ml concentration by 2.75, 2.85, and 2.85 compared with chloramphenicol which gives inhibition zones 1, 1.1, and 1.6, respectively. Crystalized KA had anticancer activity versus three types of cancer cell lines (Mcf-7, HepG2, and Huh7) and demonstrated high cytotoxic capabilities on HepG-2 cells that propose strong antitumor potent of KA versus hepatocellular carcinoma. The antibacterial and anticancer modes of action were illustrated using the molecular docking technique. Crystalized kojic acid from a biological source represented a promising microbial metabolite that could be utilized as an alternative antibacterial and anticancer agent effectively.

Characterization of tea (Camellia sinensis L.) flower extract and insights into its antifungal susceptibilities of Aspergillus flavus.

BACKGROUND: Tea (Camellia sinensis L.) flowers will compete with tea leaves in nutrition and are abandoned as an undesirable by-product. In this study, the biological efficacy of tea flowers was investigated. Further exploration of its antifungal activity was explained. METHODS: Tea flowers harvested from China were characterized in term of component, antioxidant ability, tyrosinase inhibition, and antifungal ability. Chemical compounds of tea flowers were analyzed by LC-MS. Disinfectant compounds were identified in tea flowers, and 2-ketobutyric acid exhibited antifungal activity against Aspergillus flavusCCTCC AF 2023038. The antifungal mechanism of 2-ketobutyric acid was further investigated by RNA-seq. RESULTS: Water-soluble tea flower extracts (TFEs) exhibited free radical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) as well as a high ferric-reducing ability. However, no inhibition of tyrosinase activity was observed. In the antifungal test, 6.4 mg/mL TFE reached 71.5% antifungal rate and the electrical conductivity of the culture broth increased with increasing concentration of TFE, implying that it damaged the fungal cell membrane by the TFE. Several disinfectants were identified in TFE by LC-MS, and 2-ketobutyric acid was also confirmed to be capable of fungal inhibition. Propidium iodide (PI) staining indicated that 2-ketobutyric acid caused damage to the cell membrane. RNA-seq analysis revealed that 3,808 differentially expressed genes (DEGs) were found in A. flavus CCTCC AF 2023038 treated by 2-ketobutyric acid, and more than 1,000 DEGs involved in the integral and intrinsic component of membrane were affected. Moreover, 2-ketobutyric acid downregulated aflatoxin biosynthesis genes and decreased the aflatoxin production. CONCLUSIONS: Overall, TFE exhibited excellent antioxidant ability and fungal inhibition against A. flavus CCTCC AF 2023038 due to its abundant disinfectant compounds. As a recognized food additive, 2-ketobutyric acid is safe to use in the food industry and can be utilized as the basis for the research and development of strong fungicides.

Aneuploidy Formation in the Filamentous Fungus Aspergillus flavus in Response to Azole Stress.

Aspergillus flavus is a mycotoxigenic fungus that contaminates many important agricultural crops with aflatoxin B1, the most toxic and carcinogenic natural compound. This fungus is also the second leading cause of human invasive aspergillosis, after Aspergillus fumigatus, a disease that is particularly prevalent in immunocompromised individuals. Azole drugs are considered the most effective compounds in controlling Aspergillus infections both in clinical and agricultural settings. Emergence of azole resistance in Aspergillus spp. is typically associated with point mutations in cyp51 orthologs that encode lanosterol 14α-demethylase, a component of the ergosterol biosynthesis pathway that is also the target of azoles. We hypothesized that alternative molecular mechanisms are also responsible for acquisition of azole resistance in filamentous fungi. We found that an aflatoxin-producing A. flavus strain adapted to voriconazole exposure at levels above the MIC through whole or segmental aneuploidy of specific chromosomes. We confirm a complete duplication of chromosome 8 in two sequentially isolated clones and a segmental duplication of chromosome 3 in another clone, emphasizing the potential diversity of aneuploidy-mediated resistance mechanisms. The plasticity of aneuploidy-mediated resistance was evidenced by the ability of voriconazole-resistant clones to revert to their original level of azole susceptibility following repeated transfers on drug-free media. This study provides new insights into mechanisms of azole resistance in a filamentous fungus. IMPORTANCE Fungal pathogens cause human disease and threaten global food security by contaminating crops with toxins (mycotoxins). Aspergillus flavus is an opportunistic mycotoxigenic fungus that causes invasive and noninvasive aspergillosis, diseases with high rates of mortality in immunocompromised individuals. Additionally, this fungus contaminates most major crops with the notorious carcinogen, aflatoxin. Voriconazole is the drug of choice to treat infections caused by Aspergillus spp. Although azole resistance mechanisms have been well characterized in clinical isolates of Aspergillus fumigatus, the molecular basis of azole resistance in A. flavus remains unclear. Whole-genome sequencing of eight voriconazole-resistant isolates revealed that, among other factors, A. flavus adapts to high concentrations of voriconazole by duplication of specific chromosomes (i.e., aneuploidy). Our discovery of aneuploidy-mediated resistance in a filamentous fungus represents a paradigm shift, as this type of resistance was previously thought to occur only in yeasts. This observation provides the first experimental evidence of aneuploidy-mediated azole resistance in the filamentous fungus A. flavus.

Preservation of Mimosa tenuiflora Antiaflatoxigenic Activity Using Microencapsulation by Spray-Drying.

Mimosa tenuiflora aqueous extract (MAE) is rich in phenolic compounds. Among them, condensed tannins have been demonstrated to exhibit a strong antioxidant and antiaflatoxin B1 activities in Aspergillus flavus. Since antioxidant capacity can change with time due to environmental interactions, this study aimed to evaluate the ability of encapsulation by spray-drying of Mimosa tenuiflora aqueous extract to preserve their biological activities through storage. A dry formulation may also facilitate transportation and uses. For that, three different wall materials were used and compared for their efficiency. Total phenolic content, antioxidant activity, antifungal and antiaflatoxin activities were measured after the production of the microparticles and after one year of storage at room temperature. These results confirmed that encapsulation by spray-drying using polysaccharide wall materials is able to preserve antiaflatoxin activity of Mimosa tenuiflora extract better than freezing.

Study on inhibitory activity and mechanism of chitosan oligosaccharides on Aspergillus Flavus and Aspergillus Fumigatus.

Chitosan oligosaccharides (COS) are a derivative of low molecular weight chitosan and are potent natural antimicrobial agents. The antimicrobial activity of COS against Aspergillus flavus and Aspergillus fumigatus was evaluated by minimum inhibitory concentration (MIC) and inhibition of mycelial growth. The MICs of COS against these two fungi were 31.2 and 15.6 mg/mL, respectively. COS treatment rendered fungal mycelia wrinkled and deformed with a fractured appearance. COS also increased cellular permeability leading to a significant leakage of cellular components indicating membrane damage. This compound also dose-dependently reduced chitin production and enhanced chitinase activity while enhancing the accumulation of reactive oxygen species (ROS). These characteristics suggested that COS has inhibitory effects against food spoilage fungi and acts on the cell wall and membrane and alters cellular metabolism. COS shows promise for food industry applications since it is non-toxic to higher organisms.

Effect of the essential oils of Satureja montana L., Myristica fragrans H. and Cymbopogon flexuosus S. on mycotoxin-producing Aspergillus flavus and Aspergillus ochraceus antifungal properties of essential oils.

Essential oils can be a useful alternative to the use of synthetic fungicides because they have biological potential and are relatively safe for food and agricultural products. The objectives of the present study were to evaluate the antifungal and antimycotoxigenic activities of the essential oils from Satureja montana L., Myristica fragrans H. and Cymbopogon flexuosus S. against Aspergillus flavus and Aspergillus ochraceus, as well as their effects on ergosterol synthesis and membrane morphology. The antifungal potential was evaluated by mycelial growth analysis and scanning electron microscopy. Fungicidal effects against A. flavus, with MFC of 0.98, 15.62 and 0.98 µL/mL, respectively, were observed for the essential oils from S. montana, M. fragrans and C. flexuosus. Aspergillus ochraceus did not grow in the presence of concentrations of 3.91, 15.62 and 0.98 µL/mL of the essential oils from S. montana, M. fragrans and C. flexuosus, respectively. The essential oils significantly inhibited the production of ochratoxin A by the fungus A. ochraceus. The essential oils also inhibited the production of aflatoxin B1 and aflatoxin B2. The biosynthesis of ergosterol was inhibited by the applied treatments. Biological activity in the fungal cell membrane was observed in the presence of essential oils, given that deleterious effects on the morphologies of the fungi were detected. The essential oils under study are promising as food preservatives because they significantly inhibit toxigenic fungi that contaminate food. In addition, the essential oils hindered the biosynthesis of mycotoxins.

In vivo synergism of free miltefosine or in alginate-based nanocarrier combined with voriconazole on aspergillosis.

Aim: To evaluate the activity of miltefosine (MFS), in its free form or loaded-alginate nanoparticles (MFS-AN), alone or combined with voriconazole (VRC) on Aspergillus fumigatus and Aspergillus flavus. Materials & methods: A broth microdilution assay was used for the susceptibility testing of Aspergillus isolates, and the antifungal efficacy was assessed using the aspergillosis model in Galleria mellonella larvae. Results: The in vitro synergistic effect of MFS with VRC was observed only against A. fumigatus, whereas both combined therapies (MFS + VRC and MFS-AN + VRC) showed synergism in reducing the larval mortality rate and fungal burden in the larvae infected by A. fumigatus and A. flavus. Conclusions: MFS and MFS-AN combined with VRC may be an important strategy for improving antifungal therapy against aspergillosis.

EGCG Alleviates Oxidative Stress and Inhibits Aflatoxin B1 Biosynthesis via MAPK Signaling Pathway.

Aflatoxin biosynthesis has established a connection with oxidative stress, suggesting a prevention strategy for aflatoxin contamination via reactive oxygen species (ROS) removal. Epigallocatechin gallate (EGCG) is one of the most active and the richest molecules in green tea with well-known antioxidant effects. Here, we found EGCG could inhibit aflatoxin B1 (AFB1) biosynthesis without affecting mycelial growth in Aspergillus flavus, and the arrest occurred before the synthesis of toxin intermediate metabolites. Further RNA-seq analysis indicated that multiple genes involved in AFB1 biosynthesis were down-regulated. In addition, EGCG exposure facilitated the significantly decreased expression of AtfA which is a bZIP (basic leucine zipper) transcription factor mediating oxidative stress. Notably, KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis indicated that the MAPK signaling pathway target transcription factor was down-regulated by 1 mg/mL EGCG. Further Western blot analysis showed 1 mg/mL EGCG could decrease the levels of phosphorylated SakA in both the cytoplasm and nucleus. Taken together, these data evidently supported that EGCG inhibited AFB1 biosynthesis and alleviated oxidative stress via MAPK signaling pathway. Finally, we evaluated AFB1 contamination in soy sauce fermentation and found that EGCG could completely control AFB1 contamination at 8 mg/mL. Conclusively, our results supported the potential use of EGCG as a natural agent to prevent AFB1 contamination in fermentation industry.

Formulation, optimization, and nephrotoxicity evaluation of an antifungal in situ nasal gel loaded with voriconazole‒clove oil transferosomal nanoparticles.

Fungal infections of the paranasal cavity are among the most widely spread illnesses nowadays. The aim of the current study was to estimate the effectiveness of an in situ gel loaded with voriconazole‒clove oil nano-transferosomes (VRC-CO-NT) in enhancing the activity of voriconazole against Aspergillus flavus, which causes rhinosinusitis. The nephrotoxic side effects of voriconazole may be reduced through the incorporation of the clove oil, which has antioxidant activity that protects tissue. The Box‒Behnken design was applied to formulate the VRC-CO-NT. The particle size, entrapment efficiency, antifungal inhibition zone, and serum creatinine concentration were considered dependent variables, and the soybean lecithin, VRC, and CO concentrations were considered independent ones. The final optimized formulation was loaded into a deacetylated gellan gum base and evaluated for its gelation, rheological properties, drug release profile, permeation capabilities, and in vivo nephrotoxicity. The optimum formulation was determined to be composed of 50 mg/mL lecithin, 18 mg/mL VRC, and 75 mg/mL CO, with a minimum particle size of 102.96 nm, an entrapment efficiency of 71.70%, an inhibition zone of 21.76 mm, and a serum creatinine level of 0.119 mmol/L. The optimized loaded in situ gel released 82.5% VRC after 12 hours and resulted in a 5.4-fold increase in drug permeation. The in vivo results obtained using rabbits resulted in a nonsignificant differentiation among the renal function parameters compared with the negative control group. In conclusion, nasal in situ gel loaded with VRC-CO-NT is considered an efficient novel carrier with enhanced antifungal properties with no signs of nephrotoxicity.

Aspergillus flavus Infections in Children With Leukemia Despite Liposomal Amphotericin-B Prophylaxis.

Liposomal amphotericin-B (L-AmB) prophylaxis is used in children with leukemia when azoles are contraindicated, but its effect is debated. We reviewed cases of invasive aspergillosis despite L-AmB 2.5 mg/kg twice weekly in children with high-risk leukemia during 2012-2019. Ten (16%) of 62 children had proven or probable aspergillosis. Thus, L-AmB prophylaxis offered insufficient protection for Aspergillus, in particular for Aspergillus flavus.

Mimosa tenuiflora Aqueous Extract: Role of Condensed Tannins in Anti-Aflatoxin B1 Activity in Aspergillus flavus.

Aflatoxin B1 (AFB1) is a potent carcinogenic mycotoxin that contaminates numerous crops pre- and post-harvest. To protect foods and feeds from such toxins without resorting to pesticides, the use of plant extracts has been increasingly studied. The most interesting candidate plants are those with strong antioxidative activity because oxidation reactions may interfere with AFB1 production. The present study investigates how an aqueous extract of Mimosa tenuiflora bark affects both the growth of Aspergillus flavus and AFB1 production. The results reveal a dose-dependent inhibition of toxin synthesis with no impact on fungal growth. AFB1 inhibition is related to a down-modulation of the cluster genes of the biosynthetic pathway and especially to the two internal regulators aflR and aflS. Its strong anti-oxidative activity also allows the aqueous extract to modulate the expression of genes involved in fungal oxidative-stress response, such as msnA, mtfA, atfA, or sod1. Finally, a bio-guided fractionation of the aqueous extract demonstrates that condensed tannins play a major role in the anti-aflatoxin activity of Mimosa tenuiflora bark.

Inhibition of Aspergillus flavus growth and aflatoxins production on peanuts over α-Fe2O3 nanorods under sunlight irradiation.

Peanut is an important resource of edible oil and digestible protein in daily life, which is rich in the nutriments and antioxidants such as vitamins, minerals and polyphenols. However, peanut is susceptible to the contamination of Aspergillus flavus (A. flavus), which can produce highly carcinogenic toxins that brings serious threats to human health and food safety. Exploring green and effective methods to control A. flavus is meaningful. Herein, a green and economical way to control A. flavus on peanuts was demonstrated. It was found that the growth of A. flavus hyphae and germination of its spores could be inhibited in the presence of α-Fe2O3 nanorods under sunlight irradiation according to the agar diffusion method, flat colony counting method and fluorescence-based live/dead test. The diameter of inhibition zone was 22.3 ± 0.2 mm and the inhibition rate of spores germination was about 60 ± 5%, when the concentration of α-Fe2O3 was 10 mg/mL for 7 h sunlight irradiation. Most important, α-Fe2O3 showed the photocatalytic inhibition of A. flavus on peanuts under sunlight irradiation with the inhibition rate of about 90 ± 5%, and the production of aflatoxin B1 and aflatoxin B2 were reduced by 90 ± 2% and 70 ± 3%, respectively. By comparing the fat contents, protein contents, acid value, peroxide value and antioxidative compositions of peanuts, it was found that there was no obvious effect on the quality of peanuts after inhibition treatment. The findings provide a green, safe and economical strategy to control A. flavus on peanuts, which may be as a promising way to be used in food and agro-food preservation.

Application of clove and dill oils as an alternative of salphos for chickpea food seed storage.

Mycological investigations of 25 samples of stored chickpea food seeds (Cicer arietinum L.) from grocery stores of Gurgaon and Gorakhpur revealed occurrence of seventeen fungal species belonging to genus viz., Alternaria, Aspergillus, Chaetomium, Colletotrichum, Curvularia, Fusarium, Penicillium, Rhizopus, Rhizoctonia, and Sclerotium. In these Aspergillus flavus, A. niger, Fusarium oxysporum had dominance in terms of per cent occurrence. Only one species of Bruchid (Callosobruchus chinensis L.) occurred in all the 25 samples. The biodeterioration of seeds inoculated with fungi: A. flavus, A. niger, F. oxysporum and the insect-C. chinensis, revealed their role in seed deterioration. For chickpea food seed protection essential oils were extracted from edible commodity(clove(Lavang and dill(sowa) leaf). Clove(Lavang) oil registered highest antifungal activity inhibiting (100%) mycelial growth of fungi, viz. species Aspergillus flavus, A. niger, Fusarium oxysporum at 300 ppm but was fungicidal at 400 ppm. Dill (Sowa) oil showed complete inhibition at 400 ppm and was fungicidal at 500 ppm. While mixture of both the oils (clove and dill) showed complete inhibition (100%) and fungicidal action at 400 ppm against the dominant fungi. The oils showed 100% insect repellent activity and were found fungicidal at 0.02 ml dose and also insecticidal. The mixture of oils was cidal at 0.02 ml dose. The mixture of oils showed a broad antifungal spectrum at 500 ppm while only 70-93% inhibitory activity at 300 ppm. The oils' mixture's activity was not affected by temp, storage and autoclaving up to 150 days. Oils physico-chemical properties were studied. GC-MS analysis of clove(Lavang) oil depicted major components: 75.63%eugenol while dill(sowa) leaf oil had 25.14% apiole. Formulation of Mixture of oils was more effective showing complete seed protection i.e.no growth of fungi and insects upto 150 days storage than salphos (150 days). While salphos controlled only maximum three fungi (A. terreus, C. dematium, F. moniliforme). The formulated oils mixture did not have any adverse effect on the chickpea seeds and increased their shelf life.

Rhamnolipids inhibit aflatoxins production in Aspergillus flavus by causing structural damages in the fungal hyphae and down-regulating the expression of their biosynthetic genes.

Aflatoxins are hepatotoxic and carcinogenic fungal secondary metabolites that usually contaminate crops and represent a serious health hazard for humans and animals worldwide. In this work, the effect of rhamnolipids (RLs) produced by Pseudomonas aeruginosa #112 on the growth and aflatoxins production by Aspergillus flavus MUM 17.14 was studied in vitro. At concentrations between 45 and 1500 mg/L, RLs reduced the mycelial growth of A. flavus by 23-40% and the production of aflatoxins by 93.9-99.5%. Purified mono-RLs and di-RLs exhibited a similar inhibitory activity on fungal growth. However, the RL mixture had a stronger inhibitory effect on aflatoxins production at concentrations up to 190 mg/L, probably due to a synergistic effect resulting from the combination of both congeners. Using transmission electron microscopy, it was demonstrated that RLs damaged the cell wall and the cytoplasmic membrane of the fungus, leading to the loss of intracellular content. This disruptive phenomenon explains the growth inhibition observed. Furthermore, RLs down-regulated the expression of genes aflC, aflE, aflP and aflQ involved in the aflatoxins biosynthetic pathway (6.4, 44.3, 38.1 and 2.0-fold, respectively), which is in agreement with the almost complete inhibition of aflatoxins production. Overall, the results herein gathered demonstrate for the first time that RLs could be used against aflatoxigenic fungi to attenuate the production of aflatoxins, and unraveled some of their mechanisms of action.

Impact of encapsulated orange essential oil with ß-cyclodextrin on technological, digestibility, sensory properties of wheat cakes as well as Aspergillus flavus spoilage.

BACKGROUND: The majority of studies with essential oils in foods focus mainly on improving the shelf life of products; however, the present study goes further and demonstrates not only the effect of essential oil on conservation properties, but also the effect of free and encapsulated orange essential oil (OEO) on the technological, sensorial and digestibility properties of bakery products. RESULTS: OEO was encapsulated into ß-cyclodextrin (ß-CD) by inclusion complex formation (ß-CD/OEO 97.4% of encapsulation efficiency). OEO demonstrated in vitro antifungal activity against Aspergillus flavus (inhibition zone of 11.33 mm on mycelial growth). In situ antifungal activity against A. flavus confirmed that free OEO can effectively delay the fungal growth, unlike encapsulated OEO. Regarding texture profile and starch digestibility: cake with ß-CD/OEO showed lower hardness (31.64 N) and lower starch digestibility (69.10%) than cake with free OEO (44.30 N; 82.10%, respectively) and the addition of OEO (both free and encapsulated) decreased the adhesiveness of the cakes. Cake with free OEO showed a higher intensity of orange aroma, being preferred by 60% of panelists, whereas cake with ß-CD/OEO presented a very slight orange taste and aroma. CONCLUSION: The encapsulation of OEO into ß-CD improved the crumb texture of cakes and promoted a lower starch digestibility in the cakes. On the other hand, the encapsulation process was not effective under the conditions tested (OEO concentration and baking temperatures), compromising the action of the OEO as a natural flavoring and preservative agent. © 2021 Society of Chemical Industry.

Putative invasive pulmonary aspergillosis within medical wards and intensive care units: a 4-year retrospective, observational, single-centre study.

Blot and colleagues have proposed putative invasive pulmonary aspergillosis (PIPA) definitions for troublesome diagnosis in suspected patients outside the classical criteria of immunosuppression. We retrospectively included in the study all admitted patients with an Aspergillus spp. positive culture within lower airway samples. Overall, Aspergillus spp. positivity in respiratory samples was 0.97 every 1000 hospital admissions (HA): 4.94 and 0.28/1000/HA, respectively, in intensive care units (ICUs) and medical wards (MW). 66.6% fulfilled PIPA criteria, and 33.4% were defined as colonized. 69.2% of PIPA diagnosis occurred in the ICU. Antifungal therapy was appropriate in 88.5% of subjects with PIPA and 37.5% of colonized, confirming the comparison between deads and lives. Patients with PIPA in the ICUs had more frequent COPD, sepsis or septic shock, acute kidney injury (AKI), needed more surgery, mechanical ventilation (MV), vasopressors, hemodialysis, blood or platelets transfusions. PIPA in MW had associated with a history of smoking, interstitial lung disease and inhaled steroid therapy. Overall mortality within 21 days was 50%: 54.2% in ICU, 36,8% in MW. Factors associated with death were length of hospitalization, influenza, pneumonia, liver transplant, AKI, ARDS, sepsis and septic shock. PIPA in the ICU had higher disease severity and needed more organ support than MW cases, despite that cases of PIPA in MW are emerging with trends difficult to demonstrate given the problematic diagnosis.

Untangling the multi-regime molecular mechanism of verbenol-chemotype Zingiber officinale essential oil against Aspergillus flavus and aflatoxin B1.

Aflatoxin B1 (AFB1), the natural polyketide produced by Aspergillus flavus, has a potent carcinogenic effect on humans as well as animals. In the present study, the antifungal and anti-aflatoxigenic B1 activity of chemically characterized Zingiber officinale essential oil (ZOEO) was investigated via in vitro analysis aided with molecular dynamics (MD) approaches. The GC-MS results revealed verbenol (52.41%) as the major component of oil. The antifungal and anti-aflatoxigenic activity of ZOEO was found to be 0.6 µl/ml and 0.5 µl/ml respectively. In-vitro analysis targeting the cell membrane, mitochondria and carbohydrate catabolism elucidated the probable antifungal mode of action. Further, docking and MD simulation results confirmed the inhibitory action of verbenol on the structural gene products (Nor-1, Omt-1, and Vbs) of aflatoxin biosynthetic machinery. Biochemical assays revealed the fungitoxic potential of the ZOEO while, computational results infers the stabilizing effects on the gene products upon verbenol binding leads to the impairment in its functionality. This is the first attempt to assess the multi-regime anti-AFB1 mechanism of verbenol chemotype-ZOEO targeting the Nor-1, Omt-1, and Vbs via computational approaches.

The Development of a Phytopathogenic Fungi Control Trial: Aspergillus flavus and Aspergillus niger Infection in Jojoba Tissue Culture as a Model.

After introducing the idea of using concentrations equal to or less than the minimum inhibition concentration (MIC) of some active chemical compounds for evacuating microbial cells, different types of microbes were evacuated. The original protocol was given the name sponge-like protocol and then was reduced and modified from a microorganism to another to prepare microbial ghosts for various applications such as immunological applications, drug delivery, and isolation of DNA and protein. Fungal pathogens that infect plants critically affect cost effectiveness, quality, and quantity of their production. They kill plant cells and/or cause plant stress. Plant fungal infections can originate from many sources such as infected soil, seeds, or crop debris causing diseases and quality losses around the world with billions of US dollars annually as costs of the associated productivity loss. This study focused on the application of the sponge-like protocol in protecting in vitro tissue cultures of plants against fungal pathogens. This can be useful for research purposes or may be developed to be introduced in field applications. Aspergillus flavus and Aspergillus niger infection in tissue culture of jojoba (Simmondsia chinensis (Link) Schn.) was used as a model to establish the employment of this protocol to control plant fungal diseases. The best conditions for A. flavus and A. niger ghosts production previously mapped by randomization experimental design (reduced Plackett-Burman experimental design) were used to prepare fungal ghosts. SDS, NaOH, NaHCO3, and H2O2 were used in their MIC (+1 level) or minimum growth concentration (MGC, -1 level) according to the determined optimal experimental design. The release of both of DNA and protein from the fungal cells was evaluated spectrophotometrically at 260nm and 280nm, respectively, as an indicator for cell loss of their cytoplasm. Fungal ghost cells were also examined by transmission electron microscopy. After confirming the preparation of high-quality fungal ghost cells, the same conditions were mimicked to control plant fungal infection. Jojoba grown in tissue culture was sprayed with fungal cells (about 103 CFU) as a control experiment or fungal cells followed by treatment with solution (a) represents the fungal ghost cells formation calculated critical concentration (FGCCC) of SDS, NaOH, and NaHCO3 and then treatment with solution (b) represents H2O2 FGCCC. The plant was examined on day 0 (plant grown before any infection or infection followed by treatment), day 5 (plant at day 5 after infection or infection followed by treatment), and day 10 (plant at day 10 after infection or infection followed by treatment). We observed fungal growth in case of control experiments at days 5 and 10 on the tissue culture medium, as well as plant, and the absence of any fungal growth in case of plant treated with FGCCC even after day 10. We recommend using this FGCCC in the form of chemical spraying formulation to treat the plants aiming to control different plant fungal infections in in vitro tissue culture systems or applied in field.

Oligosaccharides Derived from Tramesan: Their Structure and Activity on Mycotoxin Inhibition in Aspergillus flavus and Aspergillus carbonarius.

Food and feed safety are of paramount relevance in everyday life. The awareness that different chemicals, e.g., those largely used in agriculture, could present both environmental problems and health hazards, has led to a large limitation of their use. Chemicals were also the main tool in a control of fungal pathogens and their secondary metabolites, mycotoxins. There is a drive to develop more environmentally friendly, "green", approaches to control mycotoxin contamination of foodstuffs. Different mushroom metabolites showed the potential to act as control agents against mycotoxin production. The use of a polysaccharide, Tramesan, extracted from the basidiomycete Trametes versicolor, for controlling biosynthesis of aflatoxin B1 and ochratoxin A, has been previously discussed. In this study, oligosaccharides obtained from Tramesan were evaluated. The purified exopolysaccharide of T. versicolor was partially hydrolyzed and separated by chromatography into fractions from disaccharides to heptasaccharides. Each fraction was individually tested for mycotoxin inhibition in A. flavus and A. carbonarius. Fragments smaller than seven units showed no significant effect on mycotoxin inhibition; heptasaccharides showed inhibitory activity of up to 90% in both fungi. These results indicated that these oligosaccharides could be used as natural alternatives to crop protection chemicals for controlling these two mycotoxins.

Synthetic antimicrobial agents inhibit aflatoxin production.

Antimicrobial peptides (AMPs) are biologically active molecules that can eradicate bacteria by destroying the bacterial membrane structure, causing the bacteria to rupture. However, little is known about the extent and effect of AMPs on filamentous fungi. In this study, we synthesized small molecular polypeptides by an inexpensive heat conjugation approach and examined their effects on the growth of Aspergillus flavus and its secondary metabolism. The antimicrobial agents significantly inhibited aflatoxin production, conidiation, and sclerotia formation in A. flavus. Furthermore, we found that the expression of aflatoxin structural genes was significantly inhibited, and the intracellular reactive oxygen species (ROS) level was reduced. Additionally, the antimicrobial agents can change membrane permeability. Overall, our results demonstrated that antimicrobial agents, safe to mammalian cells, have an obvious impact on aflatoxin production, which indicated that antimicrobial agents may be adopted as a new generation of potential agents for controlling aflatoxin contamination.