Antifungal Mechanism Research Articles

UV Spectrophotometric Analysis of Apigenin in Topical Fungal Formulation containing Extract of Leonotis nepetaefolia (L) R. Br.

Fungal infections continue to endanger human health, and the problem is only becoming worse. Combination therapy may be an optional strategy for the treatment of invasive fungal illnesses, and the potential antifungal mechanisms provide new insights into the development of innovative antifungal medications. Numerous investigations have shown that combining plant extract showed significant antifungal activity. In the present work, a topical fungal formulation containing hydroalcoholic extract of Leonotis nepetaefolia (L) R.Br. (TFF) in form of a cream was estimated for apigenin. A technique has been created and validated using UV spectrophotometric analysis in order to evaluate the aforementioned drugs simultaneously in the formulation

Design, Synthesis, and Biological Evaluation of Novel Camphor-Based Hydrazide and Sulfonamide Derivatives as Laccase Inhibitors against Plant Pathogenic Fungi/Oomycetes.

To discover novel natural product-based fungicidal agrochemicals, 41 novel camphanic acid hydrazide and camphor sulfonamide derivatives were designed, synthesized, and tested for their antifungal profile against four plant pathogenic fungi and three oomycetes. As a result, some derivatives presented pronounced inhibitory activities toward Botryosphaeria dothidea, Fusarium graminearum, Phytophthora capsici, and Phytophthora nicotianae. Especially, compound 4b demonstrated the most potent anti-B. dothidea activity (EC50 = 1.28 mg/L), much stronger than positive control chlorthalonil. The in vivo assay showed that 4b displayed significant protective and curative effects on apple fruits infected by B. dothidea. The primary antifungal mechanism study revealed that 4b could obviously enhance the cell membrane permeability, destroy the mycelial surface morphology and the cell ultrastructure, and reduce the ergosterol and exopolysaccharide contents of B. dothidea. Further, 4b showed potent laccase inhibitory activity in vitro with an IC50 value of 11.3 μM, superior to positive control cysteine. The molecular docking study revealed that 4b could dock well into the active site of laccase by forming multiple interactions with the key residues in the pocket. The acute oral toxicity test in rats presented that 4b had slight toxicity with an LD50 value of 849.1 mg/kg bw (95% confidence limit: 403.9-1785.3 mg/kg bw). This research identified that the camphanic acid hydrazide derivatives could be promising leads for the development of novel laccase-targeting fungicides.

Optimization of the Fermentation Conditions of Metarhizium robertsii and Its Biological Control of Wolfberry Root Rot Disease.

Fusarium solani is the main pathogenic fungus causing the root rot of wolfberry (Lycium barbarum). The endophytic fungus Metarhizium robertsii has been widely used for the biocontrol of plant pathogenic fungi, but the biocontrol effects of this fungus on wolfberry root rot and its antifungal mechanism against F. solani have not been reported. In this study, the antagonism of endophytic fungus M. robertsii against F. solani was verified. Further, we optimized the fermentation conditions of M. robertsii fermentation broth based on the inhibition rate of F. solani. In addition, the effects of M. robertsii fermentation broth on the root rot of wolfberry and its partial inhibition mechanism were investigated. The results showed that M. robertsii exhibited good antagonism against F. solani. Glucose and beef extracts were the optimal carbon and nitrogen sources for the fermentation of M. robertsii. Under the conditions of 29 °C, 190 rpm, and pH 7.0, the fermentation broth of M. robertsii had the best inhibition effect on F. solani. Furthermore, the fermentation broth treatment decreased the activities of superoxide dismutase, catalase, and peroxidase of F. solani; promoted the accumulation of malondialdehyde; and accelerated the leakage of soluble protein and the decrease in soluble sugar. In addition, inoculation with M. robertsii significantly reduced the decay incidence and disease index of wolfberry root rot caused by F. solani. These results indicate that M. robertsii could be used as a biological control agent in wolfberry root rot disease management.

Transcriptomic and biochemical analyses revealed antifungal mechanism of trans-anethole on Aspergillus flavus growth.

Plant volatile compounds have great potential for preventing and controlling fungal spoilage in post-harvest grains. Recently, we have reported the antifungal effects of trans-anethole, the main volatile constituent of the Illicium verum fruit, on Aspergillus flavus. In this study, the inhibitory mechanisms of trans-anethole against the growth of A. flavus mycelia were investigated using transcriptomic and biochemical analyses. Biochemical and transcriptomic changes in A. flavus mycelia were evaluated after exposure to 0.2 μL/mL trans-anethole. Scanning electron microscopy showed that trans-anethole treatment resulted in the surface wrinkling of A. flavus mycelia, and calcofluor white staining confirmed that trans-anethole treatment disrupted the mycelial cell wall structure. Annexin V-fluorescein isothiocyanate/propidium iodide double staining suggested that trans-anethole induced apoptosis in A. flavus mycelia. Reduced mitochondrial membrane potential and DNA damage were observed in trans-anethole-treated A. flavus mycelia using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine and 4',6-diamidino-2-phenylindole staining, respectively. 2',7'- Dichloro-dihydro-fluorescein diacetate staining and biochemical assays demonstrated that trans-anethole treatment cause the accumulation of reactive oxygen species in the A. flavus mycelia. Transcriptome results showed that 1673 genes were differentially expressed in A. flavus mycelia exposed to trans-anethole, which were mainly associated with multidrug transport, oxidative phosphorylation, citric acid cycle, ribosomes, and cyclic adenosine monophosphate signaling. We propose that trans-anethole can inhibit the growth of A. flavus mycelia by disrupting the cell wall structure, blocking the multidrug transport process, disturbing the citric acid cycle, and inducing apoptosis. This study provides new insights into the inhibitory mechanism of trans-anethole on A. flavus mycelia and will be helpful for the development of natural fungicides. KEY POINTS: • Biochemical analyses of A. flavus mycelia exposed to trans-anethole were performed • Transcriptomic changes in trans-anethole-treated A. flavus mycelia were analyzed • An inhibitory mechanism of trans-anethole on the growth of A. flavus mycelia was proposed.

Deciphering the antifungal mechanism and functional components of cinnamomum cassia essential oil against Candida albicans through integration of network-based metabolomics and pharmacology, the greedy algorithm, and molecular docking

Ethnopharmacological relevanceFungal pathogens can cause deadly invasive infections and have become a major global public health challenge. There is an urgent need to find new treatment options beyond established antifungal agents, as well as new drug targets that can be used to develop novel antifungal agents. Cinnamomum cassia is a tropical aromatic plant that has a wide range of applications in traditional Chinese medicine, especially in the treatment of bacterial and fungal infections. Aim of the studyThe present study aimed to explore the mechanism of action and functional components of Cinnamomum cassia essential oil (CEO) against Candida albicans using an integrated strategy combining network-based metabolomics and pharmacology, the greedy algorithm and molecular docking. Materials and methodsCEO was extracted using hydrodistillation and its chemical composition was identified by GC-MS. Cluster analysis was performed on the compositions of 19 other CEOs from the published literature, as well as the sample obtained in this study. The damages of C. albicans cells upon treatment with CEO was observed using a scanning electron microscope. The mechanisms of its antifungal effect at a subinhibitory concentration of 0.1 × MIC were determined using microbial metabolomics and network analysis. The functional components were studied using the greedy algorithm and molecular docking. ResultsA total of 69 compounds were identified in the chemical analysis of CEO, which accounted for 90% of the sample. The major compounds were terpenoids (34.04%), aromatic compounds (4.52%), aliphatic compounds (0.9%), and others. Hierarchical cluster analysis of the compositions of 20 essential oils extracted from Cinnamomum cassia grown in different geographical locations showed a wide diversity of chemical composition with four major chemotypes. CEO showed strong antifungal activity and caused destruction of cell membranes in a concentration-dependent way. Metabolic fingerprint analysis identified 29 metabolites associated with lipid metabolism, which were mapped to 23 core targets mainly involved in fatty acid biosynthesis and metabolism. Six antifungal functional components of CEO were identified through network construction, greedy algorithm and molecular docking, including trans-cinnamaldehyde, δ-cadinol, ethylcinnamate, safrole, trans-anethole, and trans-cinnamyl acetate, which showed excellent binding with specific targets of AKR1B1, PPARG, BCHE, CYP19A1, CYP2C19, QPCT, and CYP51A1. ConclusionsThis study provides a systematic understanding of the antifungal activity of CEO and offers an integrated strategy for deciphering the potential metabolism and material foundation of complex component drugs.

The efficient activity of plant essential oils for inhibiting Botrytis cinerea and Penicillium expansum: Mechanistic insights into antifungal activity

Botrytis cinerea and Penicillium expansum produce deterioration in fruit quality, causing losses to the food industry. Thus, plant essential oils (EOs) have been proposed as a sustainable alternative for minimizing the application of synthetic fungicides due to their broad-spectrum antifungal properties. This study investigated the efficacy of five EOs in suppressing the growth of B. cinerea and P. expansum and their potential antifungal mechanisms. EOs of Mentha × piperita L., Origanum vulgare L., Thymus vulgaris L., Eucalyptus globules Labill., and Lavandula angustifolia Mill., were screened for both fungi. The results showed that the EO of T. vulgaris and O. vulgare were the most efficient in inhibiting the growth of B. cinerea and P. expansum. The concentration increase of all EO tested increased fungi growth inhibition. Exposure of fungi to EOs of T. vulgaris and O. vulgare increased the pH and the release of constituents absorbing 260 nm and soluble proteins, reflecting membrane permeability alterations. Fluorescence microscopic examination revealed that tested EOs produce structural alteration in cell wall component deposition, decreasing the hypha width. Moreover, propidium iodide and Calcein-AM stains evidenced the loss of membrane integrity and reduced cell viability of fungi treated with EOs. Fungi treated with EOs decreased the mitochondria activity and the respiratory process. Therefore, these EOs are effective antifungal agents against B. cinerea and P. expansum, which is attributed to changes in the cell wall structure, the breakdown of the cell membrane, and the alteration of the mitochondrial activity.

Exploring the antifungal mechanism of β-1,3-glucanase for effectively inhibiting the food contamination by Aspergillus flavus and Aspergillus fumigatus

Eco-friendly strategies are needed to solve the global public health problem of food fungal contamination. In this study, the mutant N54W of β-1,3-glucanase BglS27 with a 3.6-fold higher specific activity was constructed according to the structure comparison analysis. Molecular dynamics simulation revealed that Trp54 promoted and stabilized substrate binding, and facilitated the nucleophilic attack of catalytic residues. The mutant N54W exhibited higher antifungal activity against Aspergillus flavus and Aspergillus fumigatus. The assay of cell membrane integrity and mycelium hydrolysis elucidated that the enzyme hydrolyzes the structural β-1,3-glucans of the fungal cell to glucose and oligosaccharides, leading to cell rupture and death. Furthermore, the mutant N54W decreased the contamination rate of A. flavus and A. fumigatus in maize by 86.1% and 88.9%, respectively. Further optimization of the stability and efficiency of this enzyme in the actual food industry environment will contribute to its industrial application. These results provide meaningful information on the antifungal property of the mutant N54W at the molecular level and a promising efficient green antifungal preservative in the food industry.

Inhibitory effects of epiphytic Kluyveromyces marxianus from Indian senna (Cassia angustifolia Vahl.) on growth and aflatoxin production of Aspergillus flavus

Aspergillus flavus infection and subsequent aflatoxin contamination are considered the major constraints in senna (Cassia angustifolia Vahl.) export. Using native epiphytic yeast to control phytopathogens is a successful strategy for managing plant diseases. In the present investigation, we exploited the antagonistic potential of epiphytic yeast isolates obtained from senna against A. flavus growth and aflatoxin B1 (AFB1) production. Four Kluyveromyces marxianus strains (YSL3, YSL16, YSP12, and YSF9) exhibited vigorous antagonistic activity with a maximum inhibition of 64 %. In vivo evaluation of senna pods showed that K. marxianus strains effectively reduced A. flavus colonization with a population range of 5.87 to 7.08 log10 CFU/g. In contrast, the untreated senna pods were found to have severe fungal colonization with a population of 7.84 log10 CFU/g. In addition, HPLC analysis showed that aflatoxin B1 in senna pods was drastically reduced upon yeast treatment up to 14 DAI. Furthermore, we demonstrated the antifungal action mechanisms of K. marxianus, such as surface colonizing ability on pods, production of antifungal volatiles (VOCs), siderophores, extracellular lytic enzymes, and cell wall binding ability to AFB1. All four strains of K. marxianus showed rapid colonization on the senna pod, and YSP12 reached the maximum population of 7.18 log10 CFU/pod at 9 days after inoculation (DAI). The exposure of A. flavus to K. marxianus VOCs significantly reduced the growth by up to 99 and 93.2 % at 7 and 14 DAI, respectively. Scanning electron microscopic images demonstrated severe mycelial damage and hyphal deformities of A. flavus. In addition, yeast VOCs can reduce aflatoxin biosynthesis in A. flavus by up to 99 and 93.2 % at 7 and 14 DAI, respectively. Gas chromatography–mass spectrometry analysis confirmed the presence of antimicrobial compounds such as dimethyl trisulfide, ethyl acetate, ethanol, 3-methyl butanal, 2-methyl-1-butanol, and 3-methyl-1-butanol in the volatiles. K. marxianus strains produced siderophores and hydrolytic enzymes such as chitinase and β-1,3-glucanase. A higher AFB1 binding ability was observed in the heat-killed cells (47.5 to 70.65 %) than in the viable cells (43.16 to 60.98 %) of K. marxianus. The current study demonstrated that epiphytic K. marxianus isolated from senna could be a successful biocontrol source to reduce aflatoxin contamination in senna pods.

Genome-wide association study of maize resistance to Pythium aristosporum stalk rot.

Stalk rot, a severe and widespread soil-borne disease in maize, globally reduces yield and quality. Recent documentation reveals that Pythium aristosporum has emerged as one of the dominant causal agents of maize stalk rot. However, a previous study of maize stalk rot disease resistance mechanisms and breeding had mainly focused on other pathogens, neglecting P. aristosporum. To mitigate crop loss, resistance breeding is the most economical and effective strategy against this disease. This study involved characterizing resistance in 295 inbred lines using the drilling inoculation method and genotyping them via sequencing. By combining with population structure, disease resistance phenotype, and genome-wide association study (GWAS), we identified 39 significant single-nucleotide polymorphisms (SNPs) associated with P. aristosporum stalk rot resistance by utilizing six statistical methods. Bioinformatics analysis of these SNPs revealed 69 potential resistance genes, among which Zm00001d051313 was finally evaluated for its roles in host defense response to P. aristosporum infection. Through virus-induced gene silencing (VIGS) verification and physiological index determination, we found that transient silencing of Zm00001d051313 promoted P. aristosporum infection, indicating a positive regulatory role of this gene in maize's antifungal defense mechanism. Therefore, these findings will help advance our current understanding of the underlying mechanisms of maize defense to Pythium stalk rot.

Senkyunolide B exhibits broad-spectrum antifungal activity against plant and human pathogenic fungi via inhibiting spore germination and destroying the mature biofilm.

Aspergillus infection seriously jeopardize the health and safety of life of immunocompromised patients. The emergences of antifungal resistance highlight a demand to find new effective antifungal drugs. Angelica sinensis is a medicine-food herb and phthalides are its characteristic components. A few of phthalides have been reported to display satisfactory antifungal activities against plant pathogenic fungi. However, the structure activity relationships and antifungal action mechanism of phthalides remain to be further explored and elucidated. The antifungal activities of five natural phthalides and four artificial analogues were investigated, and their structure activity relationships were preliminarily elucidated in current study. The benzene ring moiety played an essential role in their antifungal activities; the oxygen-containing substituents on the benzene ring obviously impact their activities, the free hydroxyl was favorable to the activity. Typical phthalide senkyunolide B (SENB) exhibited broad antifungal activities against human and plant pathogenic fungi, especially, Aspergillus fumigatus. SENB affected the spore germination and hyphae growth of A. fumigatus via downregulating phosphatidylinositol-PKC-calcineurin axis and the expression of ENG genes. Moreover, SENB disturbed the oxidation-reduction process in A. fumigatus to destroy the mature biofilms. In vivo experiments indicated SENB significantly prolonged survival and decreased fungal burden in mouse model of invasive pulmonary aspergillosis. Phthalides could be considered as the valuable leads for the development of antifungal drug to cure plant and human disease. This article is protected by copyright. All rights reserved.

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

BackgroundTea (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.MethodsTea 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.ResultsWater-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.ConclusionsOverall, 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.

Ethanol mediates the interaction between Caenorhabditis elegans and the nematophagous fungus Purpureocillium lavendulum.

Accurately recognizing pathogens by the host is vital for initiating appropriate immune response against infecting microorganisms. Caenorhabditis elegans has no known receptor to recognize pathogen-associated molecular pattern. However, recent studies showed that nematodes have a strong specificity for transcriptomes infected by different pathogens, indicating that they can identify different pathogenic microorganisms. However, the mechanism(s) for such specificity remains largely unknown. In this study, we showed that the nematophagous fungus Purpureocillium lavendulum can infect the intestinal tract of the nematode C. elegans and the infection led to the accumulation of reactive oxygen species (ROS) in the infected intestinal tract, which suppressed fungal growth. Co-transcriptional analysis revealed that fungal genes related to anaerobic respiration and ethanol production were up-regulated during infection. Meanwhile, the ethanol dehydrogenase Sodh-1 in C. elegans was also up-regulated. Together, these results suggested that the infecting fungi encounter hypoxia stress in the nematode gut and that ethanol may play a role in the host-pathogen interaction. Ethanol production in vitro during fungal cultivation in hypoxia conditions was confirmed by gas chromatography-mass spectrometry. Direct treatment of C. elegans with ethanol elevated the sodh-1 expression and ROS accumulation while repressing a series of immunity genes that were also repressed during fungal infection. Mutation of sodh-1 in C. elegans blocked ROS accumulation and increased the nematode's susceptibility to fungal infection. Our study revealed a new recognition and antifungal mechanism in C. elegans. The novel mechanism of ethanol-mediated interaction between the fungus and nematode provides new insights into fungal pathogenesis and for developing alternative biocontrol of pathogenic nematodes by nematophagous fungi. IMPORTANCE Nematodes are among the most abundant animals on our planet. Many of them are parasites in animals and plants and cause human and animal health problems as well as agricultural losses. Studying the interaction of nematodes and their microbial pathogens is of great importance for the biocontrol of animal and plant parasitic nematodes. In this study, we found that the model nematode Caenorhabditis elegans can recognize its fungal pathogen, the nematophagous fungus Purpureocillium lavendulum, through fungal-produced ethanol. Then the nematode elevated the reactive oxygen species production in the gut to inhibit fungal growth in an ethanol dehydrogenase-dependent manner. With this mechanism, novel biocontrol strategies may be developed targeting the ethanol receptor or metabolic pathway of nematodes. Meanwhile, as a volatile organic compound, ethanol should be taken seriously as a vector molecule in the microbial-host interaction in nature.

Biochemical and metabolomic insights into antifungal mechanism of berberine against Candida glabrata.

An unprecedented expansion of antifungal therapy failure incidences in healthcare settings of Candida glabrata is the matter of global concern that needs to be addressed efficiently and effectively. In this pursuit, the present study has investigated the antifungal mechanism of benzylisoquinoline alkaloid berberine using biochemical, metabolic, and gene expression analysis, with the aim to delineate its therapeutic activity against C. glabrata and differentially fluconazole-responsive clinical isolates. Interestingly, the clinical isolates were found to be highly susceptible to berberine. Berberine was found to control the surface properties like hydrophobicity and charge of the cells. The cell membrane composition was altered by berberine, where the ergosterol and fatty acids were affected. The efflux pump activity was inhibited, and osmotic stress was generated in C. glabrata cells upon berberine exposure. The berberine has also generated oxidative stress and activated antioxidant system in C. glabrata cells. Furthermore, these observations were supported by the transcriptional expression study of C. glabrata cell genes (CDR1, RLM1, SLT2, SUR4, KRE1) and metabolomics analysis. Based on fold change analysis, the study identified 20 differential metabolites upon berberine treatment, which belong to central carbon, amino acids, and nucleotide pathways. The checkerboard analysis revealed the potentiation of some classically used antifungal drugs by berberine, thus suggesting it as a combinatorial nutraceutical adjuvant for the eradication of fungal infections. KEY POINTS: • Berberine exhibited better potency against azole-resistant clinical isolates • Berberine modulated metabolites of different pathways • Berberine generated oxidative stress and blocked efflux pump activity.

In vitro and in situ antifungal properties of a Trichoderma asperelloides SKRU-01 against aflatoxigenic aspergillus species

This research investigates the potential of using the culture filtrates of Trichoderma asperelloides SKRU-01 as a peanut preservative and/or fungicide against two mycotoxin-producing fungi, Aspergillus flavus and A. parasiticus. The study assessed the effectiveness of culture filtrates SKRU-01 compared to two commercial preservatives and chemical fungicides, both in vitro and in peanuts. In addition, the study investigated the antifungal and antiaflatoxigenic mechanisms of culture filtrates SKRU-01 against mycotoxin-producing strains. The in vitro results demonstrated that culture filtrates SKRU-01 had a significantly stronger inhibitory effect on the growth and spore germination of the two mycotoxin-producing strains compared to sodium benzoate, sodium propionate, and metalaxyl®, but less inhibition than prochloraz®. The study also explored the mode of action of culture filtrates SKRU-01 and found that it impeded the biosynthesis of ergosterol, indicating its antifungal activity was directed towards the plasma membrane as a potential target site. Furthermore, the culture filtrates SKRU-01 inhibited the biosynthesis of methylglyoxal (AFB1 inducer) in a manner that was dependent on the concentration, which represents a novel mechanism of action against aflatoxins. Additionally, the study observed significant impairment of both enzymatic and non-enzymatic antioxidant defense molecules in aflatoxigenic cells, indicating a potential biochemical mechanism of action. The study tested the potential application of the culture filtrates SKRU-01 in eliminating fungal spoilage in peanuts as a model. The results showed that it had a significant effect in preventing both mold density and AFB1 production, compared to commercial preservatives and chemical fungicides. The antiaflatoxigenic property with AFB1 production level was below the public health concern threshold of 20 μg/kg. Overall, the findings suggest that the culture filtrate of T. asperelloides SKRU-01 contained effective biological control agents possessing preservative and fungicidal properties.

Dehydrocostus lactone inhibits Candida albicans growth and biofilm formation

Candida albicans infections are threatening public health but there are only several antifungal drugs available. This study was to assess the effects of dehydrocostus lactone (DL) on the Candida albicans growth and biofilms Microdilution assays revealed that DL inhibits a panel of standard Candida species, including C. albicans, as well as 9 C. albicans clinical isolates. The morphological transition of C. albicans in RPMI-1640 medium and the adhesion to polystyrene surfaces can also be decreased by DL treatment, as evidenced by microscopic, metabolic activity and colony forming unit (CFU) counting assays. The XTT assay and microscopy inspection demonstrated that DL can inhibit the biofilms of C. albicans. Confocal microscopy following propidium iodide (PI) staining and DCFH-DA staining after DL treatment revealed that DL can increase the membrane permeability and intracellular reactive oxygen species (ROS) production. N-acetyl-cysteine could mitigate the inhibitory effects of DL on growth, morphological transition and biofilm formation, further confirming that ROS production induced by DL contributes to its antifungal and antibiofilm effects. This study showed that DL demonstrated antifungal and antibiofilm activity against C. albicans. The antifungal mechanisms may involve membrane damage and ROS overproduction. This study shows the potential of DL to fight Candida infections.

(E)-2-Octenal suppresses the growth of a prochloraz-resistant Penicillium italicum strain and its potential antifungal mechanisms

Blue mold caused by Penicillium italicum is one of the most devastating postharvest diseases in Citrus fruit. This work aims to determine the antifungal potential of (E)-2-octenal, a component of plant essential oil, on the mycelial growth and disease incidence of a prochloraz-resistant strain of P. italicum isolated from an orchard in Luxi, Hunan Province, China. An in vitro assay revealed that the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of (E)-2-octenal were 0.25 and 0.50 mL L−1, respectively. An in vivo assay showed that wax + 4 × MFC (E)-2-octenal treatment could reduce the blue mold incidence in Ponkan fruit inoculated with P. italicum without impairing fruit quality. Scanning electron microscopy analysis illustrated that the mycelia of P. italicum were anomalously twisted and shrunken upon treatment with (E)-2-octenal. The primary antifungal targets were attributed to the cell membrane instead of the cell wall based on propidium iodide and calcofluor white staining results. In addition, (E)-2-octenal induced massive accumulation of reactive oxygen species in hyphae in a dose-dependent manner, leading to oxidative stress and dysfunction of energy and respiration metabolism. At the same time, the activities of phosphofructokinase (PFK) and pyruvate kinase (PK) were decreased in (E)-2-octenal-treated samples. Taken together, our present study suggested that (E)-2-octenal could exert its antifungal activity against P. italicum by damaging the function of mitochondria and may be a potential antifungal substance for an alternative to chemical fungicides.

Laurus nobilis essential oil nanoemulsion-infused chitosan: A safe and effective antifungal agent for masticatory preservation

The present study reports the first time investigation on encapsulation of Laurus nobilis essential oil into chitosan nanoemulsion (CS-Ne-LNEO) and assessment of its efficacy to inhibit fungal infestation and aflatoxin B1 (AFB1) contamination in stored masticatories food system. Gas chromatography mass spectrometry (GC-MS) analysis revealed m-Eugenol (46.23%) and D-Limonene (8.89%) as the most abundant components of LNEO. The CS-Ne-LNEO was physico-chemically characterized through scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FTIR), and x-ray diffractometry (XRD) analyses. The CS-Ne-LNEO exhibited broad range of antifungal activity against food contaminating fungi including inhibition of toxigenic Aspergillus flavus (AF-LHP-PE-4) and AFB1 production at lower concentrations as compared to unencapsulated LNEO. The CS-Ne-LNEO caused impairment in ergosterol biosynthesis and enhancement in leakage of Ca2+, Mg2+, K+ ions and 260, 280 nm absorbing materials along with inhibition of methylglyoxal production suggesting the antifungal and antiaflatoxigenic mechanism of action. The DPPH antioxidant activity of CS-Ne-LNEO was noted with IC50 value of 0.004 µL/mL. In addition, the CS-Ne-LNEO caused complete protection of stored Phyllanthus emblica (model masticatories) fruit samples against fungal and AFB1 contamination without altering their sensory characteristics and exhibited high LD50 value (13,504 µL/Kg body weight) mammalian system. Overall, these results indicated that LNEO loaded chitosan nanoemulsion could be promoted as an eco-friendly preservative for complete protection of stored plant masticatories against fungal and AFB1 contamination.

Encapsulation of carvone in chitosan nanoemulsion as edible film for preservation of slice breads against Aspergillus flavus contamination and aflatoxin B1 production

Aspergillus flavus is a common fungus causing bread spoilage by aflatoxin B1 (AFB1) production. Essential oil components are considered as effective antifungal agent; however, volatility and oxidative-instability limited their practical applications. The aim of this study was to fabricate novel chitosan nanoemulsion film incorporating carvone (carvone-Ne) for protection of bread slices against A. flavus and AFB1 contamination in storage conditions. The nanoemulsion was characterized by SEM, DLS, XRD, and FTIR analyses accompanying with sustained delivery of carvone. The carvone-Ne displayed better inhibition of A. flavus (0.5 µL/mL) and AFB1 production (0.4 µL/mL) over unencapsulated carvone along with promising antioxidant activity (p < 0.05). Destruction of ergosterol, mitochondrial-membrane-potential, ions leakage, deformities in methylglyoxal biosynthesis, and in-silico interaction of carvone with Afl-R protein emphasized the antifungal and antiaflatoxigenic mechanisms of action. Further, in-situ preservation potentiality of Carvone-Ne in bread slices with improved gas compositions, and acceptable sensory qualities strengthen its application as innovative packaging material for food preservation.

Candida parapsilosis sensu stricto Antifungal Resistance Mechanisms and Associated Epidemiology.

Fungal diseases cause millions of deaths per year worldwide. Antifungal resistance has become a matter of great concern in public health. In recent years rates of non-albicans species have risen dramatically. Candida parapsilosis is now reported to be the second most frequent species causing candidemia in several countries in Europe, Latin America, South Africa and Asia. Rates of acquired azole resistance are reaching a worrisome threshold from multiple reports as in vitro susceptibility testing is now starting also to explore tolerance and heteroresistance to antifungal compounds. With this review, the authors seek to evaluate known antifungal resistance mechanisms and their worldwide distribution in Candida species infections with a specific focus on C. parapsilosis.

Effect of Potato Glycoside Alkaloids on Energy Metabolism of Fusarium solani.

Fusarium solani is one of the primary pathogens causing root rot of wolfberry. The aims of this study were to investigate the inhibitory effect of potato glycoside alkaloids (PGA) on F. solani and its energy metabolism. In this study, the effects of PGA treatment on the growth and development of F. solani were investigated and the changes in the glycolytic pathway (EMP), ATPase activity, mitochondrial complex activity, mitochondrial structure, and energy charge level were analyzed to elucidate the possible antifungal mechanism of PGA on F. solani. The results showed that PGA treatment inhibited the colony growth, biomass, and spore germination of F. solani. PGA treatment reduced the glucose content and Hexokinase (HK) activity of F. solani, but increased the activity of Fructose-6-Phosphate Kinase (PFK) and Pyruvate Kinase (PK) and promoted the accumulation of pyruvic acid. In addition, PGA treatment inhibited the activities of H+-ATPase, Ca2+-ATPase, and mitochondrial complex IV, increased the mitochondrial inner membrane Ca2+ content and mitochondrial membrane permeability transition pore, and decreased the contents of ATP, ADP, and AMP as well as the energy charge. These results indicate that PGA treatment inhibits the growth and development of F. solani, activates the glycolysis pathway, inhibits ATPase activity and mitochondrial complex activity, and destroys the structure and function of mitochondrial membrane, resulting in a lower energy charge level.