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ABSTRACT Survival factor 1 (Svf1) protein has been described in some ascomycetous fungi where it was found to be contributing to several essential physiological processes, such as response to osmotic, oxidative and cold stresses, sphingolipid biosynthesis, morphogenesis, sporulation, antifungal resistance, and pathogenicity. It was also suggested that it can be a novel central regulator affecting the expression of various genes. In the present study, function of this protein and the encoding genes is described for the first time in a fungus (i.e., in Mucor lusitanicus) belonging to the order Mucorales. M. lusitanicus has two putative svf1 genes named svf1a and svf1b. Expression of both genes was proven. Although the expression of svf1a was affected by several environmental stresses and knocking out the gene affected adaptation to low temperatures and the sporulation ability, the main survival factor functions, such as participation in the maintenance of the viability, the response to oxidative and cold stresses, and the sphingolipid biosynthesis, could be associated with Svf1b, suggesting a central regulatory role to this protein. Interestingly, knockout of both genes affected the pathogenicity of the fungus in a Drosophila model.IMPORTANCEMucor lusitanicus is a widely used model organism to study various biological processes in the basal fungal group Mucorales. Several members of this group can be agents of mucormycosis, an opportunistic fungal infection, which is associated with high mortality, rapid progression, and wide resistance to the commonly used antifungal agents. Svf1 proteins have so far only been identified in fungi, where they have been involved in pathogenicity and resistance to antifungal agents in many cases. Only a limited number of factors affecting the stress response, antifungal resistance, and virulence of Mucorales fungi have been revealed. Elucidating the function of a fungus-specific protein that may regulate these processes may bring us closer to understanding the pathogenesis of these fungi.

The climate-change-coupled fungal burden in crop management and the need to reduce chemical pesticide usage highlight the importance of finding sustainable ways to control Aspergillus flavus. This study examines the effectiveness of 50 Pseudomonas isolates obtained from corn rhizospheres against A. flavus in both solid and liquid co-cultures. The presence and quantity of aflatoxin B1 (AFB1) and AFB1-related compounds were determined using high-performance liquid chromatography–high resolution mass spectrometry analysis. Various enzymatic- or non-enzymatic mechanisms are proposed to interpret the decrease in AFB1 production, accompanied by the accumulation of biosynthetic intermediates (11-hydroxy-O-methylsterigmatocystin, aspertoxin, 11-hydroxyaspertoxin) or degradation products (the compounds C16H10O6, C16H14O5, C18H16O7, and C19H16O8). Our finding implies the upregulation or enhanced activity of fungal oxidoreductases and laccases in response to bacterial bioactive compound(s). Furthermore, non-enzymatic reactions resulted in the formation of additional degradation products due to acid accumulation in the fermented broth. Three isolates completely inhibited AFB1 or any AFB1-related compounds without significantly affecting fungal growth. These bacterial isolates supposedly block the entire pathway for AFB1 production in the fungus during interaction. Apart from identifying effective Pseudomonas isolates as potential biocontrol agents, this work lays the foundation for exploring new bacterial bioactive compounds.

The primary aim of the present study was to identify bacterial isolates with yield-enhancing potential for application as biofertilisers in the cultivation of sweet potato. Therefore, endophytic and rhizospheric strains were isolated from sweet potato plantations in Hungary to identify bacterial strains with plant growth-promoting and antifungal potential. In total, seven Bacillus licheniformis strains were identified and subjected to detailed ecophysiological investigations. Experiments have been conducted to investigate the tolerance of selected strains to different limiting factors such as pH, temperature, and water activity, which affect survivability in various agricultural environments. The majority of tested B. licheniformis strains exhibited plant growth-promoting potential (e.g., production of indole-3-acetic acid up to 40.42 μg mL−1, production of ammonia up to 0.87 mg mL−1, phosphorus solubilising activity, siderophore production), with two strains (SZMC 27713 and SZMC 27715) demonstrating inhibitory activity (ranging between 7% and 38%) against plant pathogenic fungi prevalent in sweet potato cultivation. Furthermore, strain SZMC 27715 induced accelerated germination and a significantly higher germination rate in tomato seeds compared to the control. In a field study, it was observed that strain SZMC 27715 had a potent yield enhancing effect in sweet potato, where a significant yield per plant increase was observed in all treatments (1.13, 1.09 and 1.40 kg) compared to the control plants (0.92 kg). The highest yield per plant was observed when the cuttings were soaked combined with two additional foliar treatments. To the best of our knowledge, this is the first report of the successful utilisation of the B. licheniformis strain as a biofertiliser for yield enhancement in sweet potato cultivation. Based on our results, strain SZMC 27715 has potential for application as a biofertiliser in sweet potato cultivation either as a standalone option or in a microbial consortium.

This study explores various potential substitutes for gypsum in the production of compost for white button mushrooms (Agaricus bisporus). During compost preparation, calcium sulfate (CaSO4) was replaced with calcium carbonate (CaCO3), ammonium sulfate ((NH4)2SO4), and monocalcium phosphate (Ca(H2PO4)2). Complete replacement of gypsum with calcium carbonate led to a significant pH increase during the second phase of composting, adversely affecting mushroom mycelium growth. Compost parameters were observed to be similar in scenarios where calcium sulfate was supplemented with calcium carbonate in 8:2 and 6:4 ratios, both with and without the presence of ammonium sulfate, and in 3:1 and 1:1 mixtures of calcium sulfate and monocalcium phosphate, when compared to traditional gypsum-based processes. All experimental compost mixtures yielded comparable mushroom crops in cultivation trials. Notably, the 8:2 mixture of calcium sulfate and calcium carbonate demonstrated superior performance in cultivation trials relative to the 6:4 mixture. However, supplementing these mixtures with ammonium sulfate resulted in similar crop yields. Monocalcium phosphate also emerged as a promising partial gypsum substitute, showing comparable crop production in both 3:1 and 1:1 ratios to the technological optimum. The exploration of alternative calcium sources like calcium carbonate and monocalcium phosphate reflects the adaptability of the industry in response to resource availability challenges. The potential use of byproducts like ammonium sulfate from the composting process itself offers a cost-effective and environmentally friendly approach to compost formulation, underscoring its worldwide relevance.

Integrated disease management and plant protection have been discussed with much fervor in the past decade due to the rising environmental concerns of using industrially produced pesticides. Members of the genus Trichoderma are a subject of considerable research today due to their several properties as biocontrol agents. In our study, the peptaibol production of Trichoderma longibrachiatum SZMC 1775, T. longibrachiatum f. bissettii SZMC 12546, T. reesei SZMC 22616, T. reesei SZMC 22614, T. saturnisporum SZMC 22606 and T. effusum SZMC 22611 were investigated to elucidate structure-activity relationships (SARs) between the properties of peptaibols and their 3D structures. The effects of peptaibol mixtures obtained from every Trichoderma strain were examined against nine commonly known bacteria. The lowest minimum inhibitory concentrations (MIC, mg ml−1) were exerted by T. longibrachiatum f. bissettii SZMC 12546 against Gram-positive bacteria, which was also able to inhibit the plant pathogenic Gram-negative Rhizobium radiobacter. Accelerated molecular dynamics (aMD) simulations were performed in aqueous solvent to explore the folding dynamics of 12 selected peptaibol sequences. The most characteristic difference between the peptaibols from group A and B relies in the ‘Gly-Leu-Aib-Pro’ and ‘Gly-Aib-Aib-Pro’ motifs (‘Aib’ stands for α-aminoisobutyric acid), which imparted a significant effect on the folding dynamics in water and might be correlated with their expressed bioactivity. In our aMD simulation experiments, Group A peptaibols showed more restricted folding dynamics with well-folded helical conformations as the most stable representative structures. This structural stability and dynamics may contribute to their bioactivity against the selected bacterial species.

Purpose: To investigate the dematiaceous fungal profile of patients with ocular mycoses attending a tertiary eye care hospital in Coimbatore, India Methods: The identification of dematiaceous fungus based on their morphology, their genotypes, and the measurement of the minimum inhibitory concentrations (MICs) using microdilution method of routinely used antifungal drugs were all compared. Results: A total of 148 dematiaceous fungi were isolated during a study period of 27 months. Isolates were confirmed as Curvularia spp. (n = 98), Exserohilum spp. (n = 32), Alternaria spp. (n = 14), Exophiala spp. (n = 2), Cladosporium sp. (n = 1) and Aureobasidium sp. (n = 1). Out of 50 well grown isolates characterized genotypically based on the amplification and sequencing of the ITS region of the ribosomal RNA gene cluster and subsequent BLAST analysis, Curvularia lunata (n = 24), C. aeria (n = 1), C. spicifera (n = 8), C. hawaiiensis (n = 1), C. maydis (n = 2), C. papendorfii (n = 2), C. geniculata (n = 3), C. tetramera (n = 2) and Exs. rostratum (n = 7) were identified. In vitro antifungal susceptibilities of the most tested dematiaceous isolates showed that voriconazole had a MIC50 of 0.25 μg ml−1, while amphotericin B had a MIC50 of 0.25 μg ml−1 for Curvularia spp. and Alternaria spp. Conclusion: Voriconazole proved to be the most effective drug against the pigmented filamentous fungi, followed by amphotericin B, itraconazole and econazole.

Cryptococcosis, a life-threatening fungal infection, frequently occurs in patients suffering from AIDS. The treatment of the disease is hampered by the limited number of the effective drugs and the increasing resistance; therefore, to find new active substances is needed. As meningitis is the most serious infection affecting the AIDS patients, effective drugs have to be capable of entering to the central nervous system. Ecdysteroids are natural bioactive molecules with considerable anabolic activity and without toxic side effects on humans. The aim of this work was to study the anti-cryptococcal activity of a natural ecdysteroid, 20E, and its three semi-synthetic derivatives obtained by structural modification of the original molecule. We established the minimum inhibitory concentration of the compounds with microdilution method and demonstrated their fungicidal activity by flow cytometry and cultivation of the drug-treated cells. The interaction of the compounds with each other and efflux transporter inhibitors was assessed by checkerboard titration method. Two derivatives, 20E-EOx and 20E-ZOx, inhibited the growth of Cryptococcus neoformans with minimum inhibitory concentration 2 mg/mL and 1 mg/mL, respectively; both compounds possess fungicidal effect. A combination of the ecdysteroids with each other and verapamil resulted in additive interaction. This study confirmed that structural modification of an originally non-antimicrobial molecule can enhance its effectiveness.

Abstract The mammalian HMGB1 is a high-mobility-group B protein, which is both an architectural and functional element of chromatin. Nhp6p, the extensively studied fungal homologue of HMGB1 in Saccharomyces cerevisiae has pleiotropic physiological functions. Despite the existence of Nhp6p orthologues in filamentous ascomycetes, little is known about their physiological roles besides their contribution to sexual development. Here we study the function of HmbA, the Aspergillus nidulans orthologue of Nhp6p. We show that HmbA influences the utilization of various carbon- and nitrogen sources, stress tolerance, secondary metabolism, hyphae elongation and maintenance of polarized growth. Additionally, by conducting heterologous expression studies, we demonstrate that HmbA and Nhp6p are partially interchangeable. HmbA restores SNR6 transcription and fitness of nhp6AΔBΔ mutant and reverses its heat sensitivity. Nhp6Ap complements several phenotypes of hmbAΔ, including ascospore formation, utilization of various carbon- and nitrogen-sources, radial growth rate, hypha elongation by polarized growth. However, Nhp6Ap does not complement sterigmatocystin production in a hmbAΔ strain. Finally, we also show that HmbA is necessary for the normal expression of the endochitinase chiA, a cell wall re-modeller that is pivotal for the normal mode of maintenance of polar growth.

Genes involved in mycoremediation were identified by comparative genomics analysis in 10 armillarioid species and selected groups of white-rot Basidiomycota (14) and soft-rot Ascomycota (12) species to confine the distinctive bioremediation capabilities of the armillarioids. The genomes were explored using phylogenetic principal component analysis (pPCA), searching for genes already documented in a biocatalysis/biodegradation database. The results underlined a distinct, increased potential of aromatics-degrading genes/enzymes in armillarioids, with particular emphasis on a high copy number and diverse spectrum of benzoate 4-monooxygenase [EC:1.14.14.92] homologs. In addition, other enzymes involved in the degradation of various monocyclic aromatics were more abundant in the armillarioids than in the other white-rot basidiomycetes, and enzymes involved in the degradation of polycyclic aromatic hydrocarbons (PAHs) were more prevailing in armillarioids and other white-rot species than in soft-rot Ascomycetes. Transcriptome profiling of A. ostoyae and A. borealis isolates confirmed that several genes involved in the degradation of benzoates and other monocyclic aromatics were distinctively expressed in the wood-invading fungal mycelia. Data were consistent with armillarioid species offering a more powerful potential in degrading aromatics. Our results provide a reliable, practical solution for screening the likely fungal candidates for their full biodegradation potential, applicability, and possible specialization based on their genomics data.

ABSTRACT Certain members of the order Mucorales can cause a life-threatening, often-fatal systemic infection called mucormycosis. Mucormycosis has a high mortality rate, which can reach 96 to 100% depending on the underlying condition of the patient. Mucorales species are intrinsically resistant to most antifungal agents, such as most of the azoles, which makes mucormycosis treatment challenging. The main target of azoles is the lanosterol 14α-demethylase (Erg11), which is responsible for an essential step in the biosynthesis of ergosterol, the main sterol component of the fungal membrane. Mutations in the erg11 gene can be associated with azole resistance; however, resistance can also be mediated by loss of function or mutation of other ergosterol biosynthetic enzymes, such as the sterol 24-C-methyltransferase (Erg6). The genome of Mucor lusitanicus encodes three putative erg6 genes (i.e., erg6a, erg6b, and erg6c). In this study, the role of erg6 genes in azole resistance of Mucor was analyzed by generating and analyzing knockout mutants constructed using the CRISPR-Cas9 technique. Susceptibility testing of the mutants suggested that one of the three genes, erg6b, plays a crucial role in the azole resistance of Mucor. The sterol composition of erg6b knockout mutants was significantly altered compared to that of the original strain, and it revealed the presence of at least four alternative sterol biosynthesis pathways leading to formation of ergosterol and other alternative, nontoxic sterol products. Dynamic operation of these pathways and the switching of biosynthesis from one to the other in response to azole treatment could significantly contribute to avoiding the effects of azoles by these fungi. IMPORTANCE The fungal membrane contains ergosterol instead of cholesterol, which offers a specific point of attack for the defense against pathogenic fungi. Indeed, most antifungal agents target ergosterol or its biosynthesis. Mucormycoses-causing fungi are resistant to most antifungal agents, including most of the azoles. For this reason, the drugs of choice to treat such infections are limited. The exploration of ergosterol biosynthesis is therefore of fundamental importance to understand the azole resistance of mucormycosis-causing fungi and to develop possible new control strategies. Characterization of sterol 24-C-methyltransferase demonstrated its role in the azole resistance and virulence of M. lusitanicus. Moreover, our experiments suggest that there are at least four alternative pathways for the biosynthesis of sterols in Mucor. Switching between pathways may contribute to the maintenance of azole resistance.

ABSTRACT Armillaria ostoyae, a species among the destructive forest pathogens from the genus Armillaria, causes root rot disease on woody plants worldwide. Efficient control measures to limit the growth and impact of this severe underground pathogen are under investigation. In a previous study, a new soilborne fungal isolate, Trichoderma atroviride SZMC 24276 (TA), exhibited high antagonistic efficacy, which suggested that it could be utilized as a biocontrol agent. The dual culture assay results indicated that the haploid A. ostoyae-derivative SZMC 23085 (AO) (C18/9) is highly susceptible to the mycelial invasion of TA. In the present study, we analyzed the transcriptome of AO and that of TA in in vitro dual culture assays to test the molecular arsenal of Trichoderma antagonism and the defense mechanisms of Armillaria. We conducted time-course analysis and functional annotation and analyzed enriched pathways and differentially expressed genes including biocontrol-related candidate genes from TA and defense-related candidate genes from AO. The results indicated that TA deployed several biocontrol mechanisms when confronted with AO. In response, AO initiated multiple defense mechanisms to protect against the fungal attack. To our knowledge, the present study offers the first transcriptome analysis of a biocontrol fungus attacking AO. Overall, this study provides insights that aid the further exploration of plant pathogen-biocontrol agent interaction mechanisms. IMPORTANCE Armillaria species can survive for decades in the soil on dead woody debris, develop rapidly under favorable conditions, and harmfully infect newly planted forests. Our previous study found Trichoderma atroviride to be highly effective in controlling Armillaria growth; therefore, our current work explored the molecular mechanisms that might play a key role in Trichoderma-Armillaria interactions. Direct confrontation assays combined with time course-based dual transcriptome analysis provided a reliable system for uncovering the interactive molecular dynamics between the fungal plant pathogen and its mycoparasitic partner. Furthermore, using a haploid Armillaria isolate allowed us to survey the deadly prey-invading activities of the mycoparasite and the ultimate defensive strategies of its prey. Our current study provides detailed insights into the essential genes and mechanisms involved in Armillaria defense against Trichoderma and the genes potentially involved in the efficiency of Trichoderma to control Armillaria. In addition, using a sensitive haploid Armillaria strain (C18/9), with its complete genome data already available, also offers the opportunity to test possible variable molecular responses of Armillaria ostoyae toward diverse Trichoderma isolates with various biocontrol abilities. Initial molecular tests of the dual interactions may soon help to develop a targeted biocontrol intervention with mycoparasites against plant pathogens.

Lemongrass is a medicinal plant that produces essential oil with a variety of therapeutic properties. Although lemongrass essential oil (LGEO) is promising in clinical applications, the existing knowledge on the efficacy and safety of LGEO remains limited. This scoping review aimed to identify, summarize, and synthesize existing literature related to the clinical applications of LGEO to provide an overview of its potential therapeutic benefits for patients. Three databases (PubMed, Web of Science, Scopus) were used following the PRISMA-ScR guidelines to find articles published between 1 January 2013, and 1 November 2022. A total of 671 records were identified and 8 articles were included in this scoping review. The majority of patients received oromucosal and topical treatment. The results of the studies suggest that LGEO might be a useful tool in the treatment of periodontitis, gingivitis and oral malodour, with similar efficacy to chlorhexidine (anti-gingivitis effect) and doxycycline (periodontitis). Additionally, LGEO has the potential for treating pityriasis versicolor and preventing skin aging and may have anti-dandruff effects. These findings not only underscore the diverse clinical potential of LGEO but also emphasize its comparable efficacy to established treatments. Further research is imperative to comprehensively evaluate LGEO’s effectiveness, safety, mechanisms of action, potential interactions with other medications, and its long-term tolerability across diverse populations.

A novel nicotinate degradation pathway is described in Aspergillus nidulans, with metabolic products identified that were not previously found in prokaryotic species. This is the first such pathway to be described in a eukaryote.

Fluconazole resistance is commonly encountered in Candida auris, and the yeast frequently displays resistance to other standard drugs, which severely limits the number of effective therapeutic agents against this emerging pathogen. In this study, we aimed to investigate the effect of acquired azole resistance on the viability, stress response, and virulence of this species. Fluconazole-, posaconazole-, and voriconazole- resistant strains were generated from two susceptible C. auris clinical isolates (0381, 0387) and compared under various conditions. Several evolved strains became pan-azole-resistant, as well as echinocandin-cross-resistant. While being pan-azole-resistant, the 0381-derived posaconazole-evolved strain colonized brain tissue more efficiently than any other strain, suggesting that fitness cost is not necessarily a consequence of resistance development in C. auris. All 0387-derived evolved strains carried a loss of function mutation (R160S) in BCY1, an inhibitor of the PKA pathway. Sequencing data also revealed that posaconazole treatment can result in ERG3 mutation in C. auris. Despite using the same mechanisms to generate the evolved strains, both genotype and phenotype analysis highlighted that the development of resistance was unique for each strain. Our data suggest that C. auris triazole resistance development is a highly complex process, initiated by several pleiotropic factors.

We previously reported on a novel peptaibol, named Tripleurin XIIc (TPN), an 18-residue long sequence produced by the fungus Trichoderma pleuroti. We elucidated its 3D structure via classical and accelerated molecular dynamics simulation (aMD) methods and reported the folding dynamics of TPN in water and chloroform solvents. Peptaibols, in general, are insoluble in water, as they are amphipathic and may prefer hydrophobic environments like transmembrane regions. In this study, we attempted to use aMD simulations to model an all-atom bacterial membrane system while placing a TPN molecule in its vicinity. The results highlighted that TPN was able to introduce some disorder into the membrane and caused lipid clustering. It could also enter the transmembrane region from the water-bilayer interface. The structural dynamics of TPN in the transmembrane region revealed a single energetically stable conformation similar to the one obtained from water and chloroform solvent simulations reported by us previously. However, this linear structure was found to be at the local energy minimum (stable) in water but at a metastable intermediate state (higher energy) in chloroform. Therefore, it could be said that the water solvent can be successfully used for folding simulations of peptaibols.

ABSTRACT Mucormycosis is an invasive fungal infection caused by certain members of the fungal order of Mucorales. The species most frequently identified as the etiological agents of mucormycosis belong to the genera Rhizopus, Lichtheimia, and Mucor. The frequency of systemic mucormycosis has been increasing, mainly because of increasing numbers of susceptible patients. Furthermore, Mucorales display intrinsic resistance to the majority of routinely used antifungal agents (e.g., echinocandins and short-tailed azoles), which limits the number of possible therapeutic options. All the above-mentioned issues urge the improvement of molecular identification methods and the discovery of new antifungal targets and strategies. Spore coat proteins (CotH) constitute a kinase family present in many pathogenic bacteria and fungi and participate in the spore formation in these organisms. Moreover, some of them can act as virulence factors being receptors of the human GRP78 protein during Rhizopus delemar-induced mucormycosis. We identified 17 cotH-like genes in the Mucor lusitanicus genome database. Successful disruption of five cotH genes in Mucor was performed using the CRISPR-Cas9 system. The CotH3 and CotH4 proteins play a role in adaptation to different temperatures as well as in developing the cell wall structure. We also show CotH4 protein is involved in spore wall formation by affecting the total chitin content and, thus, the composition of the spore wall. The role of CotH3 and CotH4 proteins in virulence was confirmed in two invertebrate models and a diabetic ketoacidosis (DKA) mouse model. IMPORTANCE Current treatment options for mucormycosis are inadequate, resulting in high mortality rates, especially among immunosuppressed patients. The development of novel therapies for mucormycosis has been hampered by lack of understanding of the pathogenetic mechanisms. The importance of the cell surface CotH proteins in the pathogenesis of Rhizopus-mediated mucormycosis has been recently described. However, the contribution of this family of proteins to the virulence of other mucoralean fungi and their functionality in vital processes remain undefined. Through the use of the CRISPR-Case9 gene disruption system, we demonstrate the importance of several of the CotH proteins to the virulence of Mucor lusitanicus by using three infection models. We also report on the importance of one of these proteins, CotH4, to spore wall formation by affecting chitin content. Therefore, our studies extend the importance of CotH proteins to Mucor and identify the mechanism by which one of the CotH proteins contributes to the development of a normal fungal cell wall, thereby indicating that this family of proteins can be targeted for future development of novel therapeutic strategies of mucormycosis.

Candida infections are the most prevalent cause of serious human mycoses and are the third most common pathogens isolated from bloodstream infections in hospitalized patients. C. parapsilosis is a member of the non-albicans spp., which have a predilection for causing life-threatening disease in neonates and hospitalized pediatric patients. In this study, we utilized a Drosophila melanogaster infection model to analyze the immunological responses to C. parapsilosis. Our results demonstrate that the Toll pathway in Drosophila controls C. parapsilosis proliferation as the Toll signaling mutant MyD88−/− flies are highly susceptible to C. parapsilosis. We also confirmed that the MyD88−/− fly is a convenient invertebrate animal model to analyze virulence properties of different species and strains from the C. parapsilosis sensu lato complex as C. orthopsilosis, C. metapsilosis proved to be less virulent than C. parapsilosis sensu stricto and the N-mannan deficient C. parapsilosis och1Δ/Δ strain showed attenuated pathogenicity in this immunodeficient Drosophila background. We also found that Persephone protease is not required for detection and activation of Toll pathway during C. parapsilosis infection. Furthermore, we observed that Drosophila β-glucan receptor deficient flies where more sensitive to C. parapsilosis compared to wild-type flies; however, we could not find a clear dependence on the recognition of this receptor and the cell wall β-glucan exposure-induced host response. These studies establish this D. melanogaster infection model as an efficient tool in deciphering immune responses to C. parapsilosis as well as for assessing virulence factors produced by this emerging fungal predator.

The utilization of microorganisms with biocontrol activity against fungal and bacterial pathogens of plants is recognized as a promising, effective, and environment-friendly strategy to protect agricultural crops. We report the glyphosate-tolerant Pseudomonas resinovorans SZMC 25872 isolate as a novel strain with antagonistic potential towards the plant pathogenic bacterium Agrobacterium tumefaciens. In our studies, the growth of the P. resinovorans SZMC 25872 and A. tumefaciens SZMC 14557 isolates in the presence of 74 different carbon sources, and the effect of 11 carbon sources utilized by both strains on the biocontrol efficacy was examined. Seven variations of media with different carbon sources were selected for the assays to observe the biocontrol potential of the P. resinovorans strain. Also, 50% concentrations of the cell-free culture filtrates (CCF) obtained from medium amended with L-alanine or succinic acid as sole carbon source were found to be effective for the growth suppression of A. tumefaciens by 83.03 and 56.80%, respectively. The effect of 7 media on siderophore amount and the activity of extracellular trypsin- and chymotrypsin-like proteases, as well as esterases were also evaluated. Significant positive correlation was found between the siderophore amount and the percentage of inhibition, and the inhibitory effect of the CCFs obtained from medium amended with succinic acid was eliminated in the presence of an additional iron source, suggesting that siderophores produced by P. resinovorans play an important role in its antagonistic potential. The metabolic profile analysis of the P. resinovorans SZMC 25872 strain, performed by high performance liquid chromatography - high resolution mass spectrometry (HPLC-HRMS), has identified several previously not reported metabolites that might play role in the antagonistic effect against A. tumefaciens. Based on our findings we suggest that the possible inhibition modes of A. tumefaciens SZMC 14557 by P. resinovorans SZMC 25872 include siderophore-mediated suppression, extracellular enzyme activities and novel bioactive metabolites.

The zinc restriction and zinc toxicity are part of host defence, called nutritional immunity. The crucial role of zinc homeostasis in microbial survival within a host is established, but little is known about these processes in the opportunistic human fungal pathogen Candida parapsilosis. Our in silico predictions suggested the presence of at least six potential zinc transporters (ZnTs) in C. parapsilosis—orthologues of ZRC1, ZRT3 and ZRT101—but an orthologue of PRA1 zincophore was not found. In addition, we detected a species-specific gene expansion of the novel ZnT ZRT2, as we identified three orthologue genes in the genome of C. parapsilosis. Based on predictions, we created homozygous mutant strains of the potential ZnTs and characterized them. Despite the apparent gene expansion of ZRT2 in C. parapsilosis, only CpZRT21 was essential for growth in a zinc-depleted acidic environment, in addition we found that CpZrc1 is essential for zinc detoxification and also protects the fungi against the elimination of murine macrophages. Significantly, we demonstrated that C. parapsilosis forms zincosomes in a Zrc1-independent manner and zinc detoxification is mediated by the vacuolar importer CpZrc1. Our study defines the functions of C. parapsilosis ZnTs, including a species-specific survival and zinc detoxification system.

The cultivation and consumption of sweet potato (Ipomoea batatas) are increasing globally. As the usage of chemical fertilizers and pest control agents during its cultivation may lead to soil, water and air pollution, there is an emerging need for environment-friendly, biological solutions enabling increased amounts of healthy crop and efficient disease management. Microbiological agents for agricultural purposes gained increasing importance in the past few decades. Our goal was to develop an agricultural soil inoculant from multiple microorganisms and test its application potential in sweet potato cultivation. Two Trichoderma strains were selected: Trichoderma ghanense strain SZMC 25217 based on its extracellular enzyme activities for the biodegradation of plant residues, and Trichoderma afroharzianum strain SZMC 25231 for biocontrol purposes against fungal plant pathogens. The Bacillus velezensis strain SZMC 24986 proved to be the best growth inhibitor of most of the nine tested strains of fungal species known as plant pathogens, therefore it was also selected for biocontrol purposes against fungal plant pathogens. Arthrobacter globiformis strain SZMC 25081, showing the fastest growth on nitrogen-free medium, was selected as a component with possible nitrogen-fixing potential. A Pseudomonas resinovorans strain, SZMC 25872, was selected for its ability to produce indole-3-acetic acid, which is among the important traits of potential plant growth-promoting rhizobacteria (PGPR). A series of experiments were performed to test the selected strains for their tolerance to abiotic stress factors such as pH, temperature, water activity and fungicides, influencing the survivability in agricultural environments. The selected strains were used to treat sweet potato in two separate field experiments. Yield increase was observed for the plants treated with the selected microbial consortium (synthetic community) in comparison with the control group in both cases. Our results suggest that the developed microbial inoculant has the potential to be used in sweet potato plantations. To the best of our knowledge, this is the first report about the successful application of a fungal-bacterial consortium in sweet potato cultivation.

Natural compounds are a suitable alternative to synthetic food preservatives due to their natural origin and health-promoting properties. In the current study, phenolic–phenolic and phenolic–synthetic combinations were tested for their antibiofilm formation, anti-planktonic growth, and anti-adhesion properties against Debaryomyces hansenii, Wickerhamomyces anomalus (formerly Pichia anomala), Schizosaccharomyces pombe, and Saccharomyces cerevisiae. The phenolics were vanillin and cinnamic acid, while the synthetic preservatives were sodium benzoate, potassium sorbate, and sodium diacetate. The vanillin–cinnamic acid combination had synergistic effect in all the tested yeasts for the biofilm inhibition with a fractional inhibitory concentration index (FICI) of ≤0.19 for W. anomalus, 0.25 for S. pombe, 0.31 for S. cerevisiae, and 0.5 for D. hansenii. Most of the phenolic–synthetic combinations had indifferent interaction regarding biofilm formation. The vanillin–cinnamic acid combination also had higher activity against spoilage yeasts adhesion on the abiotic surface and planktonic growth compared to the phenolic–synthetic combinations. For the phenolic–synthetic anti-planktonic activity, synergistic interaction was present in all the vanillin–synthetic combinations in S. pombe, vanillin–sodium benzoate and vanillin–potassium sorbate in S. cerevisiae, vanillin–sodium benzoate in W. anomalus, and cinnamic acid–sodium diacetate in S. pombe. These results suggest a novel antimicrobial strategy that may broaden the antimicrobial spectrum and reduce compound toxicity against food spoilage yeasts.

Abstract Background Dimorphism and biofilm formation are important virulence factors of some opportunistic human pathogenic yeasts. Such species commensally colonize skin or mucosal surfaces generally in yeast form, but under particular circumstances, convert into virulent hyphae and disseminate internal organs or cause mucocutaneous infections. The yeast-to-hypha shape-conversion promotes the development of a biofilm, a thick extracellular matrix with sessile cells within. The biofilm is capable to prevent the penetration of antifungal drugs, rendering the surviving biofilm-resident cells intrinsic sources of recurrent infections. The aim of this study was to evaluate the ability of silver nanoparticles (AgNPs) to attenuate the morphological switch and biofilm formation of several opportunistic pathogenic yeasts and to determine whether this feature depends on the nanoparticle size. Results AgNPs in three different sizes were prepared by chemical reduction approach and characterized by transmission electron microscopy, ultraviolet–visible spectroscopy and dynamic light scattering. The antifungal activity was evaluated by the microdilution method, the inhibitory capacity on biofilm formation and the biofilm degradation ability of differently sized AgNPs was assessed by viability assay. The morphological state of opportunistic pathogenic yeast cells in monoculture and in co-culture with human keratinocytes in the presence of AgNPs was examined by flow cytometry and scanning electron microscopy. All the three AgNPs inhibited the growth of the examined opportunistic pathogenic yeasts, nevertheless, AgNPs with the smallest diameter exhibited the most prominent toxic activities. AgNPs attenuated the biofilm formation in a nanoparticle size-dependent manner; however, their biofilm destruction capacity was negligible. AgNPs with the smallest size exerted the most significant effect on suppressing the morphological change of pathogens in monoculture as well as in a co-culture with keratinocytes. Conclusions Our results confirm that AgNPs are capable to hinder yeast-to-hypha morphological conversion and biofilm formation of opportunistic pathogens and this biological effect of AgNPs is size-dependent.

The aim of the study was investigate the DNA polymorphisms of Aspergillus flavus and Aspergillus tamarii isolated from regional fungal keratitis cases based on random amplified polymorphic DNA (RAPD) analyses. Thirty-two A. flavus and six A. tamarii isolates were identified using conventional macroscopic and microscopic features. Five decamer primers were used to amplify the genomic DNA sequences of the test isolates, and 140 total RAPD patterns were generated among them. From each of the 38 isolates, a range of 15–30 RAPD types were revealed. All of these were isolated from male farmers from Tamil Nadu. In the future, RAPD analyses could be employed as a reliable and rapid technique to differentiate among keratitis isolates of fungal pathogens. Keywords: RAPD PCR, Eye infection, Aspergillus flavus, Corneal ulcer, Molecular typing, Keratitis

The development of novel antibiotics with desirable properties from naturally available organisms is an on-going process in the field of microbial biotechnology. Mangroves are not extensively exploited for the isolation and screening of antimicrobial agents of the genus Streptomyces. The emergence of multidrug-resistant pathogens necessitates continuous search for novel antimicrobial agents. Also, searching of novel strains from pristine environments is an important approach for obtaining novel bioactive molecules. Streptomyces sp. AS4 was isolated from the mangroves. Antibiotic production was performed using substrates such as, apple pomace, pine apple peel, orange peel, rice bran, wheat bran, green gram husk, banana peel, pomegranate peel and black gram husk in solid state fermentation. Antibacterial activity was performed using Gram positive bacterial pathogen. The nutrient sources (carbon, nitrogen and ion) were optimized initially by one-variable-at-a-time approach and two level full factorial designs. In this study 129 actinomycetes were isolated from the mangroves. The isolates were assessed for their antibacterial activity. Among these isolates, Streptomyces sp. AS4 showed very strong antibacterial activity. Among these substrates, wheat bran showed enhanced production of antibiotics (209 U/g), whereas pomegranate peel supported very less quantity of antibiotics production (43 U/g) in a two level full factorial design. The process parameters were screened using traditional methods and statistical approach. Among the tested factors, moisture, yeast extract and CaCl2 significantly influenced the antibiotics production. Central composite design and response surface methodology enhanced antibiotics production by Streptomyces sp. AS4 by 10 fold than non-optimized medium. Streptomyces sp. AS4, a mangrove isolate showed hyper production of antibiotics. The strain AS4 utilized various agro-wastes including wheat bran for antibiotics production. Wheat bran proved to be a potent solid substrate for the production of antibiotics in an industrial scale. The actinomycetes strain, Streptomyces sp. AS4 and its antibiotics may have great application against various drug resistant organisms. Keywords: Antibiotics, Drug resistant, Solid state fermentation, Streptomyces

ABSTRACT Oral squamous cell carcinoma (OSCC) is associated with oral Candida albicans infection, although it is unclear whether the fungus promotes the genesis and progression of OSCC or whether cancer facilitates fungal growth. In this study, we investigated whether C. albicans can potentiate OSCC tumor development and progression. In vitro, the presence of live C. albicans, but not Candida parapsilosis, enhanced the progression of OSCC by stimulating the production of matrix metalloproteinases, oncometabolites, protumor signaling pathways, and overexpression of prognostic marker genes associated with metastatic events. C. albicans also upregulated oncogenes in nonmalignant cells. Using a newly established xenograft in vivo mouse model to investigate OSCC-C. albicans interactions, oral candidiasis enhanced the progression of OSCC through inflammation and induced the overexpression of metastatic genes and significant changes in markers of the epithelial-mesenchymal transition. Finally, using the 4-nitroquinoline 1-oxide (4NQO) murine model, we directly correlate these in vitro and short-term in vivo findings with the progression of oncogenesis over the long term. Taken together, these data indicate that C. albicans upregulates oncogenes, potentiates a premalignant phenotype, and is involved in early and late stages of malignant promotion and progression of oral cancer. IMPORTANCE Oral squamous cell carcinoma (OSCC) is a serious health issue worldwide that accounts for 2% to 4% of all cancer cases. Previous studies have revealed a higher yeast carriage and diversity in oral cancer patients than in healthy individuals. Furthermore, fungal colonization in the oral cavity bearing OSCC is higher on the neoplastic epithelial surface than on adjacent healthy surfaces, indicating a positive association between oral yeast carriage and epithelial carcinoma. In addition to this, there is strong evidence supporting the idea that Candida contributes to carcinogenesis events in the oral cavity. Here, we show that an increase in Candida albicans burden promotes an oncogenic phenotype in the oral cavity.

Aflatoxins (AFs) are a group of secondary metabolites that cause several diseases in both animals and humans. Since the discovery of this group of toxins, several effects were revealed, such as hepatic changes, carcinoma, failure, and cancer of the liver. In the European Union, there are concentration limits for this group of mycotoxins in food and feed products; thus, these substances are required in their pure forms to prepare reference standards or certified reference materials. In our present work, a liquid–liquid chromatographic method utilizing a toluene/acetic acid/water ternary system was improved. In order to enhance the purification and gain a higher amount of pure AFs in one separation run, a scale-up of the previous separation was carried out. In several scale-up steps—including the determination of the maximum concentration and volume to load on a 250 mL rotor via a loop and via a pump as well, and the quadruplication of the entire separation procedure to a 1000 mL rotor—an efficient scale-up was achieved. Utilizing a 250 mL rotor in an 8-hour workday, altogether approximately 2.2 g of total AFs could be purified with 8.2 liters of solvent, while on a 1000 mL column, approximately 7.8 g AFs could be prepared, utilizing around 31 liters of solvents.

In routine measurements, the length of the analysis time and nfumber of samples analysed during a time unit are crucial parameters, which are especially important for the food analysis, particularly in the case of mycotoxin determinations. High-resolution equipment, including time-of-flight or Orbitrap analyzators, can provide stable instrumental background for high-throughput analyses. In this report, a short, 1 min MS-based multi-mycotoxin method was developed with the application of a short column as a reduced chromatographic separation, taking advantages of the multiplexing and high-resolution capability of the QExactive Orbitrap MS possessing sub-1 ppm mass accuracy. The performance of the method was evaluated regarding selectivity, LOD, LOQ, linearity, matrix effect, and recovery, and compared to a UHPLC-MS/MS method. The final multiplexing method was able to quantify 11 mycotoxins in defined ranges (aflatoxins (corn, 2.8–600 μg/kg; wheat, 1.5–350 μg/kg), deoxynivalenol (corn, 640–9600 μg/kg; wheat, 128–3500 μg/kg), fumonisins (corn, 20–1500 μg/kg; wheat, 30–3500 μg/kg), HT-2 (corn, 64–5200 μg/kg; wheat, 61–3500 μg/kg), T-2 (corn, 10–800 μg/kg; wheat, 4–250 μg/kg), ochratoxin (corn, 4.7–600 μg/kg; wheat, 1–1000 μg/kg), zearalenone (corn, 64–4800 μg/kg; wheat, 4–500 μg/kg)) within one minute in corn and wheat matrices at the MRL levels stated by the European Union.

Surfactins are cyclic lipopeptides consisting of a β-hydroxy fatty acid of variable chain length and a peptide ring of seven amino acids linked together by a lactone bridge, forming the cyclic structure of the peptide chain. These compounds are produced mainly by Bacillus species and are well regarded for their antibacterial, antifungal, and antiviral activities. For their surfactin production profiling, several Bacillus strains isolated from vegetable rhizospheres were identified by their fatty acid methyl ester profiles and were tested against phytopathogen bacteria and fungi. The isolates showed significant inhibition against of E. amylovora, X. campestris, B. cinerea, and F. culmorum and caused moderate effects on P. syringae, E. carotovora, A. tumefaciens, F. graminearum, F. solani, and C. gloeosporioides. Then, an HPLC-HESI-MS/MS method was applied to simultaneously carry out the quantitative and in-depth qualitative characterisations on the extracted ferment broths. More than half of the examined Bacillus strains produced surfactin, and the MS/MS spectra analyses of their sodiated precursor ions revealed a total of 29 surfactin variants and homologues, some of them with an extremely large number of peaks with different retention times, suggesting a large number of variations in the branching of their fatty acid chains.

In Aspergillus nidulans a regulon including 11 hxn genes (hxnS, T, R, P, Y, Z, X, W, V, M and N) is inducible by a nicotinate metabolic derivative, repressible by ammonium and under stringent control of the nitrogen-state-sensitive GATA factor AreA and the specific transcription factor HxnR. This is the first report in a eukaryote of the genomic organization of a possibly complete pathway of nicotinate utilization. In A. nidulans the regulon is organized in three distinct clusters, this organization is variable in the Ascomycota. In some Pezizomycotina species all 11 genes map in a single cluster; in others they map in two clusters. This variable organization sheds light on cluster evolution. Instances of gene duplication followed by or simultaneous with integration in the cluster, partial or total cluster loss, and horizontal gene transfer of several genes (including an example of whole cluster re-acquisition in Aspergillus of section Flavi) were detected, together with the incorporation in some clusters of genes not found in the A. nidulans co-regulated regulon, which underlie both the plasticity and the reticulate character of metabolic cluster evolution. This study provides a comprehensive phylogeny of six members of the cluster across representatives of all Ascomycota classes.

The presence of viruses is less explored in Mucoromycota as compared to other fungal groups such as Ascomycota and Basidiomycota. Recently, more and more mycoviruses are identified from the early-diverging lineages of fungi. We have determined the genome of 11 novel dsRNA viruses in seven different Umbelopsis strains with next-generation sequencing (NGS). The identified viruses were named Umbelopsis ramanniana virus 5 (UrV5), 6a (UrV6a); 6b (UrV6b); 7 (UrV7); 8a (UrV8a); 8b (UrV8b); Umbelopsis gibberispora virus 1 (UgV1); 2 (UgV2) and Umbelopsis dimorpha virus 1a (UdV1a), 1b (UdV1b) and 2 (UdV2). All the newly identified viruses contain two open reading frames (ORFs), which putatively encode the coat protein (CP) and the RNA-dependent RNA polymerase (RdRp), respectively. Based on the phylogeny inferred from the RdRp sequences, eight viruses (UrV7, UrV8a, UrV8b, UgV1, UgV2, UdV1a, UdV1b and UdV2) belong to the genus Totivirus, while UrV5, UrV6a and UrV6b are placed into a yet unclassified but well-defined Totiviridae-related group. In UrV5, UgV1, UgV2, UrV8b, UdV1a, UdV2 and UdV1b, ORF2 is predicted to be translated as a fusion protein via a rare +1 (or −2) ribosomal frameshift, which is not characteristic to most members of the Totivirus genus. Virus particles 31 to 32 nm in diameter could be detected in the examined fungal strains by transmission electron microscopy. Through the identification and characterization of new viruses of Mucoromycota fungi, we can gain insight into the diversity of mycoviruses, as well as into their phylogeny and genome organization.

ABSTRACT The spleen tyrosine kinase (Syk) and the downstream adaptor protein CARD9 are crucial signaling molecules in antimicrobial immunity. Candida parapsilosis is an emerging fungal pathogen with a high incidence in neonates, while Candida albicans is the most common agent of candidiasis. While signaling through Syk/CARD9 promotes protective host mechanisms in response to C. albicans, its function in immunity against C. parapsilosis remains unclear. Here, we generated Syk−/− and CARD9−/− bone marrow chimeric mice to study the role of Syk/CARD9 signaling in immune responses to C. parapsilosis compared to C. albicans. We demonstrate various functions of this pathway (e.g., phagocytosis, phagosome acidification, and killing) in Candida-challenged, bone marrow-derived macrophages with differential involvement of Syk and CARD9 along with species-specific differences in cytokine production. We report that Syk−/− or CARD9−/− chimeras rapidly display high susceptibility to C. albicans, while C. parapsilosis infection exacerbates over a prolonged period in these animals. Thus, our results establish that Syk and CARD9 contribute to systemic resistance to C. parapsilosis and C. albicans differently. Additionally, we confirm prior studies but also detail new insights into the fundamental roles of both proteins in immunity against C. albicans. Our data further suggest that Syk has a more prominent influence on anti-Candida immunity than CARD9. Therefore, this study reinforces the Syk/CARD9 pathway as a potential target for anti-Candida immune therapy. IMPORTANCE While C. albicans remains the most clinically significant Candida species, C. parapsilosis is an emerging pathogen with increased affinity to neonates. Syk/CARD9 signaling is crucial in immunity to C. albicans, but its role in in vivo responses to other pathogenic Candida species is largely unexplored. We used mice with hematopoietic systems deficient in Syk or CARD9 to comparatively study the function of these proteins in anti-Candida immunity. We demonstrate that Syk/CARD9 signaling has a protective role against C. parapsilosis differently than against C. albicans. Thus, this study is the first to reveal that Syk can exert immune responses during systemic Candida infections species specifically. Additionally, Syk-dependent immunity to a nonalbicans Candida species in an in vivo murine model has not been reported previously. We highlight that the contribution of Syk and CARD9 to fungal infections are not identical and underline this pathway as a promising immune-therapeutic target to fight Candida infections.

A relatively new topic within the field of immunology involves the role of miRNAs in innate as well as adaptive immune response regulation. In recent years, posttranscriptional regulation of host-pathogenic fungal interactions through miRNAs was also suggested.

Hydrolysis of olive, rapeseed, linseed, almond, peanut, grape seed and menhaden oils was performed with commercial lipases of Aspergillus niger, Rhizopus oryzae, Rhizopus niveus, Rhizomucor miehei and Candida rugosa. In chromogenic plate tests, olive, rapeseed, peanut and linseed oils degraded well even after 2 h of incubation, and the R. miehei, A. niger and R. oryzae lipases exhibited the highest overall action against the oils. Gas chromatography analysis of vegetable oils hydrolyzed by R. miehei lipase revealed about 1.1 to 38.4-fold increases in the concentrations of palmitic, stearic, oleic, linoleic and α-linolenic acids after the treatment, depending on the fatty acids and the oil. The major polyunsaturated fatty acids produced by R. miehei lipase treatment from menhaden oil were linoleic, α-linolenic, hexadecanedioic, eicosapentaenoic, docosapentaenoic and docosahexaenoic acids, with yields from 12.02 to 52.85 µg/mL reaction mixture. Folin–Ciocalteu and ferric reducing power assays demonstrated improved antioxidant capacity for most tested oils after the lipase treatment in relation to the concentrations of some fatty acids. Some lipase-treated and untreated samples of oils, at 1.25 mg/mL lipid concentration, inhibited the growth of food-contaminating bacteria. The lipid mixtures obtained can be reliable sources of extractable fatty acids with health benefits.

Lipid mediators, derived from arachidonic acid metabolism, play an important role in immune regulation. The functions of bioactive eicosanoids range from modulating cytokine signaling and inflammasome formation to anti-inflammatory and pro-resolving activities. Human pathogenic fungi such as Candida albicans, Candida parapsilosis, Cryptococcus neoformans and Aspergillus fumigatus have been shown to produce such lipid mediators, associated with their virulence. To date, investigations into the molecular mechanisms of fungal eicosanoid biosynthesis in different species have revealed that several genes are associated with prostaglandin production. However, these routes remain uncharacterized in C. parapsilosis with early results suggesting it uses pathways distinct from those found in C. albicans. Therefore, we aimed to identify and characterize C. parapsilosis genes involved in eicosanoid biosynthesis. Following arachidonic acid treatment of C. parapsilosis cells, we identified several genes interfering with prostaglandin production. Out of the identified genes, homologues of a multi copper oxidase (FET3), an Acyl-CoA thiolase (POT1) and an Acyl-CoA oxidase (POX1-3) were found to play a significant role in prostaglandin synthesis. Furthermore, all three genes were confirmed to enhance C. parapsilosis pathogenicity, as the corresponding deletion mutants were cleared more efficiently by human macrophages and induced higher levels of pro-inflammatory cytokines. In addition, the mutants were less virulent than the wild-type strain in a mouse model of systemic infection. Taken together, we identified three genes that regulate eicosanoid biosynthesis in C. parapsilosis and impact the fungus’ virulence.

Mucormycosis is a life-threatening opportunistic infection caused by certain members of the fungal order Mucorales. This infection is associated with high mortality rate, which can reach nearly 100% depending on the underlying condition of the patient. Treatment of mucormycosis is challenging because these fungi are intrinsically resistant to most of the routinely used antifungal agents, such as most of the azoles. One possible mechanism of azole resistance is the drug efflux catalyzed by members of the ATP binding cassette (ABC) transporter superfamily. The pleiotropic drug resistance (PDR) transporter subfamily of ABC transporters is the most closely associated to drug resistance. The genome of Mucor circinelloides encodes eight putative PDR-type transporters. In this study, transcription of the eight pdr genes has been analyzed after azole treatment. Only the pdr1 showed increased transcript level in response to all tested azoles. Deletion of this gene caused increased susceptibility to posaconazole, ravuconazole and isavuconazole and altered growth ability of the mutant. In the pdr1 deletion mutant, transcript level of pdr2 and pdr6 significantly increased. Deletion of pdr2 and pdr6 was also done to create single and double knock out mutants for the three genes. After deletion of pdr2 and pdr6, growth ability of the mutant strains decreased, while deletion of pdr2 resulted in increased sensitivity against posaconazole, ravuconazole and isavuconazole. Our result suggests that the regulation of the eight pdr genes is interconnected and pdr1 and pdr2 participates in the resistance of the fungus to posaconazole, ravuconazole and isavuconazole.

Mucor lusitanicus and some other members of the fungal order Mucorales display the phenomenon of morphological dimorphism. This means that these fungi aerobically produce filamentous hyphae, developing a coenocytic mycelium, but they grow in a multipolar yeast-like form under anaerobiosis. Revealing the molecular mechanism of the reversible yeast-hyphal transition can be interesting for both the biotechnological application and in the understanding of the pathomechanism of mucormycosis. In the present study, transcriptomic analyses were carried out after cultivating the fungus either aerobically or anaerobically revealing significant changes in gene expression under the two conditions. In total, 539 differentially expressed genes (FDR < 0.05, |log2FC| ≥ 3) were identified, including 190 upregulated and 349 downregulated transcripts. Within the metabolism-related genes, carbohydrate metabolism was proven to be especially affected. Anaerobiosis also affected the transcription of transporters: among the 14 up- and 42 downregulated transporters, several putative sugar transporters were detected. Moreover, a considerable number of transcripts related to amino acid transport and metabolism, lipid transport and metabolism, and energy production and conversion were proven to be downregulated when the culture had been transferred into an anaerobic atmosphere.

ABSTRACT The number of invasive infections caused by Candida species is increasing worldwide. The incidence of candidiasis cases caused by non-albicans Candida species, such as Candida parapsilosis, is also increasing, and non-albicans Candida species are currently responsible for more invasive infections than C. albicans. Additionally, while the development of azole resistance during invasive disease with C. albicans remains uncommon, azole-resistant C. parapsilosis strains are frequently isolated in the hospital setting. In this study, we applied direct selection to generate azole-adapted and azole-evolved C. parapsilosis strains in order to examine the effect of azole resistance development on fungal viability and pathogenesis progression. Depending on the drug applied, the different evolved strains developed distinct cross-resistance patterns: the fluconazole-evolved (FLUEVO) and voriconazole-evolved (VOREVO) strains gained resistance to fluconazole and voriconazole only, while posaconazole evolution resulted in cross-resistance to all azoles and the posaconazole-evolved (POSEVO) strains showed higher echinocandin MIC values than the FLUEVO and VOREVO strains. Whole-genome sequencing results identified the development of different resistance mechanisms in the evolved strains: the FLUEVO and VOREVO strains harbored amino acid substitutions in Mrr1p (A808T and N394Y, respectively), and the POSEVO strain harbored an amino acid change in Erg3p (D14Y). By revealing increased efflux pump activity in both the FLUEVO and the VOREVO strains, along with the altered sterol composition of the POSEVO strain, we now highlight the impact of the above-mentioned amino acid changes in C. parapsilosis azole resistance development. We further revealed that the virulence of this species was only slightly or partially affected by fluconazole and voriconazole adaptation, while it significantly decreased after posaconazole adaptation. Our results suggest that triazole adaptation can result in azole cross-resistance and that this process may also result in virulence alterations in C. parapsilosis, depending on the applied drug. IMPORTANCE Candida parapsilosis causes life-threatening fungal infections. In the last 2 decades, the increasing number of azole-resistant C. parapsilosis clinical isolates has been attributable to the overuse and misuse of fluconazole, the first-line antifungal agent most commonly used in several countries. To date, the range of applicable antifungal drugs is limited. As a consequence, it is essential to understand the possible mechanisms of antifungal resistance development and their effect on virulence in order to optimize antifungal treatment strategies in the clinical setting. Our results revealed that the prolonged exposure to azoles resulted not only in azole resistance but also in cross-resistance development. Our data further indicate that resistance development may occur through different mechanisms that can also alter the virulence of C. parapsilosis. These results highlight the consequences of prolonged drug usage and suggest the need for developing alternative antifungal treatment strategies in clinical practice.

ABSTRACT Among all the essential micronutrients, iron plays an important role in mammalian biology. It is also essential for pathogens infecting mammalian hosts, including bacteria, fungi, and protozoans. As the availability of accessible iron is limited within the mammalian host, several human-pathogenic fungal pathogens, such as Candida albicans, Cryptococcus neoformans, Candida glabrata, and Aspergillus fumigatus, have developed various iron uptake mechanisms. Although Candida parapsilosis is the second or third most common non-albicans Candida species associated with systemic and superficial Candida infections in immunocompromised patients, the mechanisms of iron uptake and homoeostasis remain unknown in this fungus. In the current report, we show that a homologue of the multicopper oxidase gene FET3 is present in the genome of C. parapsilosis (CPAR2_603600) and plays a significant role in iron acquisition. We found that homozygous deletion mutants of CPAR2_603600 showed defects under low-iron conditions and were also sensitive to various stressors. Our results also revealed that the levels of pseudohypha formation and biofilm formation were reduced in the null mutants compared to the wild type. This phenotypic defect could be partially rescued by supplementation with excess iron in the growth medium. The expression levels of the orthologues of various iron metabolism-related genes were also altered in the mutants compared to the parental strain. In conclusion, our report describes the role of CPAR2_603600 in iron homoeostasis maintenance as well as morphology and biofilm formation regulation in this pathogenic fungus. IMPORTANCE C. parapsilosis is the second or third most common opportunistic human-pathogenic Candida species, being responsible for severe fungal infections among immunocompromised patients, especially low-birth-weight infants (0 to 2 years of age). Among the major virulence factors that pathogenic fungi possess is the ability to compete with the host for essential micronutrients, including iron. Accessible iron is required for the maintenance of several metabolic processes. In order to obtain accessible iron from the host, pathogenic fungi have developed several iron acquisition and metabolic mechanisms. Although C. parapsilosis is a frequent cause of invasive candidiasis, little is known about what iron metabolic processes this fungus possesses that could contribute to the species’ virulent behavior. In this study, we identified the multicopper oxidase FET3 gene that regulates iron homeostasis maintenance and also plays important roles in the morphology of the fungus as well as in biofilm formation, two additional factors in fungal virulence.

White button mushroom—Agaricus bisporus (J.E.Lange) Imbach—is among the most popular cultivated mushrooms worldwide. The most serious challenge in industrial mushroom production is the green mold disease caused by Trichoderma species. Our aim was to isolate and examine bacterial strains from mushroom casing material for their potential use as biocontrol agents. Twenty-seven bacterial strains were isolated and tested against mold pathogens of white button mushroom. The Bacillus velezensis strain SZMC 25431 was selected for further examination and tested under simulated Agaricus cultivation conditions against T. aggressivum SZMC 23834 in a 1200-L Fitotron SGC120 standard plant growth chamber. Our results showed that the bacterial treatment was effective against the pathogen in all cases, but the best results were achieved at an application concentration of 105 cells mL−1. Industrial-scale experiments were also carried out in Agaricus growing houses with a bearing surface of 480 m2: the bacterial suspension was mixed in water tanks applied for daily irrigation. The results suggest that the bacterial treatment may even increase the crop yield of A. bisporus. Based on our results, we concluded that the selected B. velezensis strain may potentially be used for biological and integrated treatment in Agaricus cultivation.

(1) Background: Bacillus velezensis and Bacillus amyloliquefaciens are closely related members of the “operational group B. amyloliquefaciens”, a taxonomical unit above species level within the ”Bacillus subtilis species complex”. They have similar morphological, physiological, biochemical, phenotypic, and phylogenetic characteristics. Thus, separating these two taxa from each another has proven to be difficult to implement and could not be pushed easily into the line of routine analyses. (2) Methods: The aim of this study was to determine whether whole FAME profiling could be used to distinguish between these two species, using both type strains and environmental isolates. Initially, the classification was determined by partial sequences of the gyrA and rpoB genes and the classified isolates and type strains were considered as samples to develop the identification method, based on FAME profiles. (3) Results: The dissimilarities in 16:0, 17:0 iso, and 17:0 FA components have drawn a distinction between the two species and minor differences in FA 14:0, 15:0 iso, and 16:0 iso were also visible. The statistical analysis of the FA profiles confirmed that the two taxa can be distinguished into two separate groups, where the isolates are identified without misreading. (4) Conclusions: Our study proposes that the developed easy and fast-automated identification tool based on cellular FA profiles can be routinely applied to distinguish B. velezensis and B. amyloliquefaciens.

Abstract Invasive candidiasis is among the most life-threatening infections in patients in intensive care units. Although Candida albicans is the leading cause of candidaemia, the incidence of Candida parapsilosis infections is also rising, particularly among the neonates. Due to differences in their biology, these species employ different antifungal resistance and virulence mechanisms and also induce dissimilar immune responses. Previously, it has been suggested that core virulence effecting transcription regulators could be attractive ligands for future antifungal drugs. Although the virulence regulatory mechanisms of C. albicans are well studied, less is known about similar mechanisms in C. parapsilosis. In order to search for potential targets for future antifungal drugs against this species, we analyzed the fungal transcriptome during host-pathogen interaction using an in vitro infection model. Selected genes with high expression levels were further examined through their respective null mutant strains, under conditions that mimic the host environment or influence pathogenicity. As a result, we identified several mutants with relevant pathogenicity affecting phenotypes. During the study we highlight three potentially tractable signaling regulators that influence C. parapsilosis pathogenicity in distinct mechanisms. During infection, CPAR2_100540 is responsible for nutrient acquisition, CPAR2_200390 for cell wall assembly and morphology switching and CPAR2_303700 for fungal viability.

We previously screened the total nucleic acid extracts of 123 Mucor strains for the presence of dsRNA molecules without further molecular analyses. Here, we characterized five novel dsRNA genomes isolated from four different Mucor hiemalis strains with next-generation sequencing (NGS), namely Mucor hiemalis virus 1a (MhV1a) from WRL CN(M) 122; Mucor hiemalis virus 1b (MhV1b) from NRRL 3624; Mucor hiemalis virus 2 (MhV2) from NRRL 3616; and Mucor hiemalis virus 3 (MhV3) and Mucor hiemalis virus (MhV4) from NRRL 3617 strains. Genomes contain two open reading frames (ORF), which encode the coat protein (CP) and the RNA dependent RNA polymerase (RdRp), respectively. In MhV1a and MhV1b, it is predicted to be translated as a fusion protein via -1 ribosomal frameshift, while in MhV4 via a rare +1 (or−2) ribosomal frameshift. In MhV2 and MhV3, the presence of specific UAAUG pentanucleotide motif points to the fact for coupled translation termination and reinitialization. MhV1a, MhV2, and MhV3 are part of the clade representing the genus Victorivirus, while MhV4 is seated in Totivirus genus clade. The detected VLPs in Mucor strains were from 33 to 36 nm in diameter. Hybridization analysis revealed that the dsRNA molecules of MhV1a-MhV4 hybridized to the corresponding molecules.

Previously, severe green mould infections could be attributed mainly to Trichoderma aggressivum Samuels & W. Gams, as well as T. pleuroti S.H. Yu & M.S. Park and T. pleuroticola S.H. Yu & M.S. Park in the case of Agaricus bisporus (J.E. Lange) Imbach (button mushroom) and Pleurotus ostreatus (Jacq.) P. Kumm. (oyster mushroom), respectively. The purpose of our study was the examination of green mould agents deriving from the growing facilities of button mushroom, oyster mushroom and shiitake (Lentinula edodes (Berk.) Pegler) located in various countries of Europe, and initially classified into the Trichoderma harzianum Rifai species complex (THSC). Species identification was carried out using the multilocus sequence typing analysis of the internal transcribed spacer regions, as well as translation elongation factor 1-alpha, calmodulin and RNA polymerase B subunit II gene sequences. In vitro confrontation assays were applied to test the aggressiveness of the isolates towards mushrooms, while the effect of commercial fungicides on the growth of the strains was examined by the macrodilution method. Six Trichoderma species, namely T. afroharzianum P. Chaverri, F.B. Rocha, Degenkolb & Druzhin., T. atrobrunneum F.B. Rocha, P. Chaverri & Jaklitsch, T. guizhouense Q.R. Li, McKenzie & Yong Wang, T. harzianum sensu stricto, T. pollinicola F. Liu & L. Cai and T. simmonsii P. Chaverri, F.B. Rocha, Samuels, Degenkolb & Jaklitsch were detected in the different samples, with T. harzianum, T. pollinicola and T. simmonsii being the most aggressive. Prochloraz was found to have strong in vitro inhibitory effect on mycelial growth on most strains, however, T. simmonsii isolates showed remarkable tolerance to it. Our data suggest that T. harzianum and T. simmonsii may also be considered as potential causal agents of mushroom green mould.

The genus Chaetomium is a frequently occurring fungal taxon world-wide. Chaetomium and Chaetomium-like species occur in indoor environments, where they can degrade cellulose-based building materials, thereby causing structural damage. Furthermore, several species of this genus may also cause adverse effects on human health. The aims of this research were to identify Chaetomium and Chaetomium-like strains isolated from indoor environments in Hungary and Finland, two geographically distant regions of Europe with drier and wetter continental climates, respectively, and to study their morphological and physiological properties, as well as their extracellular enzyme activities, thereby comparing the Chaetomium and Chaetomium-like species isolated from these two different regions of Europe and their properties. Chaetomium and Chaetomium-like strains were isolated from flats and offices in Hungary, as well as from schools, flats, and offices in Finland. Fragments of the translation elongation factor 1α (tef1α), the second largest subunit of RNA polymerase II (rpb2) and β-tubulin (tub2) genes, as well as the internal transcribed spacer (ITS) region of the ribosomal RNA gene cluster were sequenced, and phylogenetic analysis of the sequences performed. Morphological examinations were performed by stereomicroscopy and scanning electron microscopy. Thirty-one Chaetomium sp. strains (15 from Hungary and 16 from Finland) were examined during the study. The most abundant species was Ch. globosum in both countries. In Hungary, 13 strains were identified as Ch. globosum, 1 as Ch. cochliodes, and 1 as Ch. interruptum. In Finland, 10 strains were Ch. globosum, 2 strains were Ch. cochliodes, 2 were Ch. rectangulare, and 2 isolates (SZMC 26527, SZMC 26529) proved to be representatives of a yet undescribed phylogenetic species from the closely related genus Dichotomopilus, which we formally describe here as the new species Dichotomopilus finlandicus. Growth of the isolates was examined at different temperatures (4, 15, 20, 25, 30, 37, 35, 40, and 45 °C), while their extracellular enzyme production was determined spectrophotometrically.

Phenolic compounds are natural substances that can be obtained from plants. Many of them are potent growth inhibitors of foodborne pathogenic microorganisms, however, phenolic activities against spoilage yeasts are rarely studied. In this study, planktonic and biofilm growth, and the adhesion capacity of Pichia anomala, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Debaryomyces hansenii spoilage yeasts were investigated in the presence of hydroxybenzoic acid, hydroxycinnamic acid, stilbene, flavonoid and phenolic aldehyde compounds. The results showed significant anti-yeast properties for many phenolics. Among the tested molecules, cinnamic acid and vanillin exhibited the highest antimicrobial activity with minimum inhibitory concentration (MIC) values from 500 µg/mL to 2 mg/mL. Quercetin, (−)-epicatechin, resveratrol, 4-hydroxybenzaldehyde, p-coumaric acid and ferulic acid were also efficient growth inhibitors for certain yeasts with a MIC of 2 mg/mL. The D. hansenii, P. anomala and S. pombe biofilms were the most sensitive to the phenolics, while the S. cerevisiae biofilm was quite resistant against the activity of the compounds. Fluorescence microscopy revealed disrupted biofilm matrix on glass surfaces in the presence of certain phenolics. Highest antiadhesion activity was registered for cinnamic acid with inhibition effects between 48% and 91%. The active phenolics can be natural interventions against food-contaminating yeasts in future preservative developments.

Abstract Background Scedosporium apiospermum is an emerging opportunistic filamentous fungus, which is notorious for its high levels of antifungal-resistance. It is able to cause localized cutaneous or subcutaneous infections in both immunocompromised and immunocompetent persons, pulmonary infections in patients with predisposing pulmonary diseases and invasive mycoses in immunocompromised patients. Subcutaneous infections caused by this fungus frequently show chronic mycetomatous manifestation. Case report We report the case of a 70-year-old immunocompromised man, who developed a fungal mycetomatous infection on his right leg. There was no history of trauma; the aetiological agent was identified by microscopic examination and ITS sequencing. This is the second reported case of S. apiospermum subcutaneous infections in Hungary, which was successfully treated by surgical excision and terbinafine treatment. After 7 months, the patient remained asymptomatic. Considering the antifungal susceptibility and increasing incidence of the fungus, Scedosporium related subcutaneous infections reported in the past quarter of century in European countries were also reviewed. Conclusions Corticosteroid treatment represents a serious risk factor of S. apiospermum infections, especially if the patient get in touch with manure-enriched or polluted soil or water. Such infections have emerged several times in European countries in the past decades. The presented data suggest that besides the commonly applied voriconazole, terbinafine may be an alternative for the therapy of mycetomatous Scedosporium infections.

Aspergillus tamarii appears to be an emerging aetiological agent of human keratomycoses in South India. The investigated strains were isolated from six suspected fungal keratitis patients attending a tertiary care eye hospital in Coimbatore (Tamil Nadu, India), and were initially identified by the microscopic examinations of the scrapings and the cultures. Our data suggest that A. tamarii could be easily overlooked when identification is carried out based on morphological characteristics alone, while the sequence analysis of the calmodulin gene can be used successfully to recognize this species accurately. According to the collected clinical data, ocular trauma is a common risk factor for the infection that gradually developed from mild to severe ulcers and could be healed with an appropriate combined antifungal therapy. Antifungal susceptibility testing revealed that A. tamarii strains are susceptible to the most commonly used topical or systemic antifungal agents (i.e., econazole, itraconazole and ketoconazole) except for natamycin. Moreover, natamycin proved to be similarly less effective than the azoles against A. tamarii in our drug interaction tests, as the predominance of indifferent interactions was revealed between natamycin and econazole and between natamycin and itraconazole as well. Four and five isolates of A. tamarii were confirmed to produce cyclopiazonic acid (CPA) in RPMI-1640 – which is designed to mimic the composition of human extracellular fluids – and in yeast extract sucrose (YES) medium, respectively, which is a widely used culture medium for testing mycotoxin production. Although a ten times lower mycelial biomass was recorded in RPMI-1640 than in YES medium, the toxin contents of the samples were of the same order of magnitude in both types of media. There might be a relationship between the outcome of infections and the toxigenic properties of the infecting fungal strains. However, this remains to be investigated in the future.