Mycelial Cells Research Articles

Baseline Sensitivity and Toxicity Mechanisms of Prochloraz to Alternaria alternata Strains Associated with Maize Leaf Blight in Heilongjiang Province in China.

Alternaria species are fungal pathogens that can infect maize, causing leaf blight disease and significant economic losses. This study aimed to determine the baseline sensitivity to prochloraz of A. alternata isolates obtained from diseased maize leaves collected from Heilongjiang Province by assessing the half-maximal effective concentration (EC50) values. The EC50 values of prochloraz ranged from 0.0550 to 2.3258 μg/ml, with an average of 0.9995 ± 0.5192 μg/ml. At EC50 (1.2495 μg/ml) and 2EC50 (2.4990 μg/ml), prochloraz increased the number of mycelial offshoots, disrupted the cell membrane integrity of conidia and mycelia, and resulted in a reduced ergosterol content in the mycelia. Prochloraz significantly affected the mycelial cell membrane permeability and increased the malondialdehyde content and superoxide dismutase activity. No cross-resistance was detected between prochloraz and other fungicides. These data demonstrate that prochloraz is a promising fungicide for managing maize leaf blight caused by A. alternata and provide novel insights into understanding the mechanism of prochloraz toxicity against A. alternata isolates.

Effect of low-frequency alternating magnetic field on exopolysaccharide production and antioxidant capacity of Pleurotus citrinopileatus by submerged fermentation.

The objective of this study was to investigate the effect of low-frequency alternating magnetic field (LF-AMF) on the production of extracellular polysaccharide (EPS) by submerged fermentation of Pleurotus citrinopileatus. The fermentation conditions optimized by the central composite design method were as follows: fermentation time of 6.18days, temperature of 28.28°C, shaking speed of 149.04 r/min, and inoculum amount of 8.43%. Under these conditions, a LF-AMF was applied to the submerged fermentation of P. citrinopileatus. When the intensity of LF-AMF was 40 Gs, the initial intervention time was 24h after inoculation, and the treatment time was 6h at one time, the mycelial biomass of P. citrinopileatus increased by 11.30%, and the EPS yield increased by 23.09% compared with the fermentation without LF-AMF treatment. The morphology of mycelium after LF-AMF treatment was observed by scanning electron microscopy. It was found that the surface of mycelium was wrinkled, and the structure of mycelium was loose, which might be more conducive to the production of EPS. Mycelium diameter decreased, and ATPase activity increased, indicating that LF-AMF had a positive effect on the production of EPS by P. citrinopileatus fermentation. Moreover, LF-AMF could improve the permeability of the mycelial cell membrane, facilitate the exchange of intracellular and extracellular substances, and increase the metabolic capacity of P. citrinopileatus. In vitro antioxidant test of EPS showed that LF-AMF treatment also improved its antioxidant capacity.

Design, Synthesis, and Biological Activity Evaluation of Eco-Friendly Rosin-Diamide-Based Fungicides as Potential Succinate Dehydrogenase Inhibitors for Sustainable Crop Protection.

To develop novel succinate dehydrogenase (SDH) inhibitors for sustainable crop protection, a series of dehydroabietyl-diamide-based fungicides (a total of 21) were designed. In vitro fungicidal activity measurement showed that compound 3u exhibited excellent fungicidal activity against Valsa mali (half-maximal effective concentration, EC50 = 0.195 μg/mL), surpassing that of the positive control carbendazim (EC50 = 1.35 μg/mL). The in vivo fungicidal activity assessment suggested that 3u exhibited a protective effect on apple branches (69.7-48.1%) and apples (94.6-56.6%). Furthermore, biosafety evaluation indicated that 3u was significantly environmentally friendly toward zebrafish. Subsequently, morphology, physiology, and molecular docking were investigated to elucidate the mode of action of 3u against V. mali. Results demonstrated a strong binding between 3u and SDH, resulting in decreased SDH activity (half-maximal inhibitory concentration, IC50 = 11.7 μg/mL). Moreover, 3u disrupted the mycelial cell membrane and accelerated electrolyte leakage, ultimately resulting in the death of V. mali. These findings suggest that 3u could serve as a potent SDH inhibitor for sustainable crop protection.

Nerol as a Novel Antifungal Agent: In Vitro Inhibitory Effects on Fusarium oxysporum, Pestalotiopsis neglecta, and Valsa mali and Its Potential Mechanisms against F. oxysporum.

This study explores the in vitro antifungal effects of nerol, a linear acyclic monoterpene alcohol of plant origin, on Fusarium oxysporum, Pestalotiopsis neglecta, and Valsa mali. To further investigate the antifungal mechanism of nerol against F. oxysporum, we examined changes in mycelial morphology and cell membrane integrity-related indices, as well as the activities of antioxidant and pathogenicity-related enzymes. The results demonstrated that nerol exhibited significant concentration-dependent inhibition of mycelial growth in all three fungi, with EC50 values of 0.46 μL/mL for F. oxysporum, 1.81 μL/mL for P. neglecta, and 1.26 μL/mL for V. mali, with the strongest antifungal activity observed against F. oxysporum. Scanning electron microscopy revealed that nerol severely disrupted the mycelial structure of F. oxysporum, causing deformation, swelling, and even rupture. Treatment with 0.04 μL/mL nerol led to significant leakage of soluble proteins and intracellular ions in F. oxysporum, and the Na+/K+-ATPase activity was reduced to 28.02% of the control, indicating enhanced membrane permeability. The elevated levels of hydrogen peroxide and malondialdehyde, along with propidium iodide staining of treated microconidia, further confirmed cell membrane disruption caused by nerol. Additionally, after 12 h of exposure to 0.04 μL/mL nerol, the activity of superoxide dismutase in F. oxysporum decreased to 55.81% of the control, and the activities of catalase and peroxidase were also significantly inhibited. Nerol markedly reduced the activities of pathogenicity-related enzymes, such as endo-1,4-β-D-glucanase, polygalacturonase, and pectin lyase, affecting fungal growth and virulence. In conclusion, nerol disrupts the cell membrane integrity and permeability of F. oxysporum, reduces its virulence, and ultimately inhibits fungal growth, highlighting its potential as an alternative to chemical fungicides for controlling F. oxysporum.

Discovery of Novel Cyclobutyl Oxime Ester Derivatives Containing an α,β-Unsaturated Carbonyl Moiety as Potential Mitochondrial Toxins.

As part of continuous work to explore novel and efficient fungicides originating from natural products, a series of cyclobutyl oxime ester derivatives containing an α,β-unsaturated carbonyl moiety were designed and synthesized. In line with the primary evaluation of the inhibitory effect on common pathogenic fungi causing crop failure, a systematic study on the antifungal activity of target compounds against Rhizoctonia solani was carried out. Most target compounds exhibited satisfactory antifungal activity, and 10 of them were superior to the positive control trifloxystrobin. The most notable median effective concentration (EC50) of compound 6b was 1.70 μg/mL, which was considerable for an intensive study. The control efficacy of compound 6b on potted rice against R. solani was superior to trifloxystrobin at identical concentration. The mycelial morphology and cell membrane permeability of the treated fungi were disrupted, and the meaningful enzyme activities of SDH and POD were also restrained. The reactive oxygen species, nuclear morphology, and mitochondrial membrane potential of the treated hypha reflected an apparent difference compared with the normal morphology, which represented mitochondrial function damage. In addition, chemical features essential for the activity and docking mode within the compound and cytochrome bc1 complex were accessed by computer-aided technology. This study provided insights into the development of new green and efficient fungicides targeting the mitochondria.

Isolation, antioxidant and antifungal activity of furanocoumarins from the leaves of Heracleum vicinum Boiss.

In this work, coumarin compounds were extracted by ultrasonic-microwave synergistic extraction from the leaves of Heracleum vicinum Boiss.. The five furanocoumarins—pimpinellin, isopimpinellin, bergapten, xanthotoxin, and imperatorin—that were isolated from the leaves of Heracleum vicinum Boiss. were quantified using HPLC and GC, and their quantities in the aboveground sections was determined. The antioxidant activities of five furanocoumarins were assessed with ABTS radicals and DPPH radicals, with the best scavenging effects of xanthotoxin and bergapten, with half maximal inhibitory concentration (IC50) of xanthotoxin for ABTS radicals and DPPH radicals of 1.85 mg/mL and 3.95 mg/mL, respectively, and bergapten for ABTS radicals and DPPH radicals IC50 of 1.68 mg/mL and 4.61 mg/mL, respectively. The antifungal activities of five furanocoumarins against trichophyton rubrum were evaluated. The minimum inhibitory concentration (MIC) of xanthotoxin (200 μg/mL) and imperatorin (12.5 μg/mL) showed the highest inhibitory effects against trichophyton rubrum. The inhibitory mechanism of xanthotoxin was discussed. When trichophyton rubrum was treated with 200 μg/mL of xanthotoxin, the spore germination rate was 34.92%, the nucleic acid leakage was 52.22%, the inhibition of radial growth of mycelium could reach 87.87%, and the content of ergosterol was reduced by 37.37%. It indicates that xanthotoxin was found to be able to damage mycelium shape, prevent spore germination and mycelium growth, kill mycelium cells and release intracellular materials, and obstruct the production of ergosterol. These results indicate that furanocoumarin is a potent active component that has an important role in Heracleum vicinum Boiss. leaves.

Biochemical mechanism of chlorine dioxide fumigation in inhibiting Ceratocystis fimbriata and black rot in postharvest sweetpotato

The inhibitory properties and underlying mechanism of chlorine dioxide (ClO2) fumigation on the pathogen Ceratocystis fimbriata (C. fimbriata) and resultant sweetpotato black rot were investigated in vitro and in vivo. Results revealed that the ClO2 fumigation effectively inhibited fungal growth and induced obvious morphological variation of C. fimbriata mycelia. Furthermore, the mycelial membrane suffered damage, as evidenced by a significant increase in malondialdehyde content and the leakage of protein and nucleic acid from mycelia cells, accompanied by a marked decrease in ergosterol content. Additionally, ClO2 fumigation caused spores cell membrane damage, a notable decrease in spore viability, and induced cell apoptosis as indicated by reductions in spore germination rate, two fluorescence staining observations, and flow cytometry analysis. Moreover, the decay diameter of sweetpotato black rot lesions decreased significantly after ClO2 fumigation, and the growth of C. fimbriata was also inhibited. These findings present a novel and effective technology for inhibiting the progression of sweetpotato black rot.

Fusiform nanoparticle boosts efficient genetic transformation in Sclerotinia sclerotiorum

BackgroundSclerotinia sclerotiorum is a highly destructive phytopathogenic fungus that poses a significant threat to a wide array of crops. The current constraints in genetic manipulation techniques impede a thorough comprehension of its pathogenic mechanisms and the development of effective control strategies.ResultsHerein, we present a highly efficient genetic transformation system for S. sclerotiorum, leveraging the use of fusiform nanoparticles, which are synthesized with FeCl3 and 2,6-diaminopyrimidine (DAP). These nanoparticles, with an average longitude length of 59.00 nm and a positively charged surface, facilitate the direct delivery of exogenous DNA into the mycelial cells of S. sclerotiorum, as well as successful integration with stable expression. Notably, this system circumvents fungal protoplast preparation and tedious recovery processes, streamlining the transformation process considerably. Furthermore, we successfully employed this system to generate S. sclerotiorum strains with silenced oxaloacetate acetylhydrolase-encoding gene Ss-oah1.ConclusionsOur findings demonstrate the feasibility of using nanoparticle-mediated delivery as a rapid and reliable tool for genetic modification in S. sclerotiorum. Given its simplicity and high efficiency, it has the potential to significantly propel genetic research in filamentous fungi, offering new avenues for elucidating the intricacies of pathogenicity and developing innovative disease management strategies.Graphical

Plant-derived citronellol can significantly disrupt cell wall integrity maintenance of Colletotrichum camelliae

Anthracnose, a fungal disease, commonly infects tea plants and severely impacts the yield and quality of tea. One method for controlling anthracnose is the application of citronellol, a plant extract that exhibits broad-spectrum antimicrobial activity. Herein, the physiological and biochemical mechanism by which citronellol controls anthracnose caused by Colletotrichum camelliae was investigated. Citronellol exhibited excellent antifungal activity based on direct and indirect mycelial growth inhibition assays, with EC50 values of 76.88 mg/L and 29.79 μL/L air, respectively. Citronellol also exhibited good control effects on C. camelliae in semi-isolated leaf experiments. Optical and scanning electron microscopy revealed that citronellol caused C. camelliae mycelia to thin, fracture, fold and deform. Transmission electron microscopy revealed that the mycelial cell walls collapsed inward and separated, and the organelles became blurred after treatment with citronellol. The sensitivity of C. camelliae to calcofluor white staining was significantly enhanced by citronellol, while PI staining showed minimal fluorescence, and the relative conductivity of mycelia were not significantly different. Under citronellol treatment, the expression levels of β-1,3-glucanase, chitin synthase, and chitin deacetylase-related genes were significantly decreased, while the expression levels of chitinase genes were increased, leading to lower chitinase activity and increased β-1,3-glucanase activity. Therefore, citronellol disrupted the cell wall integrity of C. camelliae and inhibited normal mycelial growth.

Discovery of natural rosin-based preservative candidates to control Phytophthora capsici for postharvest disease management of solanaceae vegetables

To mitigate the postharvest harm of pathogens on solanaceous vegetables, a series of natural rosin-1,2,4-triazole derivatives (totally 24) have been designed and developed. In vivo fungicidal activity evaluation showed that Co. 5 f could inhibit the growth of Phytophthora capsici (EC50 = 0.22 mg/L). In vivo activity suggested that Co. 5 f presented certain protective effect on bell pepper (64.93 % – 32.68 %) and tomato (91.93 % – 67.85 %) fruit. Additionally, Co. 5 f prevented declines in both weight and ascorbic acid levels and reduced the malondialdehyde content of bell peppers and tomato, suggesting its efficacy in vegetable preservation during storage. The physiological research revealed that Co. 5 f disrupted the mycelial cell membrane, accelerated the accumulation of reactive oxygen species, and decreased the enzyme activity of peroxidase (POD) and catalase (CAT) protein activities. Subsequently, quantum chemical calculations and molecular docking successfully predicted the active site of Co. 5 f. Metabolomics was then employed to elucidate the mechanism of physiological disorders, suggesting that differential metabolites were primarily enriched in the biosynthesis of amino acid and secondary metabolites. Transcriptomics was also utilized to further uncover the mechanism of mycelial metabolic disorders, revealing that differential genes were primarily enriched in the biosynthesis of secondary metabolites and amino acid. Additionally, the integration of physiology, metabolomics, transcriptomics, and computer-aided drug design unveiled the action mechanism of Co. 5 f against P. capsici, indicating that the regulation of POD and CAT biosynthesis involved six core genes, while the regulation of ARG, HIS, and TRP biosynthesis involved four core genes. Finally, the expression of these ten core genes was validated using quantitative real-time polymerase chain reaction (qRT-PCR). In summary, the above research integrated physiology, metabolomics, transcriptomics and computer-aided drug design to reveal the action mechanism of Co. 5 f, providing a certain theoretical support for the postharvest disease management of bell pepper and tomato fruit.

Rhamnolipids and fengycins interact differently with biomimetic lipid membrane models of Botrytis cinerea and Sclerotinia sclerotiorum: Lipidomics profiles and biophysical studies

Rhamnolipids (RLs) and Fengycins (FGs) are biosurfactants with very promising antifungal properties proposed to reduce the use of synthetic pesticides in crops. They are amphiphilic molecules, both known to target the plasma membrane. They act differently on Botrytis cinerea and Sclerotinia sclerotiorum, two close Sclerotiniaceae phytopathogenic fungi. RLs are more efficient at permeabilizing S. sclerotiorum, and FGs are more efficient at permeabilizing B. cinerea mycelial cells. To study the link between the lipid membrane composition and the activity of RLs and FGs, we analyzed the lipid profiles of B. cinerea and S. sclerotiorum. We determined that unsaturated or saturated C18 and saturated C16 fatty acids are predominant in both fungi. We also showed that phosphatidylethanolamine (PE), phosphatidic acid (PA), and phosphatidylcholine (PC) are the main phospholipids (in this order) in both fungi, with more PA and less PC in S. sclerotiorum. The results were used to build biomimetic lipid membrane models of B. cinerea and S. sclerotiorum for all-atom molecular dynamic simulations and solid-state NMR experiments to more deeply study the interactions between RLs or FGs with different compositions of lipid bilayers. Distinctive effects are exerted by both compounds. RLs completely insert in all the studied model membranes with a fluidification effect. FGs tend to form aggregates out of the bilayer and insert individually more easily into the models representative of B. cinerea than those of S. sclerotiorum, with a higher fluidification effect. These results provide new insights into the lipid composition of closely related fungi and its impact on the mode of action of very promising membranotropic antifungal molecules for agricultural applications.

A nanoscale γ-cyclodextrin metal-organic framework loaded with eugenol (CMFE) for controlling F. graminearum on the wheat during harvest time

The loss of grains in the production chain caused huge waste worldwide, and the control of plant pathogenic fungi during harvest time is a critical solution to mitigate the damage. Eugenol, an important component of clove oil (Syzygium aromaticum L.), has antioxidant, antifungal, and other activities. In this study, the unstable eugenol was incorporated into the nanoscale γ-cyclodextrin metal-organic framework (γ-CD-MOF), a cubic porous structure synthesized by vapor diffusion method. The investigation of morphologies, functional groups, crystalline structures and weight losing behaviors demonstrated the inclusion complex prepared from γ-CD-MOF and eugenol (CMFE). Compared with the γ-CD loaded with eugenol (CE), CMFE exhibited an improved loading rate with 4.06 % (w/w). And CMFE enhanced the stability of its inner eugenol against degradation by light and air. Moreover, the CMFE demonstrated a significant anti-F. graminearum activity both in the medium and on the wheat grains. The inhibition rate reached 83.8 % at 8 mg/mL in vitro, while the growth of the hyphae was effectively restrained at 4 mg/g in vivo. CMFE could damage the mycelial cell wall and trigger intracellular reactive oxygen species (ROS) accumulation, thus exhibiting its anti-F. graminearum activity. Our results showed that the biocompatible CMFE would be an environment-friendly substitute to chemical fungicides for the prevention of fungal contamination caused by F. graminearum.

The photoactivated antifungal activity and possible mode of action of sodium pheophorbide a on Diaporthe mahothocarpus causing leaf spot blight in Camellia oleifera

IntroductionSodium pheophorbide a (SPA) is a natural plant-derived photosensitizer, with high photoactivated antifungal activity against some phytopathogenic fungi. However, its fungicidal effect on Diaporthe mahothocarpus, a novel pathogen that causes Camellia oleifera leaf spot blight, is unclear.MethodsIn the present study, we explored its inhibitory effects on spore germination and mycelial growth of D. mahothocarpus. Then we determined its effects on the cell membrane, mycelial morphology, redox homeostasis, and cell death through bioassay. Finally, RNA-seq was used further to elucidate its mode of action at the transcriptional level.ResultsWe found that SPA effectively inhibited the growth of D. mahothocarpus, with half-maximal effective concentrations to inhibit mycelial growth and spore germination of 1.059 and 2.287 mg/mL, respectively. After 1.0 mg/mL SPA treatment, the conductivity and malondialdehyde content of D. mahothocarpus were significantly increased. Scanning electron microscopy and transmission electron microscopy indicated that SPA significantly affected the morphology and ultrastructure of D. mahothocarpus hyphae, revealing that SPA can destroy the mycelial morphology and cell structure, especially the cell membrane of D. mahothocarpus. Furthermore, transcriptome analysis revealed that SPA significantly suppressed the expression of genes involved in morphology, cell membrane permeability, and oxidative stress. Then, we also found that SPA significantly promoted the accumulation of reactive oxygen species (ROS) in of D. mahothocarpus, while it decreased the content of reduced glutathione, inhibited the enzyme activities of superoxide dismutase and catalase, and exacerbated DNA damage. Annexin V-FITC/PI staining also confirmed that 1.0 mg/mL SPA could significantly induce apoptosis and necrosis.DiscussionGenerally, SPA can induce ROS-mediated oxidative stress and cell death, thus destroying the cell membrane and hyphal morphology, and ultimately inhibiting mycelial growth, which indicates that SPA has multiple modes of action, providing a scientific basis for the use of SPA as an alternative plant-derived photoactivated fungicide against C. oleifera leaf spot blight.

Antifungal Activity of Novel Indole Derivatives Containing 1,3,4-Thiadiazole.

In this study, 24 indole derivatives containing 1,3,4-thiadiazole were discovered and synthesized. The target compounds' antifungal efficacy against 14 plant pathogenic fungal pathogens was then determined in vitro. With an EC50 value of 2.7 μg/mL, Z2 demonstrated the highest level of bioactivity among them against Botrytis cinerea (B.c.), exceeding the concentrations of the control prescription drugs azoxystrobin (Az) (EC50 = 14.5 μg/mL) and fluopyram (Fl) (EC50 = 10.1 μg/mL). Z2 underwent in vivo testing on blueberry leaves in order to evaluate its usefulness in real-world settings. A reasonable protective effect was obtained with a control effectiveness of 93.0% at 200 μg/mL, which was superior to those of Az (83.0%) and Fl (52.0%). At 200 μg/mL, this chemical had an efficacy of 84.0% in terms of curative efficacy. These figures outperformed those of Az (69.0%) and Fl (48.0%). Scanning electron microscopy (SEM) experiments and light microscopy experiments showed that Z2 altered the integrity of the cell wall and cell membrane of the pathogenic fungus B.c., which led to an increase in the content of malondialdehyde (MDA), cellular leakage, and cellular permeability. Enzyme activity assays and molecular docking studies indicated that Z2 could act as a potential succinate dehydrogenase inhibitor (SDHI). It was hypothesized that Z2 could cause disruption of mycelial cell membranes, which in turn leads to mycelial death. According to the research, indole derivatives containing 1,3,4-thiadiazole were expected to evolve into new fungicides due to their significant antifungal effects on plant fungi.

Extracellular Vesicles from Scedosporium apiospermum Mycelial Cells: Implication for Fungal-Host Interplays.

The release of extracellular vesicles (EVs) has been implicated as an alternative transport mechanism for the passage of macromolecules through the fungal cell wall, a phenomenon widely reported in yeasts but poorly explored in mycelial cells. In the present work, we have purified and characterized the EVs released by mycelia of the emerging, opportunistic, widespread and multidrug-resistant filamentous fungus Scedosporium apiospermum. Transmission electron microscopy images and light scattering measurements revealed the fungal EVs, which were observed individually or grouped with heterogeneous morphology, size and electron density. The mean diameter of the EVs, evaluated by the light scattering technique, was 179.7 nm. Overall, the structural stability of S. apiospermum EVs was preserved during incubation under various storage conditions. The lipid, carbohydrate and protein contents were quantified, and the EVs' protein profile was evidenced by SDS-PAGE, revealing proteins with molecular masses ranging from 20 to 118 kDa. Through immunoblotting, ELISA and immunocytochemistry assays, antigenic molecules were evidenced in EVs using a polyclonal serum (called anti-secreted molecules) from a rabbit inoculated with conditioned cell-free supernatant obtained from S. apiospermum mycelial cells. By Western blotting, several antigenic proteins were identified. The ELISA assay confirmed that the anti-secreted molecules exhibited a positive reaction up to a serum dilution of 1:3200. Despite transporting immunogenic molecules, S. apiospermum EVs slightly induced an in vitro cytotoxicity effect after 48 h of contact with either macrophages or lung epithelial cells. Interestingly, the pretreatment of both mammalian cells with purified EVs significantly increased the association index with S. apiospermum conidia. Furthermore, EVs were highly toxic to Galleria mellonella, leading to larval death in a typically dose- and time-dependent manner. Collectively, the results represent the first report of detecting EVs in the S. apiospermum filamentous form, highlighting a possible implication in fungal pathogenesis.

Copper ion sorption ability of Alternaria alternata (Fr.) Keissl. and Fusarium oxysporum Schltdl. micromycetes

Our analysis of metabolic and ecological features of fungi indicates a high bioremediation potential of fungal mycelium. The relevance of the problems solved in this work is conditioned by insufficiently studied mechanisms and conditions of micromycetal activity on heavy metal detoxication. The effect of copper on the development of two micromycete species, namely, melanised Alternaria alternata (Fr.) Keissl., 1912 and hyaline Fusarium oxysporum Schltdl., 1824 cultivated on Czapek agar (with 2 or 3% sucrose) was compared; the ability of mycelium to sorb copper cations when grown in a liquid culture with 0, 0.05, 0.1, 0.25, and 0.5 mg Cu2+ / L was evaluated. F. oxysporum had noticeable advantages in terms of growth rate, tolerance, conidia production on medium with Cu2+. The effective concentration of copper (EC50), indicating the resistance of the fungus, increased for F. oxysporum by 1.5 times with increasing sucrose content in the medium. The melanised culture of A. alternata was superior to F. oxysporum in terms of sorption capacity. The percentage of Cu2+ extraction by fungal mycelium from the medium reached 40% in the case of F. oxysporum and twice as much in the case of A. alternata . The vast majority of copper was sorbed by the cell walls of hyphae and washed away by water. A small amount (a maximum of 0.16 µg Cu2+ /g dry mycelium of A. alternate ) penetrated inside the mycelial cells. F. oxysporum, which had no intracellular protective melanins, accumulated Cu2+ (2 to 14 times) less. The results show that the mechanisms of resistance to Cu2+ and sorption are different in the studied species: in Fusarium they are determined mainly by the barrier functions of hyphae cell walls, while in Alternaria melanin plays a significant role in protection against Cu toxic action.

New Vision of Cell Walls in Aspergillus fumigatus from Solid-State NMR Spectroscopy.

The fungal cell wall plays a critical role in regulating cellular integrity and communication, and serves as a frontline defense against stress. It is also a prime target for the development of antifungal agents. The cell wall is comprised of diverse polysaccharides and proteins and poses a challenging target for high-resolution structural characterization. Recently, the solid-state nuclear magnetic resonance (ssNMR) analysis of intact Aspergillus fumigatus cells has provided atomic-level insights into the structural polymorphism and functional assembly principles of carbohydrate components within the cell wall. This physical perspective, alongside structural information from biochemical assays, offers a renewed understanding of the cell wall as a highly complex and dynamic organelle. Here, we summarize key conceptual advancements in the structural elucidation of A. fumigatus mycelial and conidial cell walls and their responses to stressors. We also highlight underexplored areas and discuss the opportunities facilitated by technical advancements in ssNMR spectroscopy.

Histoplasma antigens as novel players for the development of new enzyme immunoassays for the serodiagnosis of histoplasmosis: A comparative study of their analytical performance.

Definitive diagnosis of histoplasmosis relies on culture and/or cytology/histopathology; however, these procedures have limited sensitivity and cultures are time-consuming. Antibodies detection by immunodiffusion has low sensitivity in immunocompromised individuals and uses histoplasmin (HMN), a crude antigenic extract, as reagent. Novel protein antigen candidates have been recently identified and produced by DNA-recombinant techniques to obtain standardized and specific reagents for diagnosing histoplasmosis. To compare the analytical performance of novel enzyme-linked immunosorbent assays (ELISAs) for antibodies testing for diagnosing histoplasmosis using different Histoplasma capsulatum antigens as reagents. The H. capsulatum 100kDa protein (Hcp100), the M antigen and its immunoreactive fragment F1 were produced by DNA-recombinant techniques. Galactomannan was purified from both the yeast and mycelial cell walls (yGM and mGM, respectively). The analytical performance of the ELISA tests for the serological detection of antibodies against these antigens was evaluated and compared with those obtained using HMN as reagent. Antibodies detection by the Hcp100 ELISA demonstrated 90.0% sensitivity and 92.0% specificity, versus 43.3% sensitivity and 95.0% specificity of the M ELISA, 33.3% sensitivity and 84.0% specificity of the F1 ELISA, 96.7% sensitivity and 94.0% specificity of the yGM ELISA, 83.3% sensitivity and 88.0% specificity of the mGM ELISA, and 70.0% sensitivity and 86.0% specificity for the HMN ELISA. In summary, Hcp100 is proposed as the most promising candidate for the serodiagnosis of histoplasmosis. The primary immunoreactive element in HMN proved to be GM rather than the M antigen. Nevertheless, a higher incidence of cross-reactions was noted with GM compared to M.

Identification of Trichderma harzianum in postharvest Agaricus bisporus and a novel control approach using eucalyptus essential oil emulsion

Pathogenic infections have been a key factor leading to mushroom deterioration. However, few studies have focused on the isolation, identification, and control of pathogens causing infections in mushroom. In this study, the Trichoderma harzianum strain TPS2 was screened from the tissues of infected Agaricus bisporus. To inhibit the pathogenic microorganism, the eucalyptus essential oil (EEO)-tamarind gum (TG) emulsion was prepared and its antimicrobial mechanism and preservative effect of postharvest Agaricus bisporus were explored through emulsion fumigation. According to the in vitro experiment results, the EEO-TG emulsion induced the accumulation of reactive oxygen species, lipid peroxidation of the cell membrane, and destruction of cell membrane integrity. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) results further revealed that the EEO-TG emulsion damaged the membrane permeability of T. harzianum, thereby resulting in the increased leakage of mycelial cell contents and achieving the antimicrobial effect. As the essential oil concentration in the EEO-TG emulsion increased, the effect on T. harzianum intensified. The ratio of EEO to TG at 1:10 was the minimum inhibitory concentration (MIC100), and the median inhibitory concentration (MIC50) was 1:40. In situ experiments, mushrooms fumigated with the EEO-TG emulsion exhibited high firmness, high whiteness, low electrical conductivity, and low weight loss, along with effective inhibition of the respiratory intensity and reduction in the excessive loss of nutrients. Therefore, the EEO-TG emulsion is of great significance in preventing postharvest diseases symptoms and delaying the senescence of mushrooms.

Design and Synthesis of Novel Diphenyl Ether Carboxamide Derivatives To Control the Phytopathogenic Fungus Sclerotinia sclerotiorum.

Sclerotinia stem rot (SSR) caused by the phytopathogenic fungus Sclerotinia sclerotiorum has led to serious losses in the yields of oilseed rape and other crops every year. Here, we designed and synthesized a series of carboxamide derivatives containing a diphenyl ether skeleton by adopting the scaffold splicing strategy. From the results of the mycelium growth inhibition experiment, inhibition rates of compounds 4j and 4i showed more than 80% to control S. sclerotiorum at a dose of 50 μg/mL, which is close to that of the positive control (flubeneteram, 95%). Then, the results of a structure-activity relationship study showed that the benzyl scaffold was very important for antifungal activity and that introducing a halogen atom on the benzyl ring would improve antifungal activity. Furthermore, the results of an in vitro activity test suggested that these novel compounds can inhibit the activity of succinate dehydrogenase (SDH), and the binding mode of 4j with SDH was basically similar to that of the flutolanil derivative. Morphological observation of mycelium revealed that compound 4j could cause a damage on the mycelial morphology and cell structure of S. sclerotiorum, resulting in inhibition of the growth of mycelia. Furthermore, in vivo antifungal activity assessment of 4j displayed a good control of S. sclerotiorum (>97%) with a result similar to that of the positive control at a concentration of 200 mg/L. Thus, the diphenyl ether carboxamide skeleton is a new starting point for the discovery of new SDH inhibitors and is worthy of further development.