The ergosterol pathway is a prime antifungal target. The minimum inhibitory concentration (MIC) assay is a simple research tool that determines the lowest concentration at which a novel antimicrobial with limited scope to determine the mechanism of action for a drug. In this study, we show that by adding hydrogen peroxide, an oxidative stressor, or glutathione (GSH), an antioxidant, to modify a commonly performed MIC assay allowed us to screen selectively for new antifungal drugs that target ergosterol biosynthesis in fungi. A human pathogen, Microsporum gypseum, was used as a test organism. When exposed to ergosterol targeting drugs, the hydrogen peroxide treatment significantly decreased fungal survival by reducing ergosterol in the cell wall, whereas GSH increased survival of M. gypseum. Further, by performing a series of experiments with M. gypseum, it was determined that the oxidative stress from hydrogen peroxide causes cell death at different developmental stages. These findings allow us to describe a simple, high-throughput method for simultaneously screening new antifungal drugs for activity and effects on the ergosterol pathway. By using this tool, two isoquinoline alkaloids were discovered to be potent inhibitors of ergosterol biosynthesis by reducing the amount of ergosterol without affecting the expression of 1,3-β-glucan.

Cryptococcus neoformans threaten the health, causing cryptococcal meningitis and pneumonia, especially in immunosuppressed patients, which can be fatal. Recently, our research group evaluated and studied the mechanisms of action of four synthetic peptides (SP) against C. neoformans. Here, in silico and in vitro analyses help deepen understanding of peptides' mechanisms of action. The interaction of the peptides with a membrane receptor was analyzed by docking analysis, in addition to ROS overproduction and the modulation of redox metabolism, inhibition of ergosterol biosynthesis, and release of cytochrome c. Out of four, three peptides interacted with membrane receptor PHO36 altering its structure and function and leading to a higher accumulation of O₂- and H2O2. C. neoformans cells treated with SP presented a reduction in the activity of antioxidant enzymes, corroborating ROS accumulation. However, in the presence of the antioxidant ascorbic acid, some peptides could not induce this oxidative stress and have the activity against C. neoformans affected. Curiously, two of these SPs still maintained the activity against C. neoformans and even induced the membrane pore formation as revealed by propidium iodide uptake assay, revealing their mechanism of action is ROS-independent. Additionally, SPs inhibited the biosynthesis of ergosterol, which corroborates the pore formation on the membrane of C. neoformans cells, inhibited the lactate dehydrogenase activity affecting the cell metabolism, and induced the release of Cyt c from the mitochondria inducing death by apoptosis in the cryptococcal cells. Our findings strongly suggest that SPs act by multiple mechanisms, making it difficult for C. neoformans to acquire resistance highlighting the potential of SPs as alternative molecules in treating infections caused by C. neoformans.

Fusarium head blight (FHB) can cause contamination of cereal grain with mycotoxins. Winter wheat is also infected with FHB. It is more resistant than durum wheat to head infection and less than other small grain cereals. The aim of this study was to identify winter wheat lines that combine low head infection and kernel damage with low toxin contamination. Resistance to FHB of 27 winter wheat lines and cultivars was evaluated over a three-year experiment established in two locations. At the anthesis stage, heads were inoculated with Fusarium culmorum isolates. The FHB index was scored and the percentage of Fusarium-damaged kernels (FDKs) assessed. The grain was analysed for type B trichothecenes (deoxynivalenol and derivatives, nivalenol) and zearalenone content. The average FHB index was 12.9%. The proportion of FDK was 6.9% (weight) and 8.5% (number). An average content of deoxynivalenol amounted to 3.543 mg/kg and nivalenol to 2.115 mg/kg. In total, it was 5.804 m/kg of type B trichothecenes. The zearalenone content in the grain was 0.214 mg/kg. Relationships between FHB index, FDK and mycotoxin contents were highly significant for wheat lines; however, they were stronger for FDK versus mycotoxins. Breeding lines combing all types of FHB resistance were found, and five of them had resistance similar to that of wheat lines with the Fhb1 gene.

Growing acreage and changing consumer preferences cause increasing interest in the cereal products originating from organic farming. Lack of results of objective test, however, does not allow drawing conclusions about the effects of cultivation in the organic system and comparison to currently preferred conventional system. Field experiment was conducted in organic and conventional fields. Thirty modern cultivars of winter wheat were sown. They were characterized for disease infection including Fusarium head blight, seed sowing value, the amount of DNA of the six species of Fusarium fungi as well as concentration of ergosterol and trichothecenes in grain. The intensity Fusarium head blight was at a similar level in both systems. However, Fusarium colonization of kernels expressed as ergosterol level or DNA concentration was higher for the organic system. It did not reflect in an increased accumulation of trichothecenes in grain, which was similar in both systems, but sowing value of organically produced seeds was lower. Significant differences between analyzed cropping systems and experimental variants were found. The selection of the individual cultivars for organic growing in terms of resistance to diseases and contamination of grain with Fusarium toxins was possible. Effects of organic growing differ significantly from the conventional and grain obtained such way can be recommended to consumers. There are indications for use of particular cultivars bred for conventional agriculture in the case of organic farming, and the growing organic decreases plant stress resulting from intense fertilization and chemical plant protection.