The global rise of drug-resistant fungal pathogens, particularly non-albicans Candida species, necessitates the development of novel therapeutic strategies. In this study, we investigated the antifungal, antibiofilm, and mitochondrial/membrane-disruptive activities of NET1 and NET3, two novel, protease-resistant synthetic antimicrobial peptides (AMPs) composed of D- and L-amino acid combinations, against reference strains of Candida albicans, Candida krusei (Pichia kudriavzevii), Candida lusitaniae (Clavispora lusitaniae), and Candida parapsilosis. Minimum inhibitory concentration (MIC) values for both peptides ranged from 8 to 16 μg/mL across all tested strains. Notably, both peptides inhibited initial biofilm formation at sub-MIC levels in C. albicans (MBICs of 2 μg/mL for NET1 and 1 μg/mL for NET3; 8- to 16-fold below their MICs). To elucidate the cellular mechanisms accompanying fungal growth inhibition, key mitochondrial and membrane-associated changes and membrane hallmarks were evaluated following peptide exposure. At 0.5 × MIC, JC-1 fluorescent staining revealed that both NET1 and NET3 induced widespread collapse of the mitochondrial membrane potential. At 1 × MIC, subcellular fractionation and spectrophotometric analysis at 550 nm showed a marked shift of Cytochrome c from the mitochondrial to the cytosolic fraction, consistent with outer mitochondrial membrane permeabilization. Furthermore, CF®488A Annexin V and Propidium Iodide (PI) dual-staining at 0.5 × MIC revealed phosphatidylserine externalization with intact membranes (predominantly early apoptotic-like Annexin V+/PI− cells) in C. albicans and C. parapsilosis, and dual-positive staining (Annexin V+/PI+, consistent with late apoptosis-like progression or secondary membrane permeabilization) in peptide-treated C. krusei and C. lusitaniae; primary necrotic (Annexin V−/PI+) cells were rarely detected at sub-MIC levels. These results suggest that the antifungal activity of NET1 and NET3 involves a mitochondria-associated, apoptosis-like response alongside membrane disruption rather than primary necrosis alone. Overall, these protease-resistant AMPs represent promising therapeutic candidates for combating opportunistic Candida infections.
Among Candida species, Candida albicans is the leading cause of candidemia and invasive candidiasis, which can cause morbidity and mortality in immunocompromised patients. Fungi have developed resistance to the few existing antifungal drugs, which are limited in efficacy and mechanistic diversity, emphasizing the urgent need for novel antifungals and combination therapies. Developing novel antifungals is more challenging than developing antibacterial agents, since eukaryotic fungi share components and metabolism with human cells, making the discovery of compounds with low cytotoxicity difficult. Phenolic compounds from plant-based essential oils with broad-spectrum antifungal activity and minimal cytotoxicity to humans, such as carvacrol, are regaining interest as antifungals. This study investigates the combined impact of carvacrol treatment and conditional repression of ARO1, a key enzyme of the shikimate pathway, which synergistically disrupts metabolic activity, mitochondrial function, intracellular adenosine triphosphate levels, causes oxidative stress, and perturbs mitochondrial and plasma membrane potentials, as visualized by altered cell membrane and wall ultrastructure in Candida albicans. These findings indicate a potential strategy for combination therapy to address antifungal resistance.
IntroductionBlack spot disease, caused by Alternaria brassicicola, significantly reduces the yield and quality of broccoli. Growing concerns about environmental pollution and fungicide resistance have increased interest in beneficial microorganisms as sustainable biological control agents for plant disease management.MethodsThis study evaluated the in vitro and in planta antifungal activity of cell-free culture filtrates (CFFs) produced by the rice rhizosphere bacterium Burkholderia seminalis R456 against A. brassicicola and investigated candidate bioactive metabolites associated with the observed antifungal effects.Results and discussionR456 CFF suppressed mycelial growth in a fermentation-time- and concentration-dependent manner; the filtrate obtained after 5 days of fermentation showed the best activity, and 50% CFF caused complete or near-complete inhibition under the plate-assay conditions. The filtrate reduced conidial germination to 3.87%. Microscopic observations showed that CFF treatment was associated with pronounced morphological abnormalities in A. brassicicola, including hyphal and conidial deformation, shrinkage, collapse, and surface irregularities. In planta assays under controlled conditions showed that R456 CFF suppressed black spot disease, achieving control efficacies of 71.56% on detached leaves and 70.72% on whole plants. The 100% ethanol-eluted fraction showed the best activity among the fractions tested, and LC-MS/MS comparison with an authentic standard supported pyrrolnitrin as an important candidate antifungal metabolite. Functional validation with the pyrrolnitrin standard further showed concentration-dependent disease suppression, with control efficacies at 50 mg/L of 100% on detached leaves and 96.50% on whole plants. Overall, these findings support R456-derived extracellular metabolites as a potential antifungal resource and provide direct evidence that pyrrolnitrin is biologically active in the A. brassicicola-broccoli pathosystem; however, the quantitative contribution of
Soil reclamation is widely practiced in saline–alkali land areas. However, limited research has focused on how reclamation duration regulates soil microbial communities and crop yield via modifying soil environmental conditions. In this study, high-throughput sequencing, ecological network analysis, and soil physicochemical characterization were integrated to investigate variations in soil properties, microbial community composition, and crop yield across saline–alkali fields with different reclamation durations (1, 5, and 10 years). The results showed that prolonged reclamation significantly reduced soil salinity and increased soil nutrient contents as well as wheat yield. The richness of fungi and protists increased with the extension of reclamation durations, whereas bacterial richness exhibited an opposite trend. Network-associated key taxa of bacteria, fungi and protists were significantly correlated with wheat yield, soil salinity, soil organic matter (SOM), total nitrogen (TN), as well as the activities of leucine aminopeptidase (LAP) and β-glucosidase (Glu). Structural equation modeling (SEM) further revealed that soil salinity was associated with soil physicochemical traits, network-associated key taxa and enzyme activities, altered the soil microecological environment, and ultimately affected wheat yield. In conclusion, reclamation durations mitigated soil salinity, which drove the screening of microbial taxa by soil microhabitats and reconstructed the overall microbial community structure, thereby influencing wheat productivity. These findings provide theoretical and technical support for the sustainable reclamation of saline–alkali land, health management of farmland soil, and stable improvement of grain productivity.
