Journal of Fungi

Sclerotium formation represents a critical transition phase in the life cycle of morel, shifting from vegetative growth to dormant structures. The capacity for sclerotium formation directly influences the yield and stability of artificial cultivation. To elucidate the molecular regulatory mechanisms underlying this process, a combined transcriptomics and metabolomics approach was employed to analyze gene expression and metabolite dynamics during sclerotium development of Morchella eximia. A total of 2567 differentially expressed metabolites (DEMs) and 2314 differentially expressed genes (DEGs) were detected, primarily enriched in amino acid metabolism, lipid synthesis, and energy metabolism pathways. Amino acid metabolism facilitates protein synthesis and supplies carbon skeletons, while lipid metabolic networks, particularly de novo fatty acid synthesis from acetyl-CoA precursors, glycerophospholipid metabolism, sphingolipid metabolism, and unsaturated fatty acid biosynthesis, play a central role in sclerotium formation. A regulatory model was constructed, focusing on signal response, transcriptional regulation, nutrient transport and metabolism, morphology transition, lipid accumulation, and membrane system remodeling, demonstrating that lipids not only provide energy storage and membrane structural components for sclerotia but also mediate developmental transitions and environmental adaptation through signaling molecules and regulation of membrane properties. These findings systematically reveal the regulatory network governing morel sclerotium formation at the multi-omics level, with particular emphasis on the central role of lipid metabolism and membrane remodeling. The results offer a theoretical foundation for improving morel cultivation yield and stability through targeted metabolic regulation strategies.

Ophiocordyceps xuefengensis is an important medicinal fungus with considerable pharmaceutical and economic value. However, its industrial and scientific utilization has been severely limited by the lack of an efficient genetic transformation system, largely due to limited genomic information and wild growth. In this study, we established an efficient and stable plasmid transformation system within O. xuefengensis protoplasts mediated by PEG. To overcome low protoplast yield and transformation efficiency, key factors influencing protoplast preparation including enzyme composition and concentration, fungal age, and digestion conditions were systematically optimized. The optimal protocol involved digesting 4-day-old mycelia with a mixture of 1.5% lywallzyme 1 and 1.5% snailase at 34 °C and 130 rpm for 3.5 h, yielding at least 9.42 × 10⁷ CFU/mL protoplasts. Protoplast regeneration was significantly enhanced in PY medium supplemented with 0.6 M mannitol. Under these optimized conditions, a transformation efficiency of 45.5% was achieved, with stable plasmid integration confirmed over four successive generations. Furthermore, the transformation system was successfully applied to functional gene characterization by driving exogenous gene expression using the endogenous gpd1 promoter. This study provides a foundational platform for functional gene analysis and paves the way for further elucidation of growth and development mechanisms and metabolic engineering in O. xuefengensis.

Blueberry rust disease is caused by the fungal pathogen Pucciniastrum minimum (syn. Thekopsora minima). Despite its importance as a plant pathogen, there are relatively few published studies on P. minimum. This study investigated and refined methodologies to cultivate and study this obligate parasite. P. minimum was successfully cultivated on detached blueberry leaves by misting leaves with water, followed by dusting with dry urediniospores. In vitro germination of urediniospores on water agar was achieved using a spore dusting technique, and germination rates were 70% higher compared to a spore suspension. Time after leaf detachment affected urediniospore germination and highlighted the importance of the processing time for replicability between experiments. Urediniospore viability could be evaluated by co-staining with fluorescein diacetate and propidium iodide, and the assessed viability was significantly higher than germination rates achieved in vitro. In detached leaf inoculations, leaves sourced from inside the glasshouse developed more rust than those from outside; this is discussed in the context of knowledge gaps on the infection process of P. minimum. This study resolves some key methodological issues involved with studying P. minimum rust urediniospores, and the general protocols we developed can be applied to other rust species for biological survival research.