Mikania micrantha, one of the world’s most destructive invasive species, is known for causing significant ecological and economic harm. While extensive research has focused on its growth characteristics, secondary metabolites, and control measures, its chemical interactions with the environment—particularly the role of flavonoids in shaping soil microbial communities—remain underexplored. In this study, we identified and quantified ten flavonoids from M. micrantha root exudates using UPLC-MS, including Hispidulin, Isorhamnetin, and Mikanin. To examine their impact, crude flavonoid extracts were applied to soil in potted experiments, which demonstrated that these compounds significantly increased soil fungal diversity and boosted the relative abundance of arbuscular mycorrhizal fungi (AMF). Furthermore, KEGG pathway analysis revealed that flavonoid addition elevated the copy numbers of genes involved in nitrogen cycling and metabolic functions, enhancing nutrient availability and microbial activity. Additionally, crude flavonoid extracts promoted the relative abundance of beneficial soil bacteria, such as Achromobacter, as well as AMF, both of which contribute to nutrient acquisition, plant growth, and soil health. These findings indicate that M. micrantha’s flavonoids can alter soil microbial community composition, thereby creating a favorable environment that reinforces its competitive edge over native plants.

Protoplast-based transformation is a vital tool for genetic studies in fungi, yet no protoplast method existed for P. sclerotiorum-scaumcx01 before this study. Here, we optimized protoplast isolation, regeneration, and transformation efficiency. The highest protoplast yield (6.72 × 10⁶ cells/mL) was obtained from liquid mycelium after 12 h of enzymatic digestion at 28 °C using Lysing Enzymes, Yatalase, cellulase, and pectinase. Among osmotic stabilizers, 1 M MgSO₄ yielded the most viable protoplasts. Regeneration occurred via direct mycelial outgrowth and new protoplast formation, with a 1.02% regeneration rate. PEG-mediated transformation with a hygromycin resistance gene and GFP tagging resulted in stable GFP expression in fungal spores and mycelium over five generations. LC/MS-based metabolomic analysis revealed significant changes in glycerophospholipid metabolism, indicating lipid-related dynamics influenced by GFP tagging. Microscopy confirmed successful colonization of tomato roots by GFP-tagged scaumcx01, with GFP fluorescence observed in cortical tissues. Enzymatic (cellulase) seed pretreatment enhanced fungal colonization by modifying root surface properties, promoting plant–fungal interaction. This study establishes an efficient protoplast transformation system, reveals the metabolic impacts of genetic modifications, and demonstrates the potential of enzymatic seed treatment for enhancing plant–fungal interactions.