Locoweeds, including Oxytropis and Astragalus species, are globally recognized as plants containing swainsonine (SW), a neurotoxic alkaloid that induces neurological dysfunction and growth inhibition in livestock. SW is produced by endophytic fungi in plants; the pyrroline-5-carboxylate reductase (P5CR) gene is critical in the fungal SW biosynthetic pathway. In this study, a P5CR gene knockout mutant (ΔP5CR) was constructed from the endophytic fungus Alternaria oxytropis OW7.8 isolated from Oxytropis glabra. Compared to the wild-type strain (A. oxytropis OW7.8), the SW content in the ΔP5CR mycelia was significantly reduced, indicating that the P5CR gene plays a crucial role in promoting SW biosynthesis. Compared to the wild-type strain A. oxytropis OW7.8, the ΔP5CR mutant exhibited distinct morphological alterations in both colony and mycelial structures. The transcriptomic analysis of A. oxytropis OW7.8 and ΔP5CR revealed the downregulation of six genes associated with SW biosynthesis. Metabolomic profiling further demonstrated altered levels of six metabolites linked to SW synthesis. These findings provide foundational insights into the molecular mechanisms and metabolic pathways underlying SW biosynthesis in fungi. They hold significant value for future strategies to control SW in Oxytropis glabra and contribute positively to the protection and sustainable development of grassland ecosystems.

The swnN gene in the endophytic fungus Alternaria oxytropis OW 7.8 isolated from Oxytropis glabra was identified, and the gene knockout mutant ΔswnN was first constructed in this study. Compared with A. oxytropis OW 7.8, the ΔswnN mutant exhibited altered colony and mycelia morphology, slower growth rate, and no swainsonine (SW) in mycelia. SW was detected in the gene function complementation strain ΔswnN/swnN, indicating that the function of the swnN gene promoted SW biosynthesis. Six differentially expressed genes (DEGs) closely associated with SW synthesis were identified by transcriptomic analysis of A. oxytropis OW 7.8 and ΔswnN, with P5CR, swnR, swnK, swnH2, and swnH1 down-regulating, and sac up-regulating. The expression levels of the six genes were consistent with the transcriptomic analysis results. Five differential metabolites (DEMs) closely associated with SW synthesis were identified by metabolomic analysis, with L-glutamate, α-ketoglutaric acid, and L-proline up-regulating, and phosphatidic acid (PA) and 2-aminoadipic acid down-regulating. The SW biosynthetic pathways in A. oxytropis OW 7.8 were predicted and refined. The results lay the foundation for in-depth elucidation of molecular mechanisms and the SW synthesis pathway in fungi. They are also of importance for the prevention of locoism in livestock, the control and utilization of locoweeds, and the protection and sustainable development of grassland ecosystems.

Groundwater harbors phylogenetically diverse Candidate Phyla Radiation (CPR) bacteria, representing an ideal ecosystem for studying this microbial dark matter. However, no CPR strains have been successfully isolated from groundwater, severely limiting further research. This study employed a multi-metagenome approach, integrating time-resolved sampling, antibiotic/nutrient interventions, and microbial correlation networks to unravel CPR ecological roles in groundwater and provide insights into their subsequent cultivation. Through 36 metagenomes from a groundwater system containing at least 68 CPR phyla, we revealed the time-sensitive collapse of CPR communities: total abundance plummeted from 7.9% to 0.15% within 48 h post-sampling, driven by competition with rapidly dividing non-CPR bacteria, such as members of Pseudomonadota. Ampicillin (100 mg/L) stabilized CPR communities by suppressing competitors, whereas low-nutrient conditions paradoxically reversed this effect. Long-term enrichment (14 months) recovered 63 CPR phyla (0.35% abundance), revealing their survival resilience despite nutrient deprivation. Correlation networks prioritized Actinomyces, a novel Acidimicrobiaceae genus, Aestuariivirga, Baekduia and Caedimonadaceae as potential CPR partners, providing actionable targets for co-culture trials. Here, we propose actionable recommendations spanning groundwater sampling, activation status, identification of CPR symbiotic partners, and optimization of culture conditions, which bypass traditional blind cultivation and are critical for future efforts to cultivate CPR bacterial strains from groundwater. Cultivating CPR bacteria will contribute to clarifying their diversity, ecological roles, evolutionary mechanisms, metabolic pathways, and genetic potential.