This study used typical chestnut soil from Huangyuan County, Qinghai Province as the experimental soil, and selected three oat varieties, “Baiyan 7”, “Qingyan 2”, and “Qinghai 444”, for mixed sowing. A blank control (CK) and five treatments with different ratios of exogenous nutrient bag waste (PF-R-1 to PF-R-5) were established. Through soil physicochemical analysis, microbial biomass determination, RNA sequencing (RNA-Seq), and weighted gene co-expression network analysis (WGCNA), the effects of exogenous nutrient bag waste with different ratios soil nutrient availability, microbial activity and root gene expression in oats were systematically evaluated. The results showed that most treatments enhanced soil organic matter, nitrogen content, and microbial activity. Transcriptomic analysis revealed that the comparison between PF-R-3 and PF-R-2 treatments exhibited the highest total number of differentially expressed genes (DEGs), with enrichment in pathways related to ribosomal protein translation, oxidative phosphorylation energy metabolism, and DNA damage repair. These enrichment patterns suggest potential changes in energy metabolism and protein synthesis, although direct functional validation was not performed. Moreover, DEGs uniquely identified in PF-R-3 were significantly enriched in nitrogen metabolism and secondary metabolite biosynthesis pathways; WGCNA identified the coral2 module as the core regulatory module, with its genes predominantly enriched in functions related to transmembrane transport and plant hormone signal transduction, which were highly consistent with efficient root nutrient acquisition and the maintenance of metabolic homeostasis. Fluorescence staining confirmed the successful colonization of oat root tissues by Morchella mycelium. Taken together, based on the integration of soil nutrient supply stability, root gene expression regulation, and fungal colonization evidence, the PF-R-3 treatment (wheat + corncob + oat) emerged as a promising candidate among the tested formulations under the experimental conditions. However, further field trials incorporating yield and quality data are needed before a definitive recommendation can be made. This study provides a theoretical basis and technical support for the resource utilization of exogenous nutrient bag waste and the green cultivation of oat in alpine regions.

The application of exogenous nutrient bags (ENBs) is critical for achieving high yields in morel mushroom cultivation; however, the high cost and lack of locally adapted formulations have become major constraints for the sustainable expansion of this industry. This study aimed to evaluate the effects of ten treatments, including a conventional formulation control, incorporating locally available agricultural by-products (rapeseed straw, organic fertilizer, alfalfa, and oats), on soil fertility, enzyme activity, microbial community structure, and ultimately the yield and quality of cultivated Morchella sextelata in Jianzha County, Qinghai. A randomized complete block design was established, and soil physicochemical properties, microbial biomass carbon/nitrogen/phosphorus (MBC/MBN/MBP), and enzyme activities (urease, phosphatase, and catalase) were measured at 38, 62, and 137 days post-application, while bacterial and fungal community compositions were characterized via high-throughput sequencing of 16S rRNA and ITS2 genes. Our results demonstrated that the wa-60 treatment (30% wheat + 60% alfalfa) outperformed all the other formulations, delivering the highest yield (1.32 ± 0.08 kg/m²) and a 28.6% increase over the control (1.03 ± 0.06 kg/m²; p < 0.01), while maintaining a high total amino acid content (19.8 g/100 g). Soil analysis revealed that wa-60 notably enhanced urease activity (peaking at 85 µg/g/h at day 137) and alkaline phosphatase activity (322.55 µg/g/h), alongside significant increases in MBC and MBN. Microbiome profiling further demonstrated that wa-60 selectively increased the relative abundance of various taxa, particularly the bacterial phylum Bacteroidota and the fungal phylum Mortierellomycota. Correlation analyses indicated strong positive associations among these enriched taxa (primarily at the genus level), enhanced enzyme activities, and improved soil nutrient availability. Collectively, these findings establish that the wa-60 formulation, leveraging locally sourced alfalfa, represents a cost-effective and high-performance strategy for morel cultivation in the Qinghai Plateau, providing a microbial–ecological basis for optimizing ENB design and offering a practical pathway for recycling agricultural waste in edible mushroom production.