Introduction: Currently, straw biodegradation and soil improvement in rice-mushroom rotation systems have attracted much attention. However, there is still a lack of studies on the effects of rice-mushroom rotation on yield, soil properties and microbial succession.Methods: In this study, no treatment (CK), green manure return (GM) and rice straw return (RS) were used as controls to fully evaluate the effect of Stropharia rugosoannulata cultivation substrate return (SRS) on soil properties and microorganisms.Results: The results indicated that rice yield, soil nutrient (organic matter, organic carbon, total nitrogen, available nitrogen and available potassium) and soil enzyme (urease, saccharase, lignin peroxidase and laccase) activities had positive responses to the rice-mushroom rotation. At the interannual level, microbial diversity varied significantly among treatments, with the rice-mushroom rotation significantly increasing the relative alpha diversity index of soil bacteria and enriching beneficial microbial communities such as Rhizobium, Bacillus and Trichoderma for rice growth. Soil nutrients and enzymatic activities were significantly correlated with microbial communities during rice-mushroom rotation. The fungal-bacterial co-occurrence networks were modular, and Latescibacterota, Chloroflexi, Gemmatimonadota and Patescibacteria were closely related to the accumulation of nutrients in the soil. The structural equation model (SEM) showed that fungal diversity responded more to changes in soil nutrients than did bacterial diversity.Discussion: Overall, the rice-mushroom rotation model improved soil nutrients and rice yields, enriched beneficial microorganisms and maintained microbial diversity. This study provides new insights into the use of S. rugosoannulata cultivation substrates in the sustainable development of agroecosystems.

Glutathione reductase (GR) is essential for maintaining cellular redox homeostasis by sustaining reduced glutathione (GSH) levels. In Hypsizygus marmoreus, GR silencing led to impaired mycelial growth, elevated reactive oxygen species (ROS) accumulation, and disrupted antioxidant enzyme activity, ultimately hindering fruiting body development. Mitochondrial size was markedly reduced in GR-silenced strains, indicating compromised cellular metabolism. Supplementation with the reducing agent vitamin C (Vc) partially restored redox balance and enzyme activity in a developmental stage–dependent manner, alleviating the defects caused by GR suppression. Moreover, GR was found to influence lignocellulose-degrading enzyme activity, further linking redox regulation to substrate utilization. Overall, these findings demonstrate that GR plays a central role in coordinating redox balance, energy metabolism, and enzyme function in H. marmoreus, providing new insights for enhancing industrial mushroom production through antioxidant regulation.

The artificially cultivated edible mushroom Stropharia rugosoannulata is widely promoted and cultivated in China because of its ability to efficiently decompose agricultural and forestry waste. However, methods for CRISPR/Cas9 genome editing have not yet been established for S. rugosoannulata. In this study, we identified three SrU6 promoters in S. rugosoannulata and constructed the CRISPR/Cas9 expression vector GPiE-SrU6. Moreover, we found that mutant strains were obtained only when the expression of the single guide RNA (sgRNA) was driven by the SrU6-3 promoter. We subsequently employed a tandemly repeated SrU6-tRNA-sgRNA module to knock out two sites within the ura3 gene. The expression vector was introduced into the mycelium via Agrobacterium-mediated transformation (ATMT). Following dual selection with 60 μg/mL hygromycin (Hyg) and 0.2 mg/mL 5-fluoroorotic acid (5-FOA), stable transformants were obtained and subcultured. The mutation efficiency at the targeted ura3 locus was subsequently assessed. The CRISPR/Cas9 system successfully disrupted the target marker gene (ura3), achieving an editing efficiency of 14.9%. In summary, this study reports the first successful establishment of a CRISPR/Cas9 genome editing system in S. rugosoannulata. This study not only meets a future need for genetic manipulation tools for S. rugosoannulata but also provides a robust platform for engineering superior strains for eco-circular agriculture.

Spent mushroom substrate (SMS) return is a vital strategy for agricultural waste recycling and soil fertility improvement, yet its ecological impacts of duration remain poorly understood. This study employed metagenomic sequencing to explore soil fertility, microbial dynamics, and nitrogen cycling across different SMS return durations (0, 1, and 3 years) within rice–mushroom crop rotation systems. Soil nutrients (organic matter, total nitrogen, total phosphorus) initially decreased and then increased throughout the rice growth cycle. The one-year return (y1) induced early nutrient depletion, whereas the three-year return (y3) significantly enhanced late-stage nutrient accumulation. With increasing duration, bacterial and archaeal assembly shifted from stochastic toward deterministic processes, while fungal diversity and stochasticity decreased continuously. Co-occurrence network analysis demonstrated that SMS return increased network complexity and intercommunity competition. This transition was accompanied by a functional shift in keystone taxa from those responsive to exogenous organic matter in y1 to those mediating nitrogen fixation, anammox, and sulfur metabolism in y3. Nitrogen cycling in y1 increased potential N₂O emission risks through nirS upregulation and nosZ downregulation, whereas y3 mitigated inorganic nitrogen loss by upregulating gene abundances of ammonia assimilation, nitrification, and DNRA genes. Notably, the structure of nitrogen-cycling genes fluctuated in y1 but was resilient to y0 levels in y3. These findings demonstrated that while initial SMS return triggered ecological fluctuations and environmental risks, continuous return (y3) achieved functional stability by reshaping microbial niches. This study highlights the importance of SMS return duration in balancing soil fertility enhancement with environmental risk mitigation in sustainable paddy ecosystems.

Morchella is an edible and medicinal fungus valued for its distinctive flavor and nutritional properties. However, unstable yield and quality remain major constraints on its commercial cultivation. Although soybean meal is used as an organic nutrient supplement in edible mushroom production, its effects on protein accumulation and transcriptional regulation in Morchella remain unclear. This study combined fruiting-body protein measurements with transcriptome analysis to compare soybean meal supplied through soil application or exogenous nutrient bags and to characterize tissue-specific responses in the cap and stipe. Soybean meal increased protein content in an application method-, concentration-, and tissue-dependent manner. Soil application was more effective than nutrient-bag supplementation, while stipes exhibited a stronger transcriptional response than caps. RNA sequencing detected 11,982 expressed genes and revealed both shared and tissue-specific responses to the two supplementation methods. Gene Ontology analysis showed broadly similar functional categories among comparison groups, whereas KEGG enrichment analysis revealed distinct metabolic responses. In caps, soil application was associated with glutathione metabolism and amino sugar and nucleotide sugar metabolism, while nutrient-bag supplementation was associated with the pentose phosphate pathway, glyoxylate and dicarboxylate metabolism, and pentose and glucuronate interconversions. In stipes, the genes involved in ribosome and aminoacyl-tRNA biosynthesis were downregulated under soil application and nutrient-bag supplementation, respectively. Weighted gene co-expression network analysis identified three modules associated with protein content and highlighted ribosome, aminoacyl-tRNA biosynthesis, glycerophospholipid metabolism, and arginine biosynthesis as candidate pathways. The most highly connected genes in each key module were identified as hub genes, and selected expression patterns were supported by qRT-PCR. These findings provide a transcriptomic framework for understanding how soybean meal supplementation is associated with protein accumulation and tissue-specific physiological responses in Morchella.

Preharvest nutrient inputs can modify mushroom composition, but whether graded supplementation differentially affects the protein and amino acid composition of anatomically distinct fruiting-body tissues remains unclear. This study aimed to characterize the compositional and molecular responses of the cap and stipe of Morchella sextelata to additional soybean meal. Protein and 18 hydrolyzed amino acids were quantified following 0–10% supplementation, while the control, 2%, 4%, and 8% treatments were analyzed by transcriptomics and proteomics. Protein content responded nonlinearly and was highest at 8% in both tissues; total and essential amino acid contents were also highest in 8% caps. However, cap protein-normalized essential amino acid proportionality did not improve correspondingly, distinguishing compositional enrichment from amino acid balance. Caps generally exhibited more differentially expressed genes, whereas stipes contained more differentially expressed proteins, with downregulated proteins predominating in the 2 and 8% stipe comparisons. Integrated analyses associated these responses with carbon and amino acid metabolism, translation, protein homeostasis, energy metabolism, and RNA processing. These findings demonstrate tissue- and molecular-layer-dependent responses to preharvest nutrient supplementation. Future studies should determine whether the associated pathways translate into altered enzyme activities, metabolic fluxes, and sensory-relevant metabolites.